ENZYME TECHNOLOGY

Enzymes are biological catalysts. They increase the rate of chemical reactions
taking place within living cells without suffering any overall change
The use of purified enzymes for generating a useful product or service constitutes
enzyme technology.
The reactions of enzyme catalyzed reactions are termed substrate and each
enzyme is quite specific in character, acting on a particular substrate to produce a
particular product or products.
1.2 BRIEF HISTORY OF ENZYMES
The word enzyme literally means “in yeast” (en=in, zyme=yeast). This is
originated from the fast that ethyl alcohol and CO2 are produced by the enzyme ‘zymase’,
which is present in yeast cells.
The term ‘enzyme’ was introduced by kuhne in 1878, although the first
observation of enzyme activity in a test tube was reported by payen and persoz in 1833.
During 1890’s Fisher suggested the lockand key’ model of enzyme action, while a
mathematical model of enzyme action was proposed by Michaelis and Menten in 1913.
In 1926, Sumner crystallized for the first time an enzyme (urease).
The transition state theory of enzyme action was put forth by pauling in 1948,
and in 1951 pauling and corey discovered the α -helix and β -sheet structures of
enzymes.
Sanger in 1953, determined the amino acid sequence of a protein (insulin). In
1986, Cech discovered catalytic RNA, while Lerner and Schutlz developed catalytic
antibodies.
1.3 GENERAL PROPERTIES OF ENZYMES
• They are synthesized only by living cell
• Thermolabile, large molecular weight.
• They are needed in minute quantities for catalytic action.
• Their chemical nature is not altered irreversibly during the course of catalytic
activity.
• The most important property is its high reaction rate.
Enzyme catalyzed reactions are 103 to 1016 times faster than corresponding
uncatalyzed reactions and several times faster than the chemically catalyzed reaction. For
ex, the activation energy for the decomposition of hydrogen peroxide varies depending
on the type of catalysis.
H2O2 → 1/2O2 + H2O
Activation Energy – Uncatalyzed reaction – 18 kcal/mole
– Chemically catalyzed reaction – 13 kcal/mole
– Enzyme catalyzed reaction – 7 kcal//mole.
All enzymes are proteins. Some RNA molecules have catalytic action; these are called
‘ribozymes’.
Some protein enzymes require a non-protein group for their activity. This group is
either a cofactor, such as metal ions, Mg, Zn, Mn, Fe or a coenzyme, such as complex
orgonic molecule, NAD,FAD,COA, or some vitamins (Riboflanin, Vit. B12, Thiamine,
etc.)
An enzyme containing a non-protein group is called a holoenzyme.
The protein part of this is the apoenzyme.
To summarize diagrammatically,
Inactive Protein (Apoenzyme) + Cofactor

Enzyme Holoenzyme

Active Protein
Organic molecule Metal ion
(Coenzyme)
1.3.1 Coenzymes
Thermostable, low molecular weight, non-protein organic substance called as co-
enzyme.
Since the involvement of co-enzyme in a given reaction on a substrate is so
intimate that co-enzyme os often called co-substrate or second substrate.
Cofactor – Role of Metal Ions in Enzymes
The activity of many enzymes depends on the presence of certain metal ions such
as K+, Mg++, Ca++, Zn++, Cu++.
Eg. Cu2+ - Cytochome oxidase
Zn2+ - Carbonic anhydase
Mg2+ - Nexokinase.

1.3.2 Role of metal ions

- to maintain the active structural conformation of enzymes.
- To accept or donate electrons
- To make structural changes in the substrate molecule
- Formation of enzsyme-substrate complex.
1.4 CLASSIFICATION OF ENZYMES
Nonmenclature of Enzymes
At present two types of systems are in use for nomenclature of enzymes.
Accordingly enzymes have a ‘trivial name; and a systematic name.
Trivial Name
The trivial name is composed of the substrate involved, the type of the reaction
catalyzed and the endingase. For ex,
Lactate + dehydregenation + ase = lLactate dehydregenase
A number of well and long-known enzymes have retained their traditional names
(e.g. pepsin, trypsin, chymotrypsin).
Systematic Name
The systematic name for an enzyme is constructed in a more complicated manner.
It is framed from the names of substrates, the type of reaction catalyzed and the ending-
ase.
For ex. The systematic name for the enzyme lactate dehydiegenase is written as,
L – Lactate +NAD+ : oxidation –redurtien +ase = L – Lactate : NAD
oxidoreductase
A cell contains more than 3,000 enzymes. Naming of such luge number of
enzymes in a systematic way was a tedious problem. Therefore enzymes were named in
many different ways as given below.
a) On the basis of their occurrence.
 b) On the basis of substrate used
c) On the basis of type of reaction catalyzed.
d) On the basis of substrate and type of reaction catalyzed.
e) On the basis of chemical composition of enzymes
f) On the basis of overall chemical reaction
a). On the basis of their occurrence

1 Intracellular enzymes:
Most of the enzymes act usually within the cell in which are produced. These
enzymes are called interacellular enzymes or endoenzymes; eg. Most of the plant
and animal enzymes.

2 Extracellular Enzymes (exoenzymes)

Some enzymes produced by the living cells catalyze important reactions outside
the cells environment.
e.g. enzymes found in bacteria, fungi, and digestive tract.

b). On the basis of substrate used:

The substance upon which an enzyme acts is called the substrate. Enzymes are
named by adding the suffix -ase to a part or full name of the substrate.
Eg. Proteinase, maltase, surcease, urease.

c). On the basis of reaction catalyzed

In this method the suffix –ase is added after the name of the reaction catalyzed
since enzymes are highly specific in their reactions. E.g.
a) Hydrolases (hydrolysis)
b) Dehydregenases (dehydregenation)
c) Transaminases (transamination)

d). On the basis of substrate and type of reaction catalyzed

The name of the enzyme is modified to give a clue about the substrate used and
the type of reaction catalyzed.
For ex. The exyme puryvate dehydrogenase catalyzes the dehydrogenation of
puryvic acid. Similarly lactate dehydrogenase catalyses the dehydrogenation of lactic
acid.

e). On the basis of chemical composition of Enzymes

In this method, enzymes are classified into three goups on the bais of their
chemical composition as mentioned below.
(i). Enzyme molecules consisting of protein only., Eg. Pepsin, trypsin, Urease, etc.
(ii). Enzyme molecules containing a protein and a cation. Eg. Carbonic anhydrase (Zn2+
as Cation), arginas (Mn2+ as Cation)
(iii). Enzyme molecules containing a protein and a non proteinaceous organic compound.
a). Iron porphysin enzymes – Catalase, cytochrome C, peroxidase.
b). Flavoprotein enzymes – glycine oxides, pyruvate oxidase
c). Diphorphothiamine enzymes - β - Carboxylase , pyruvate mutase
d). Enxymes requiring other co –0 enzymes – phosphorylase, amino acid decarboxylase.

f). On the basis of overall reaction catalyzed

The international union of Biochemistry (IUBC) in 1961 look overall chemical
reaction into consideration as a basis for the classification and naming of enzymes.
This method is a complicated one, but it is precise, descriptive and informative.
1.5 TYPES OF ENZYMES:
Although all enzymes initially produced in the cell, some are secreted through the
cell wall and function in the cells environments. Thus we recognize two types of enzymes
on the basis of site of action.
• Intracellular enzymes or endo enzymes (functioning in the cell) and
• Extra cellular enzymes or exoenzymes (function outside of the cell)

INTRACELLULAR ENZYMES:
It is localized inside the cell where it is produced. Examples included most of the
enzymes in catabolic pathways, all enzymes in biosynthetic pathways, energy production,
reproduction etc. Intracellular enzymes may be cytosolic or membrane bound.
Glycolytic enzymes are examples for cytosolic enzymes. These soluble enzymes
can be extracted and purified by disrupting the cells in a isotonic solution. Membrane
bound enzymes are of two types.
(i). Peripherally bound or extrinsic enzymes
(ii). Intrinsic enzymes – which is integrated and spans the entire cytoplasonic membrane
width.
Peripheral enzymes are loosely bounded and can be recovered from cell
membranes by slight osmotic shock, as intrinsic enzymes are bounded to lipids as
lipoproteins.
In eukaryotic cells, intracellular enzymes are compartmentalized inside organelles
to carry out the specified functions.
Eg. All respiratory and energy producing enzymes will be inside mitochondria
and all enzymes involved in photosynthesis will be in chloroplast.
Other examples of marker enzymes related to organelles.
Plasma membrane → Adenylate cyclase phospho diesterase
Mitochrondria → Adrenylate cyclase MDH, citrate synthase, kreb’s
cycle
enzymes, electron transport chain enzymes.
Endo plasmic reticulum → Smooth → Lipid synthesis
→ Rough → Protein Synthesis
Golgi Complex → Secretary enzymes
Lysosome → Carboxy peptidase, Elastase, lipase, phospholopse,
nucleases, lysozyme, muraminidase
Nucleus → Transcription enzymes, replication enzymes,
DNA polymerase, RNA polymerase, Lelicase.
Cytosol → Glycolytic enzymes, HMP Shunt, gluconeogenesis,
Fatty acid, synthesis enzymes.
EXTRACELLULAR ENZYMES:

Extra cellular enzymes are secreted by the cell to the outer environment. The
principal function of the extra cellular enzymes is medium to allow them to enter the cell.
These enzymes catalyse reaction outside the cell and usually they are hydrolytic
enzymes. Examples are proteolytic, lipolytic or amylolytic enzymes, produced by
microorganism or in the digestive tract of higher animals. They helps in cleaving and
producing simpler monomers of food which can be transported and assiminilated inside
the cell. Eg. Trypsin, peneratic amylase, microbial proteases.
Most of the industrially used enzymes are extracellular. They are having
advantage over inter cellular enzymes as follows:
• More stable than the intracellular enzymes.
• It works in a wider range of environmental conditions than intracellular enzymes.
• Purification and recovery is very easy and thus cost effective than intracellular
enzymes.
• Extra cellular enzymes can be precipitated and recovered by a simple two step
process from the medium where the cells grown. It will be comparatively pure than
intracellular enzymes.

1.6 THE ENZYME COMSSION’S RECOMMONDATIOSN ON

NOMENCLATURE:

In common practice, many enzymes are known by name that is usually derived
from the name of its main substrate with the suffix – are added.
Some enzymes even do not have – are at their ends (Eg. Pepsin, trypin, etc). This
has led to confusion because enzymes catalyze similar but identical reactions.
So, because of the lack of consistency in the nomenclature it become apparent as
the list of known enzymes rapidly grew that there was a need for a systematic way of
naming and classifying enzymes. The enzyme names based on this method are known as
systematic names.
A commission was appointed by the International Union of Biochemistry known
as Enzyme Commission (EC) forms the basis of present accepted system. The
commission assigns to each enzyme a systematic name in addition to its existing trivial
name.
The systematic and the Enzyme Commission (EC) classification number
unambiguously describe the reaction catalyst by an enzyme. Now ever, these names are
like to be long unwisely. Trivial names may, there fore we used in a communication,
once they have been introduced and defined in terms of the systematic names and EC
number. Trivial names also inevitable used in everyday situations in the laboratory. The
enzyme commission made recommendations as to which trivial names were acceptable.
In order to have a uniformity and unambiguity in identification of enzymes.
International Union of Biochemistry (IUB) adopted a nomenclature system based on
chemical reaction type and reaction mechanism. According to this system, enzymes are
grouped in ‘six main classes’.
 • Each enzyme is characterized by a code number (enzyme code No or E.C., No)
comprising four figures (digits) separated by points, the first being that of the main
class (One of the six).
• The second figure indicates the type of group involved in the reaction.
• Third figure denotes the reaction more preciously indicating substrate on which
the group acts.
• The fourth figure is the serial number of the enzyme.
Briefly, the four digit’s characterize class, sub – class, sub – sub – class and serial
number of a particular enzyme.
The six main classes of enzymes are as follows:
First Digit Enzyme Class Type of reaction catalyzed
1 Oxidoreduetases Oxidation/ reduction reactiosn
2 Transferases Transfer of an atom or group between two molecules
3 Hydrolases Hydrolysis reactions
4 Lyases Removal of a group from substrate (Not by hydrolysis)
5 Isomerases Isomerization reactions
The systematic joining of two molecules coupled with
6 Ligase the breakdown of pyrophosphate bond in a nucleoside
eriphosphate.

Main Class 1: Oxidoreductases

These enzymes catalyze the transfer of ‘H’ atoms, ‘O’ atoms or electrons from
one substrate to another.
The second digit in the code number of odixoreductases indicates the donor of the
reducing equivalents (hydrogen or electrons) involved in the reaction. For ex,

Second Digit Hydrogen or electron donor

1 Alcohol (> CHOH)
2 Aldehyde or ketone (> C = O)
3 - CH . CH -
4 Primary amine ( - CHNH2 or – CHNH3)
5 Secondary amine (> CHNH -)
NADH or NADPH (only where some other redox
6
catalyst is the acceptor)
The third digit refers to the hydrogen or electron acceptor as follows:

Third Digit Hydrogen or electron acceptor

1 NAD+ or NADP+
2 Fe3+ (Eg. Cytochromes)
3 O2
99 An otherwise unclassified acceptor
Example:
1. L – lactate: NAD+ odixorreductase (E.C. 1.1.1.2) (trivial Name lactate
dehydrogenase) catalyses.
CH3. CH. CO2 + NAD CH3.C.CO2- + NADH + H+
L – Lactate Pyryvate
 The systematic name for an enzyme of this group is:
Donor: Acceptor – oxido reduetase. There are 17 group in the class and about 480
enzymes comes under this class.
Eg. Dehydrogenase (hydric transfer)
Oxidases (e- tranfer to O2)
Oxygenases (Oxygen atom transfer from O2)
Peroxidases (e- transfer to peroxides)

Eg.2. trivial name: D – amino acid oxidase

Systematic name: D – amino acid: oxygen reductase
IUPAC (IUB) Name: E.C. 1.4.3.3.

R – CH – CO2 – + H2O + O2 -------> R- C- CO2 – + +NH4 + H2O2

D – amino acid Oxo acid
Oxidoreductases which involve redox reactions in which hydrogen or oxygen atoms or
electrons are transferred between molecules. This extensive class includes the
dehydrogenases (hydride transfer), oxidases (electron transfer to molecular oxygen),
oxygenases (oxygen transfer from molecular oxygen) and peroxidases (electron transfer
to peroxide). For example: glucose oxidase (EC 1.1.3.4, systematic name, b-D-
glucose:oxygen 1-oxidoreductase).

[1.1]
b-D-glucose + oxygen D-glucono-1,5-lactone + hydrogen peroxide

Main Class 2: Transferases

Transferases catalyze the transfer of an atom or group of atoms like (aryl - , alkyl
– and glycosyl groups).
These catalyze reactions of the type: AX + B BX + A
But specifically exclude oxidoreducetase and hyrolase reactions. Names of
transferases ends with X – transferase where ‘X’ is the group transferred.
The second digit describes the type of group gransferred.
Second Digit Group Transferred
1 1 – Carbon group
2 Aldehyde or ketone group (> C = O)
3 Acyl group (– C – R)
4 Glycosyl (Carbohydrate) group
77 Phosphate group
The third digit further describes the group transferred. Thus for 1 – carbon group.
Third Digit Group Transferred
1 Methyl transferase (transfer – CH3)
2 Hydroxemethyl transferases (transfer – CH2 OH)
3 Carboxyl – or carbomoyl – transferases (transfer – C – OH or – C – NH2)

There is opportunity to indicate the acceptor, incase of transfer of phosphate

groups.
2.7.1. - Alcohol group as acceptor
2.7.2. - Carboxyl group as acceptor
2.7.3 - Nitorgeneous group as acceptor
Four digit denotes the ‘serial number’. The systematic name is derived based on
the following formula:
Donor : Acceptor – group transferred – transferase or
Acceptor : Donor – group transferred – transferase

Example 1: Phosphotransferases usually have a trivial name ending in – kinase.

Trivial Name: Nucleotide Monophosphate Kinase
Systematic Name: ATP: NMP phosphotransferase

ATP + NMP ----------------> ADP + NDP

Here, NMP kinase transfers a phosphoryl group from ATP to NMP to form a
nucleotide diphosphate (NDP) and ADP.IUB No. is E.C. 2.7.4.4.

Example 2: D – Hexose – 6 – phosphotransferase (E.C. 2.7.1.1)

Trivial Name: Hexokinase, which catalyses.

C5H9O5 . CH2OH + ATP----------> C5H9O5.CH2OPO32- + ADP

D – Hexose D – Hexose – 6 – Phosphase
- Here – OH group as acceptor.

Few examples of enzymes of this group are, trans aldolase, transketolase, aayl,
methyl, glycesyl, phosphyryl, transferase, kinas, phosphomutase.

Transferases which catalyse the transfer of an atom or group of atoms (e.g. acyl-, alkyl-
and glycosyl-), between two molecules, but excluding such transfers as are classified in
the other groups (e.g. oxidoreductases and hydrolases). For example: aspartate
aminotransferase (EC 2.6.1.1, systematic name, L-aspartate:2-oxoglutarate
aminotransferase; also called glutamic-oxaloacetic transaminase or simply GOT).
 [1.2]
L-aspartate + 2-oxoglutarate oxaloacetate + L-glutamate

Main Class 3: Hydrolases

Enzymes of this group catalyze the cleavage of the substrate by adding water.
Digestive enzymes and enzymes of lysosomes belong to this group. They promote
hydrolytic decomposition of large bio molecules into simpler ones.
The trivial name for hydrolases are madeup and adding the ending – ase to the
name of the substrate that is cleaved.
The systematic name is formed by including the term hydrolase with the
substrate. These enzymes catalyze the hydrolytic reactions of the form:
A – X + H2O ------> X – Oh + HA
They are classified according to the type of bond hydrolyzed. For ex.
Second Digit Bond Hydrolyzed
1 Ester
2 Glycosidic (linking carbohydrate units0
4 Peptide (– C – N – )
5 C – N bonds other than peptides
The third digit further describes the type of bond hydrolyzed.
Example 1: E.C. 3.5.3.1. – L – arginine amidinohydrolase. (The word ‘amidino’ refers to
the group that is cleaved from arginine by introduction of a molecule of water).
Trivial name is – ‘Arginase’.
In this reaction, IUPAC (IUB) No is E.C. 3.5.3.1.
Main Class (1st group): Hydrolysis - 3
nd
2 Group : Non – peptide bond (C – N) is cleaved - 5
3rd Group : bond type acted upon or the group transferred in the reaction -
3
4th Group : Serial Number - 1
Thus the whole classification number for arginase is 3.5.3.1.
Some examples of enmzymes of this group includes, Esterase, Clycosidases,
Peptidases, Phosphatases, Phospholipase, Deaminase, Ribonucleae.
Hydrolases which involve hydrolytic reactions and their reversal. This is presently the
most commonly encountered class of enzymes within the field of enzyme technology and
 includes the esterases, glycosidases, lipases and proteases. For example: chymosin (EC
3.4.23.4, no systematic name declared; also called Rennin.

k-casein + water para-k-casein + caseino macropeptide

Main Class 4: Lyases

Enzymes of this group catalyze the cleavage of substrate molecules without
oxidation or addition of water.
These enzymes catalyze the non – hydrolytic removal of groups from substrates,
often cleaving double bonds.
The trivial name usually indicates the participation of the moiety in reaction. The
systematic name is derived from the following formula:
Substrate : type of reaction + ase
The second digit in the classification indicates the bond broken for ex,
Second Digit Bond Broken
1 C–C
2 C–O
3 C–N
4 C–S
The third digit refers to the type of group removed.
Third Digit Group Removed
1 Carboxyl group (i.e. CO2)
2 Aldehyde group (i.e. – CH = O)
3 Ketoacid group (– C – CO2 – )
Example:
Trivial Name - Histidine decarboxylase
Systematic Name - L – histidine Carboxy – lyase
Classification No: - E.C. 4.1.1.22

C3N2H3.CH2.CH.NH3+ C3N2H3.CH2.CH2+NH2 + CO2

Histidine Histamine

Some examples of enzymes of this group includes, Decarboxylases, Aldolases,

Synthase, lyase.
 Lyases which involve elimination reactions in which a group of atoms is removed from
the substrate. This includes the aldolases, decarboxylases, dehydratases and some
pectinases but does not include hydrolases. For example: histidine ammonia-lyase (EC
4.3.1.3, systematic name, L-histidine ammonia-lyase; also called histidase).

[1.4]
L-histidine urocanate + ammonia

Main Class 5: Isomeraes

Enzymes catalyzing isomerization reactions are classified according to the type of
reactions involved.
The systematic name is given by the following formula:
Substrate + type of isomerization + ase + reaction
Second Digit Type of reaction
Racemization or epimerization
1
(inversion at an asymmetric carbon atom)
2 Eis – trans isomerization
3 Intramolecular oxido reductases
4 Intramolecular transfer reaction
The third digit further describes the type of molecules undergoing isomerization.
Thus for racemases and epimerases:
Third Digit Substrate
1 Amino Acids
2 Hydroxy Acids
3 Carbohydrates
Eg: 1. Alanie racemase (E.C. 5.1.1.1.), which catalyses.
L – alanine --------------> D – alanine

2. Glucose isomerase, Glucose---------->Fructose

Isomerases which catalyse molecular isomerisations and includes the epimerases,

racemases and intramolecular transferases. For example: xylose isomerase (EC 5.3.1.5,
systematic name, D-xylose ketol-isomerase; commonly called glucose isomerase).
 [1.
5]
a-D-glucopyranose a-D-fructofuranose
Main Class 6: Ligases (Synthetases)
Enzymes of this group catalyze the addition of two molecules using the energy of
phosphote bond. ATP or other nucleoside triphosphats serve as energy sources in the
synthetase catalyzed reactions.
The trivial name is formed by the addition of “kinase” to a part of the substrate
(Eg. Glucokinase, phosphofructokinase).
The syustematic name uses the following pattern:
Substrate 1: Substrate 2 + Ligase or synthetase
These enzymes catalyze the synthesis of new bonds, coupled to the breakdown of
ATP or other nucleoside triphosphates. The reactions are of the form:
X + Y + ATP X – Y + ADP + Pi Or
X + Y + ATP X – Y + AMP + (PP)i
The second digit in the code indicates the type of bond synthesized. For ex.
Second Digit Bond Synthesized
1 C–O
2 C–S
3 C–N
4 C–C
The third digit describes the bond being formed. Thus
E.C. 6.3.1. enzymes are acid – ammonia lilgases (amide ,–C–NH2,
Synthetases) and
E.C. 6.3.2. enzymes are acid – ammonia acid ligases (peptide,– C – N – ,
Synthetases)
Eg: Trivial name : glutamine synthetase
Systematic name : L – glutamate : amino ligase
Commission number : E.c. 6.3.12

O = C – CH2 . CH – CO2 – + ATP + NH3 -------------->

L – glutamate
O = C. CH2. CH2. CH. CO2 – + ADP + Pi
L – glutamine

Enzymes of this group includes synthetase, carboxylase.

Ligases, also known as synthetases, form a relatively small group of enzymes which
involve the formation of a covalent bond joining two molecules together, coupled with
the hydrolysis of a nucleoside triphosphate. For example: glutathione synthase (EC
6.3.2.3, systematic name, g-L-glutamyl-L-cysteine:glycine ligase (ADP-forming); also
called glutathione synthetase).

1.7 REGULATION OF ENZYME SYNTHESIS:

Various mechanism are present within the living cells to prevent there
unnecessary proteins.When the product of an enzymatic pathway is no longer recovered
by a cell, the enzymes that catalyze the reactions of the pathway become unnecessary,
Control mechanism that modulate the enzymatic composition of the cell can come into
play. This regulation is effected at the level of gene expression.
Based on the regulation of the enzyme synthesis, Enzymes may be divided into
two groups as follows:
(i). Constitutive Enzymes
(ii). Inducible Enzymes

(i). Constitutive Enzymes

Constitutive enzymes are those enzymes which are present in a constant
concentration in a given cell throughout its life. These enzymes are always produced by
the cell. They are found in essentially the same amount regardless of the concentration of
their substrate in the medium.
They are coded by “Constitutive or house – keeping genes”, which is transcribe
constantly. From cell to cell, concentration or quantity of constitutive enzymes may vary
in functionally different cell types, but in specific cell type its quantity will be constant.
Eg. Enzymes of glycolysis, energy synthesizing enzymes, bio synthetic enzymes
(hexokinase, DNA, polymerase, pyruvate dehydrogenase).

(ii). Inducible Enzymes

These are produced by the cell only in response to the presence of a particular
substrate: The are produced, in a sense, only when needed. The process is referred to as
enzyme induction, and the substrate responsible for evoking formation of the enzyme is
an ‘inducer’.
 Eg. (1). An example of Inducible enzyme in prokaryotes is ‘β – galaetosidase’:
its inducer is the sugar lactose.
(2). Another example in eukaryotic cell is induction of nitrous oxide synthase (iNOS).
Enzyme in macrophasges. INOS is synthesized only by the induction of cytokine – ZFN -
γ - which in term is produced as a part of immune response towards inwarding
microbes.
Regulation of the production of an enzyme is achieved during transcription of that
gene. The term ‘induction’ represents increased synthesis of an enzyme and repression
indicates decreased synthesis of enzyme. Such induction or repression of the transcription
regulates the quantity of the enzyme presenting in the cell.
Eg. Insulin induces the synthesis of glycogen synthtase enzymes, glucokinase,
phosphofructokinase, and pyruvate kinase, all these enzymes are involved in the
utilization of Glucose at the same time, insulin repress the synthesis of key enzyme
involved in gluconeogenesis. The net result is that insulin increases the utilization of
glucose.
Prokaryotic cells like bacteria are subjected to more variations in the environment
especially in nutritional aspect. So food procuring enzymes, generally hydrolytic
exoenzymes are of inducible type.
Bacteria, such as E.Coli usually rely on glucose as there source of carbon and
energy. However when glucose is scarce, E.Coli can use lactose as a carbon source even
though this disaccharide does not lie on any major metabolic pathways.
An essential enzyme in the metabolism of lactose is β - galactosidase, which
hydrolyses lactose into galatose and glucose.
The presence of lactose in the culture medium induces a large incrase in the
amount of β - galactosidase by eliciting the synthesis of new enzyme molecules.
A crucial clue to the mechanism of gene regulation was the observation that two
other proteins are synthesized in concert with β - galactosidease – namely galactoside
permease and thiogalactoside transacetylase. The permiase is required for the transport of
the lactose across the bactraial cell membrane. The transacetylase is not essential for
lactose metabolism but appears to play a role in the detoxification of compound that also
may be transported by the permease.
Thus the expression levels of a set of enzymes that all contribute to the
adaptiation to a given change in the environment change together. Such a coordinated of
gene expression is called an ‘operon’.

UNITS OF ENZYME ACTIVITY

Most convenient way to estimate the concentration of a particular enzyme in a
preparation or fluid is to determine its catalytic activity. Usually it is determined as how
much of the substrate is capable of converting to product in a given time under specified
conditions. In 1961, enzyme commission of the IUB defined Enzyme Units (later to be
known as International Unit) as the amount of enzyme causing loss of 1 micro mole
substrate per minute. Later in 1973, (kat) was introduced as the System International (SI)
unit of enzyme. One katal is the amount of enzyme causing loss of 1 mol substrate per
second under specified conditions. This is consistent with other physical units, but
obviously microkatals are more useful. One katal is equal to 6x107 micromole/min of
substrate or 6 x 107 units. One IU is 1.7 x 10-8 kat.
 IU = µ mol/min
Kat = M/Sec

The concentration of an enzyme in solution is usually expressed as units per milli

liters.

However even these units are not ideal always. For example, katal per gram of
liver could vary according to which part of the same liver was sampled and assayed. To
minimize this problem a more general unit of enzyme activity termed specific present in
the sample. It is expressed as IU/mg of protein or kat/kg of protein.
Enzyme activity
Specific activity =
Total protein
Turn over number represents the maximum number of substrate molecule per
enzyme per unit time. Turn over number of most of the enzyme lies between 1 – 10 4 per
second. Turn over number is also called molar catalytic activity or Kcal. Kcal is the
number of moles of product produced by one mole of enzyme per second and it is
expressed as katals per mole of enzymes. The value of turn over varies with different
enzymes and depends upon conditions in which the reaction is taking place.

Kcat = katal per mole of enzyme

Enzyme Turn over number per

1 Lysozyme 0.5
2 DNA pol – I 15
3 Phosphogluco mutase 20.5
4 LDH 1000
5 Acetyl Choline Esterase 25000
6 Carbonic anhydrase 60000

1.9 MECHANISMS OF ENZYME ACTION

Catalysis is a process that increases the rate at which a reaction approaches
equilibrium. Since, rate of a reaction is a function of its free energy of activation
(activation energy), a catalyst acts by lowering the height of this kinetic barrier i.e., a
catalyst stabilizes the transition state with respect to the uncatalysed reaction Mechanisms
for Enzymtic and non-enzymatic catalysis are comparable.
Enzymes are more powerful catalysts because of their specificity of substrte
binding combined with their optimal arrangement of catalyric groups. Catalytic
functional groups interact transiently with a substrate and active it for reaction. The
energy required to lower activation energy is derived from weak, nonocovalent
interactions between the substrate and the enzyme. The interaction between substrate and
enzyme in the ES complex is mediated by weak forces such as hydrogen bonds,
hydrophobic bonds and van der walls interactions. Formation of each weak interaction in
the ES complex is accompanied by a small release of free energy that provides a degree
of stability to such interaction. The energy released or derived from enzyme-substrate
interaction is called binding energy which not only stabilizes such interaction but also
acts as a major source of free enzymes to lower the activation energy of reactions.
The types of catalytic mechanisms that enzymes employ have been classified as,
• Acid-base catalysis
• Covalent catalysis
• Metal ion catalysis
• Electrostatic catalysis
• Catalysis through proximity and orientation effect.
• Preferential binding of transition state complex.
• Strain or distortion

1.9.1Acid – base catalysis

Since enzymes contain a number of amino acid side-chains that are capable of
acting as proton donors or acceptors, it is reasonable tosuppose that acid-basecatalysis
would be important in enzyme catalyzed reactions. The task of the enzyme in this
reaction is to make a potentially active group more reaction by increasing its electrophilic
or nucleophilic character simplest way to do this is by adding a removing a proton.
(electrophiles are elctron deficient substances that react with electron rich substances,
nucleophiles are electron rich substances that react with electron-deficient substance).
In simple organic reactions, acid cataysis is divided into specific acid catalysis
and general acid catalysis. In specific acid catalysis rate expression includes only
contribution from H+ and in general acid catalysis expression of rate includes.
Contributions from H+ and other groups capable of releasing protons in solution. (such
proton donors are called Bronsted acids because, its significance is first evaluated by
Bronsted & lowry) similarly specific base catalysis and general base catalysis are there.
The side chains involving in general base cataysis are shown below.
1.9.2 General acids
- COOH
- NH3+

OH

- SH
General acid /base catalysis are the major machanism in enzymatic reactions. Acid-
base catalysis can be explained by two types of reaction mechanisms.

Reaction mechanism I:
An example given below illustrates the general base-mediated catalysis. Abase
-
(OH ) accelerates hemiacetal formation.
CH3-OH + OH- CH3O: + H2O
Methanol base Nucleophile

O OCH3 OCH3
| | |
-
CH3-O : + CH3-C-H CH3-C-O- CH3-C-OH +OH-
 | |
H H
Acetadehyde Intermediate Hemiacetal

Since –OH is recycled in the reaction it can be considered as catalyst.

Reaction Mechanishm II
An example given below illustrates the acid-mediated catalysis, which involves
the formation of oxinium salt. CH3
O O+-H O
H
H+ + CH3-C-H CH3-C :O-CH3 CH3-C-OH
Acetaldehyde H H Intermediate -H+
Oxonium ion

O
CH3-C-OH
H
Hemiacetal
+
Since H is recycled in the reaction it can be considered as a catalyst.

1.9.3 Covalent Catalysis

In covalent catalysis, the speed up of reaction achieved by the formation of
intermediates (- intermediates are formed rapidly and also rapidly breaking down).
A classic example is provided by the decarboxylation of acetoacetate, which is
catalyzed by primary amine via rapid formation and breakdown of an iminic (schiff
base).
The advantage conferred by interfermediate formation in this case is that ‘schiff
base’ is readily protonated, thus providing electron-withdrawing power to aid the loss of
CO2 than would be provide by the carbonyl group itself.

1.9.4 Metal Ion catalysis

Nearly one third of all enzymes require the presence of metal ions or catalytic
activity. There are two classes of metal ion – requiring enzymes that are distinguishable
by the strength of their ion – protein interactions.

I. Metalloenzymes:

Contains tightly bound metal ions most commonly transitions metal ions such as
Fe2+, Fe3+, Cu2+, Zn2+, Mn2+ or Co6+.
II. Metal activated Enzymes:
 Loosely bind metal ions from solution. Usually the alkali and alkaline earth metal
ions – Na+, K+, Mg2+ or Ca2+.

Metal ions participate in the catalytic process of three major ways:

• By binding to substrates so as to orient them properly for reaction.

• By mediating oxidation – reduction rexetiosn through reversible changes in the
metal ion oxidation state.
• By electro statically stabilizing or shielding negative charges.
1.9.5 Electro static catalysis:

The binding of substrate generally excludes water from an enzymes active site.
The local dielectric constant of the active site therefore resembles that in an organic
solvent, where electro static interactions are much stronger. Thus ionization constant of
amino acids in protein may vary by several units from their normal value.
The charge distribution about the active site of enzymes are arranged so that to
stabilize the transition states of the catalyzed reaction. Such mode of rate enhancement
(resembles metal ion catalysis) is termed electro – static catalysis.

1.9.6 Catalysis through proximity and orientation effect:

Enzymes bind substrates in a manner that both immobilizes them and align them
so as to optimize their activities.
In multi substrate – enzyme catalyzed reactions, enzyme can hold substrates such
that reactive region of substrates are close to each other and to the enzymes active which
is known as the ‘proximity effect’. Also enzymes may hold substrates at certain positions
and angles to improve the reaction rate, which is known as the “orientation
effect”.Substrate reacts only if they have proper orientation and favourable proximity.
Estimates of the effects of activation and thus rate of reaction shows that both factors
gave rate enhancement of 108 folds.

1.9.7 Catalysis by preferential transition – state binding:

One significant mechanism of enzyme catalysis as binding of the transition state
to an enzyme with greater affinity than the corresponding substrates.

The original concept of transition state binding proposes strain or distortion effect.
In that, substrates are strained towards transition stat geometry through binding sites into
which undistorted substrate did not properly fit. This is so – called ‘rack mechanism’.
This strained reaction more closely resemble transition state./ Thus interactions that
preferentially bind the transition state increases its concentration and therefore
proportionally increase the reaction rate.

The theory that enzyme bind transition state with higher affinity than substrate has
led to a rational basis for drug design based on the understanding of specific enzyme
reaction mechanisms.
1.11 CONCEPT OF ACTIVE SITE AND ENERGETIC OF ENZYME
CATALYZED REACTION:

ACTIVE SITE:
The region which contains the binding and catalytic sites is termed the active site
or active center of the enzyme. This comprises only a small proportion of the total
volume of the enzyme and is usually at or near the surface, since it must be accessible to
substrate molecules. In some cases, X – ray diffraction studies have revealed a clearly
defined pocket or elect in the enzyme molecule into which the whole or part of each
substrate can fit.

1.12 SALIANT FEATURES OF ACTIVE SITE:

The substrate molecules are usually much smaller than the enzyme molecules.
They bind to a specific region or site of the enzyme molecule. Such sites are referred to
as active site or catalytic site, which possess the following common features.

 The existence of active site is due to the tertiary or quaternary structure of the
enzyme – protein molecules. Loss of native configuration leads to alterations of the
active site.
 The active site of the enzyme consists of a very small portion or part of the
enzyme molecule.
 The active sites are usually in the form of grooves or cervices or pockets
occupying a small region in the outer surface of the enzyme molecule.
 The active site made up of amino acids – the common amino acids found at the
active site are serine, asparate, histidine, lysine, cysteine, arginine, glutamate and
tyrosine. Among these amino acids, serine is the most frequently found.
 The arrangement of side chains in the active site is well defined. It provides
marked specifically to the enzyme molecule.
 Water molecules are usually excluded from the active site.
 The active site often includes both polar and non – polar amino acid residues,
creating an arrangement of hydrophilic and hydrophobic microenvironment not found
elsewhere on an enzyme molecule. Thus, the function of an enzyme may depend not
only on the spatial arrangement of binding and catalytic sites, bus also on the
environment in which these sites occur.
  Co – enzymes or cofactors are present as a part of the active sites in some
enzymes.
 Active site consists of two parts, namely, the substrate binding site and the
catalytic site.
 Only weak forces are used for binding of the substrate with its active site.
 The configuration of the active site changes only slightly when a substrate
approaches it for equilibrium.
The following functional groups present at the active site of the enzyme molecule
take part in catalysis:
• – COOH groups of discarboxylic amino acid and terminal – COOH group of a
polypeptide chain.
• – NH2 groups of lysine and terminal – NH2 groups of a polypeptide chain.
• Guanidine group of arginine
• Imidazole group of histidine.
• – OH group of serine and threonine
• – SH group of cystein and disulfide group of cystine
• Phenolic group of tyrosine, et,
1.13 ENERGETIC OF AN ENZYME CATALYZED REACTION:
Enzymes lower the activation energy of the reaction catalyzed by binding the
substrate and forming an enzyme – substrate complex. Fig. 2. illustrates the action of an
enzyme form the activation energy point of view.
ACTIVATION ENERGY:
The excess energy that the reactant molecules having energy less than the
threshold energy must acquire in order to react to yield products is known as ‘activation
energy’.
Activation Energy – Threshold energy – Energy actually possessed by molecules
According to this concept, non – active molecules (having energy less than the
threshold energy) can be ‘activated’ by absorption of extra energy. This extra energy is
evidently the activation energy.
1.14 ENZYMES DECREASE ACTIVATION ENERGY:
The energy required to lower activation energy is derived from weak, non-
covalent interactions between the substrate and the enzyme. The interaction between
substrate and enzyme in the ES complex was mediated by weak forces such as hydrogen
bonds, hydrophobic bonds and Vander Walls interactions. Formation of each weak
interaction in the ES complex is accompanied bny a small release of free energy that
provides a degree of stability to such interaction. The energy released or derived from
enzyme – substrate interaction is called binding energy, which not only stabilize such
interaction but also acts as a major source of free energy for enzymes to lower the
activation energy of reaction.
For example, the activation energy for the decomposition of hydrogen peroxide
varies depending on the type of catalysis. For,
• Uncatalyzed reaction – Activation Energy/18 Kilocalories per mole (Kcal/mole)
 • Chemically catalyzed reaction – 13 Kcal/ mole
• Enzymatically catalyzed reaction – 7 Kcal/ mole
That is, catalase accelerates the rate of reaction by a factor of about 108.
1.15 ENZYME – SUBSTRATE COMPLEX FORMATION:
The molecule aspects of enzyme – substrate interaction are not yet fully
understood. This interaction varies from one enzyme – substrate complex to another.
Varies studies using x – ray and Raman Spectroscopy have revealed the presence of
enzyme – substrate complex. The interaction between the enzyme and its substrate is
usually by weak forces. In most cases, Vander Waals forces and hydrogen bonding are
responsible for the formation ES complexes. The substrates is relatively small; molecule
and fits into certain region on the enzyme molecule, which is much larger molecule.
These are two models describing the ES complex formation (Fig. 3).
• Temperature or Lock – and – key Model
• Induced – Fit or Kohsland Model

FISHER’S LOCK – AND – KEY MODEL :

This model was originally proposed by ‘Fisher’ in 1980, which states that the
active site already exists in proper conformation even in absence of substrate. Thus the
active site by itself provides a rigid, preshaped template fitting with the size and shape of
the substrate molecule. Substrate fits into active site of an enzyme as the key fits into the
lock and hence it is called the lock – and – key – model. This model proposes that
substrate bind with rigid pre – existing temperature of the ative site, provides additional
groups for binding other ligands. But this cannot explain change in enzymatic activity in
presence of allosteric modulators, for enzymes which catalyzes the reversible reaction
and for multi–substrate enzyme catalyzed reaction. Fig.4.

1.15.2 INDUCED FIT OR KOSHALAND MODEL:

The lock – and – Key hypothesis explains many feature of enzyme specificity, but
takes no account of the known flexibility of proteins. Because of the restrictive nature of
the lock – and – key model, another model was proposed by Koshland in 1963, which is
known as induced – fit mode. The important features of this model is the flexibility of the
region of the active site. X – ray diffraction analysis and data from several forms of
spectroscopy, including nuclear magnetic resonance (NMR), have revealed differences in
structure between free and substrate – bound enzymes. Thus the binding of a substrate to
an enzyme may bring about a conformational change, i.e., a change in three –
dimensional structure but not in primary structure.
 The bonds formed between a substrate and its binding sites may have replaced
previously existing linkages between a substrate and its binding sites may have replaced
previously existing linkages between each binding site and neighbouring groups on the
enzyme. So the presence of a substrate at the active site may exclude water molecules and
thus make the region more than non – polar. Both of these factors could be responsible
for some degree of change in tertiary structure taking place.
Kohsland, in his induced – fit hypothesis suggested that the structure of a
substrate may be complementary to that of the active site in the enzyme – substrate
complex, but not in free enzyme; a conformational change takes place in the enzyme
during the binding of substrate which results in the required matching of structures. The
induced fit hypothesis requires the active site to be floppy and the substrate to be rigid
allowing the enzyme to warp itself around the substrate in this way bringing together the
corresponding catalytic sites a reacting groups.
In some respects, the relationship between a substrate and an active site is similar
to that between a hand and a Woollen glove; in each interaction the structure of one
component (substrate or hand) remains fixed and the shape of the second component
(active site or glove) changes to become complementary to that of the first.

Non – Productive Interactions:

Only when a binding group of the substrate is recognized by the corresponding
site of the enzyme and then binding process proceeds as the conformational change take
place which results in all the relevant groups in substrate and enzyme coming together.
Of course, a similar binding group in a substrate other than the substrate might trigger off
a conformational change, but in general this would not result in catalytic groups being
brought together in the vicinity of an appropriate reacting group, so no reaction would
take place. This would be termed non – productive binding.
1.16 SPECIFICITY OF ENZYMES:
Another important property of enzymes is their specificity. The specificity is of three
different types namely:
• Stereochemicla Specificity
• Reaction Specificity and
• Substate Specificity

Stereospecificity:
1. Optical Specificity:
There can be mainly optical isomers of a substrate. However it is only one of the
isomers which acts as a substrate for an enzyme action, e.g. for the oxidation D – and L –
amino acids, there are two types of enzyme which will act on D – and l – isomers of
amino acids. Secondly there can be a product of enzyme action which can have isomers.
However, it is only one kind of isomer which will be produced as a product, e.g. succinic
dehydrogenase while acting on succinic acid will give only fumaric acid and not malic
acid which is its isomer.
2. Reaction Specificity:
A substrate can undergo many reactiosn but in a reaction specificity one enzyme
can catalyze only one of the various reactions. For example, oxaloacetic acid can undergo
several reactions but each reaction is catalyzed but its own separate enzyme which
catalyzes only that reaction and none of the others.
3. Substrate Specificity:
The extent of substrate specificity varies from enzyme to enzyme. These are two
types of substrate specificity.
• Absolute Specificity and
• Relative Specificity
Absolute Specificity: Absolute specificity is comparatively rare sush as urease which
catalyzes the hydrolysis of urea.
Relative Substrate Specificity: Relative substrate specificity is further divided as,
• Group dependent or
• Bond dependent
Examples of group specificity are trypsin chymotrypsin. Trypsin hydrolyzes the
residues of only lysine and arginine, while chymotripsin hydrolyzes residues of only
aromatic amino acids.
4. Bond Specificity:
Bond Specificity is observed in case of proteolytic enzymes, glucosidases and
lipases which act on peptide bonds, glycosidic bonds and easter bonds respectively.

TURN OVER NUMBER:

Turn over number represent the maximum number of substrate molecule per
enzyme per unit time. Turn over number of most of the enzyme lies between 1 – 10 4 per
second. Turn over number is also called molar catalytic activity or Kcal. Kcal is the
number of moles of product produced by one mole of enzyme per second and it is
expressed and Katals per mole of enzyme.
The value of turn over varies with different enzymes and depends upon
conditions in which the reaction is taking place. It is useful in comparing the same
enzyme activities from different tissues and in comparing different isozymes. The
following table gives a liset of few enzymes and their turn over number:

Enzymes Turnover Number (Per Second)

Lysozyme 0.5
DNA polymerase 15
Chymotrypsin 100
Lactate dehydrogenase 1000
Carbonic anhydrease 6,00,000

1.18 The mechanism of enzyme catalysis

In order for a reaction to occur, reactant molecules must contain sufficient energy
to cross a potential energy barrier, the activation energy. All molecules possess varying
amounts of energy depending, for example, on their recent collision history but,
generally, only a few have sufficient energy for reaction. The lower the potential energy
barrier to reaction, the more reactants have sufficient energy and, hence, the faster the
reaction will occur. All catalysts, including enzymes, function by forming a transition
state, with the reactants, of lower free energy than would be found in the uncatalysed
reaction (Figure 1.1). Even quite modest reductions in this potential energy barrier may
produce large increases in the rate of reaction (e.g. the activation energy for the
uncatalysed breakdown of hydrogen peroxide to oxygen and water is 76 kJ M -1 whereas,
in the presence of the enzyme catalase, this is reduced to 30 kJ M-1 and the rate of
reaction is increased by a factor of 108, sufficient to convert a reaction time measured in
years into one measured in seconds).

Figure 1.1. A schematic diagram showing the free energy profile of the course of
an enzyme catalysed reaction involving the formation of enzyme-substrate (ES) and
enzyme-product (EP) complexes, i.e.

The catalysed reaction pathway goes through the transition states TSc1, TSc2 and
TSc3, with standard free energy of activation DGc*, whereas the uncatalysed reaction goes
through the transition state TSu with standard free energy of activation DGu*. In this
example the rate limiting step would be the conversion of ES into EP. Reactions
involving several substrates and products, or more intermediates, are even more
complicated. The Michaelis-Menten reaction scheme [1.7] would give a similar profile
but without the EP-complex free energy trough. The schematic profile for the uncatalysed
reaction is shown as the dashed line. It should be noted that the catalytic effect only
concerns the lowering of the standard free energy of activation from DGu* to DGc* and
has no effect on the overall free energy change (i.e.. the difference between the initial and
final states) or the related equilibrium constant.
There are a number of mechanisms by which this activation energy decrease may
be achieved. The most important of these involves the enzyme initially binding the
substrate(s), in the correct orientation to react, close to the catalytic groups on the active
enzyme complex and any other substrates. In this way the binding energy is used partially
in order to reduce the contribution of the considerable activation entropy, due to the loss
of the reactants' (and catalytic groups') translational and rotational entropy, towards the
total activation energy. Other contributing factors are the introduction of strain into the
reactants (allowing more binding energy to be available for the transition state), provision
of an alternative reactive pathway and the desolvation of reacting and catalysing ionic
groups.
 The energies available to enzymes for binding their substrates are determined
primarily by the complementarity of structures (i.e. a good 3-dimensional fit plus optimal
non-covalent ionic and/or hydrogen bonding forces). The specificity depends upon
minimal steric repulsion, the absence of unsolvated or unpaired charges, and the presence
of sufficient hydrogen bonds. These binding energies are capable of being quite large. As
examples, antibody-antigen dissociation constants are characteristically near 10-8 M (free
energy of binding is 46 kJ M-1), ATP binds to myosin with a dissociation constant of 10-13
M (free energy of binding is 75 kJ M-1) and biotin binds to avidin, a protein found in egg
white, with a dissociation constant of 10-15 M (free energy of binding is 86 kJ M-1).
However, enzymes do not use this potential binding energy simply in order to bind the
substrate(s) and form stable long-lasting complexes. If this were to be the case, the
formation of the transition state between ES and EP would involve an extremely large
free energy change due to the breaking of these strong binding forces, and the rate of
formation of products would be very slow. They must use this binding energy for
reducing the free energy of the transition state. This is generally achieved by increasing
the binding to the transition state rather than the reactants and, in the process, introducing
an energetic strain into the system and allowing more favourable interactions between the
enzyme's catalytic groups and the reactants.

1.18.1 KINETICS OF SINGLE SUBSTRATE REACTIONS OR MICHACLIS

MENTEN KINETIC OR SATURATION KINETICS:

Kinetics: The study of the rate at which an enzyme works is called enzyme kinetics.
A mathematical model of the kinetic of single – substrate enzyme catalyzed
reaction was first developed by V.C.R. Henri in 1902 and by L. Michaelis and M.Menten
in 1913. Kinetic of simple enzyme catalyzed reactions are often referred to as Michaelis –
Menten Kinetic or Saturation Kinetics.

Effects of substrate concentration on the rate of an enzyme catalyzed reaction:

The overall reaction of an enzyme catalyzed reaction is composed of two

elementary reactions in which the substrate forms a complex with the enzyme that
subsequently decomposes to products and enzyme.
K1 K2
E+S ES P+E
K –1
Here E, S, Es and P symbolize the enzyme substrate, enzyme – substrate complex
and product respectively.
These models are based on data from batch reactors with constant liquid volume
in which the initial substrate [S0] and enzyme [E0] concentrations are known. According
to this model, when the substrate concentration become high enough to entirely convert
the enzyme to the ES form, the second step of the reaction becomes rate limiting and the
overall reaction rate becomes insensitive to further increases in substrate concentration.
The general expression for the velocity (rate) of this reaction is:
d [P]
υ= K2 [ES]
dt
 The overall rate of production of [ES] is the difference between the rates of
elementary reactions leading to its appearance and those resulting in its disappearance.
d [P]
= K1 [E] [S] – K–1 [ES] – K2 [ES]
dt
This equation cannot be explicitly integrated, however without simplifying
assumptions, two possibilities are,
1. Assumption of Equilibrium:
In 1913, Leonor Michaelis and Maude Menten, building upon earlier work by
Victor Henri, assumed that K–1 >> K2, so that the first step of the reaction achieves
equilibrium.
K–1 [E] [S]
KS = =
K1 [ES]
Here KS is the dissociation constant of the first step in the enzymatic reaction. With this
assumption the equation 3 can be integrated. Although this assumption is not often
correct, in recognition of the importance of this pioneering work, the non covalently
bound enzyme- substrate complex ES is known as the ‘Michaelis Complex’.
2. Assumption of Steady State:
With the exception of the initial stage of the reaction, the so called transient phase
which is usually over with in milliseconds of mixing the enzyme and substrate, [ES]
remains approximately constant until the substrate is nearly exhausted. Hence the rate of
synthesis of ES must equal its rate of consumption over most of the course of the
d [ES]
reaction; that is [ES] maintains steady state.
= 0 One can therefore assume with a reasonable
degree of accuracy that [ES] is constant,
dt that is:
This is called steady – state assumption was first proposed by G.E. Briggs and
James B.S. Haldane.In order to be of use, kinetic expressions for overall reactions must
be formulated in terms of experimentally measurable quantities. The quantities [ES] and
[E] are not in general, directly measurable but the total (initial) enzyme concentration.
[E0] = [E] + [ES]
[E] = [E0] – [ES]
is usually determined. The rate equation for our enzymatic reaction is the derived as
followed. Combining equation 3 with the steady state assumption 5,
O = K1 [E] [S] – K–1 [ES] – K2 [ES]
Solving for [ES], we can get
[ES] (K–1 + K2) = K1 [E] [S]
Applying 6 in 7, K1 ([E0] – [ES]) [S]
[ES] =
K–1 + K2
K1 [E0] · [S] – K1 [ES] [S]
=
K–1 [ES] + K2 [ES] = K1 [E0] [S] – K1 [ES] [S]K–1 + K2
K–1 [ES] + K2 [ES] + K1 [ES] [S] = K1 [E0] [S]
[ES] (K–1 + K2 + K1 [S]) = K1 [E0] [S]
Divide both sides of the equation by K1,
K–1 + K2 [E0] [S]
[ES] + [S] = [E0] [S] OR
K1 [ES] = K–1 + K2
+ [S]
Substitute 8 into 2 yields, K1
[E0] [S]
d[P] K2
υ= K2 [ES] =
dt K–1 + K2
+ [S]
Vmax . [S] K1
υ=
Km + [S]
Equation 9 is known as Michaelis – Menten equation, is the basic equation of the enzyme
kinetics.
K–1 + K2
Where, Vmax = K2 [E0] and Km =
K1
Here υ – the rate (velocity) of the reaction
Vmax – maximum velocity of the reaction
Km – Michaclis Constant
[S] – Substrate Concentration
Three cases will illustrate how Michaclis – Menten equation behaves:
Case1: [S] Very large
[S] >> Km., The enzyme is saturated with substrate. In this case, [S] + Km = [S]
(Approximately). So, equation 9 becomes,
Vmax . [S]
υ= = Vmax
[S]
This is the rate at large substrate concentration the maximal rate for [E], which is
called Vmax and at high substrate concentration. The rate is independent of substrate
concentration.
Case2: [S] Very Small
[S] << Km., The enzyme activity is in the linear range. In this case, [S] + Km =
Km (Approximately). So, equation 9 becomes,
Vmax . [S]
υ=
Km
So, at low [S], υ is linearly proportional to [S].

Case3: [S] = Km Then equation 9 becomes,

Vmax . [S] Vmax . [S] Vmax
υ= = =
[S] + [S] 2 [S] 2

‘Km’ is defined as the[S] that results in half – maximal rate.

1.19 ESTIMATION OF MICHAELIS – MENTEN PARAMETERS:
EXPERIMENTALLY DETERMINING RATE PARAMETERS FOR MICHAELIS
– MENTION TYPE KINETICS:
The determination of values for Km and Vm with high precision can be difficult.
Typically, experimental data are obtained from initial – rate experiments. A batch reactor
is charged with a known amount of substrate [S0] and enzyme [E0]. The product (or
substrate concentration) is plotted against time. The initial slope of this curve is
estimated.
d[P] d[S]
(i.e V = = – )
dt dt
These values of ‘V’ then depend on the values of [E0] and [S0] in the charge to the
reactor. Many such experiments can be used to generate many pairs of V and [S] data.
These could be plotted as in Fig.1, but the accurate determination of Km from such a plot
is very difficult. Consequently, other methods of analyzing such data have been
suggested.

Vm

½ Vm –

V
|

[S] = Km Substrate, [S]

Vmax . [S]
V=
Km + [S]
1 1 Km 1
The equation 1 can be linearized in double reciprocal form: = +
V Vmax Vm [S]
y = c + m
x
A plot of 1/V VS 1/[S] yields a linear line with a slope of Km/Vm and y – axis
intercept of 1/Vm, as depicted in Fig.2.

1/ V
Slope = Km/Vm

← 1/ Vm

– 1/ Km 1/ [S]
Line weaver Burk Plot

A plot of V VS V/[S] results in a line of slope – Km and y – axis intercept of Vm as

depicted in
.
← Vm

V
Slope = – Km

Vm/Km
↓

V/ [S]
Eadie – Hofstee plot:
Rearrangement of 2 yields,
[S] Km 1
= + [S]
V Vm Vm
A plot of [S]/V VS [S] results in a line of slope 1/Vm and y – axis intercepts of
Km/Vm as depicted in Fig.4. This plot is used to determine Vm more accurately.

[S]
V Slope = 1/Vm

← Km/ Vm

– Km [S]
Hanes – Wooly Plot:
In this method, each observation is represented by al line, drawn from the value of
S, on the horizontal axis, through the corresponding value of v, on the vertical axis. A
series of such lines meet at a point. The horizontal and vertical coordinates drawn from
this pint are Km and Vmax.

[V]

Vmax

Km [S]
Eisenthal and Cornish – Bowden plot:

KINETICS OF MULTI-SUBSTRATE ENZYME-CATALYSED REACTIONS

Most biochemical reactions involve at least two substrates, so it is necessary to

consider the kinetics of such reactions is a (two substrate two product (bi-bi) reactions).
These are often transfer reactions of one type or another (including oxidation// reduction
reactions) and can best be represented as:
AX + B BX+A
The reaction mechanism may be a sequential one, where both substrates bind to
the enzyme to form a ternary complex before the first product is formed, or it may be
non-sequential.

PING-PONG BI-BI MECHANISM

An example of non-sequential mechanism is the ping-pong bi-bi or double

displacement.
AX + E E.AX EX.A EX + A
EX + B EX.B E.BX E + BX
At first binds to the enzyme E, forming a binary complex E.AX (X is usually a
small group and does not participate in the reaction as a free molecule, so it is not
regarded as a separate reactant). An intramolecular reorganization takes place, the bond
E-X being formed and the X-A bond being broken. The first product, A, then leaves
before the second substrate arrives. B cannot bind to the enzyme E But bind to the
modified enzyme EX. Since only one substrate is present on the enzyme at any one time
there may only be a single binding site.
Another intermolecular rearrangement takes place the bond B-X being formed and the
bond E-X being broken. The second product, BX is then liberated, leaving the enzyme in
its original form.
Cleland has devised a diagrammatic representation which shows this sequence of
events as follows:

RANDOM-ORDER MECHANISM
A random-order mechanism is one in which any substrate can bind first to the
enzyme and any product can leave first. It is a sequential mechanism and for a two-
substrate reaction involves the formation of a ternary complex (one involving enzyme
and both substrates):
COMPULSORY – ORDER MECHANISM
A compulsory –order (or simply ordered) mechanism is a sequential mechanism
where the order of binding to and leaving the enzyme is compulsory. For a two-substrate
reaction, a ternary complex will be involved. The precise order must be specified e.g.

As before, the enzyme will have a binding site for A/Ax and a separate one for B/BX.

STEADY – STATE KINETICS

The general rate equation of alberty

Many two – substrate enzyme – catalysed reactions obey the michaclis – menten
equatin with respect to one substrate at constant concentration of the other substrate. This
applies both to reactions catalysed by enzymes with just one binding site per substrate,
provided there is no interaction between the binding sites. For such reactions, Alberty
(1953) derived the general equation,
Vmax [AX] [B]
V=
KMB [AX] + KMAX [B] + [AX] [B] + [AX] [B] +KSAX. KmB
Where,
Vmax – the maximum possible V when AX and B are both saturating
KMAX – is the concentration of AX which gives ½ max when B is saturating
KMB – is the concentration of B which gives ½ Vmax when AX is saturating.
KSAX - is the dissociation constant for E +AX E.AX
The total enzyme concentration is constant and much smaller than the
concentration of the two substrates.
At very high [B] the general equation simplifies to:

V= Vmax = Vmax [AX]

AX
1 + KM /[AX] [AX] + KMAX
At constant but not saturatind [B], The general equation can be rearranged to give:

Vmax K1 [AX]
V=
[AX] +K2

Where, [B]
K1 = KMB + [B]

And KSAX KMB + KMAX [B]

K2 =
 KMB + [B]

Lincweaver – Burk equation (General)

The general rate equation,
Vmax
V=
KSA. KMB + KMA + KMB +1
[A] [B] [A] [B]

Reciprocal of this equation,

1/V = 1 (KMA + (KSA. KMB) ) 1/[A] + 1 (1 + KMB/[B])

Vmax [B] Vmax

Lineweaver – Burk plot at fixed concentration of [B] assuming no interaction

between binding sites of A and B.
2.1 ENZYME DEACTIVATION KINETICS
Enzymes are protein molecules of complex configuration that can be destabilized
by relatively weak forces. In the course of enzyme-catalysed reactions, enzyme
deactivation occurs at a rate which is dependent on the structure of the enzyme and the
reaction conditions. Environmental factors affecting enzyme stability include
temperature, pH, ionic strength, mechanical factors and presence of denaturants such as
solvents, detergents and heavy metals. Because the amount of active enzyme an decline
considerably during reaction, in many applications the kinetics of enzyme deactivation
are just as important as the kinetics of the reaction itself. In the simplest model of
enzyme deactivation, active enzyme Ea undergoes irreversible transformation to an
inactive form E;
Ea Ei
Rate of deactivation is generally considered to be first order in active enzyme
concentration.
Rd = kd ea
Where Rd is the volumetric rate of deactivation, ea is the active enzyme
concentration and kd is the aeactivation rate constant.
In a closed system where enzyme deactivation is the only process affecting the
concentration of active enzyme:
-dea/dt=rd= kd ea
Integration values of eqn. (3) gives an expression for active enzyme concentration as a
function of time:
-dea/dt=rd=kdea
-dea/ea=kd. dt, by applying limits,
ea t
-∫dea/ea = kd ∫dt
ea0 0
ea
-[ln ea] =kdt
 ea0
ln ea/ea0 = -kdt

ea = eaO.e-kdt
Where ea0 is the concentration of active enzyme at time zero. According to eqn.
(4), concentration of active enzyme decreases exponentially with time; the greatest rate of
enzyme deactivation occurs when ea is high.
Combining this deactivation model with the sample catalytic reaction sequence
used by Michaelis and Menten gives,
K1 K2
Ea + S ===== EaS Ea
K-1
K∂
Ea Ei
If we apply the reasonable assumption that the deactivation process is much
slower than reactions in equation 5, involving the quasi – steady state approximation for
(EaS) complex gives,
K2 eao . S
υ=
Km +S
Where K2eao = Vmax. eao is the total concentration of active enzyme both in free and
complexed forms. The rate of change of eao is given by,
deao = 1 – K∂ ea
–
dt deao K∂ eao . S
By applying quasi – steady assumption, =
dt 1 + S/Km
The value of Vmax for enzyme reaction depends on the amount of active enzyme
present, i.e, Vmax = K2 ea.
Therefore, as ea declines due to deactivation Vmax is also diminished.

We can estimate the variation of Vmax with time by substituting into equation 8,
the expression foe ea from 4:
Vmax = K2 eao e– K∂ t = Vmaxo e– K∂t
Where Vmaxo is the initial value of Vmax before deactivation occurs.
Stability of enzymes is frequently reported in terms of half – life. ‘Half – life’ is
the time required for half the enzyme activity to be lost as a result of deactivation; after
one half – life, the active enzyme concentration equals eao/2. Substituting ea = eao/2 into
equation 4, taking logarithms and rearranging fields the following expression:

ea = eao. e – K∂dt
ea
ln = – K∂ t eao
eao /2
ln eao = – K∂ t or ln 2 = – K∂ t
ln 2
K∂
 th =

Where th is the enzyme half – life.

Rate of enzyme deactivation is strongly dependent on temperature. This
depending is generally well described using the Arrhenius equation.

K = Ae – E∂/RT
Where A is the Arrhenius constant or frequency factor, E∂ is activation energy for
enzyme deactivation, R is the ideal gas constant and T is absolute temperature.

According to equation 11 as T increases, rate of enzyme deactivation increases

exponentially. Values of E∂ are high of the order 170 – 400 KJ g mol–1 for many
enzymes.

TYPES INHIBITION – KINETIC MODELS

ENZYME INHIBITION
The activity of an enzyme may be reduced by several means. A structural analog
of the substrate may bind in the active site, either reversibly or irreversibly, and thereby
reduce activity. Alternatively, an inhibitior may bind on some other portion of the
enzyme and induce conformational changes that may reduce to bind substrate.
We thus consider two main types of inhibition reversible and irreversible
inhibition, depending on whether activity can be restored by deduction or removal of the
inhibitor.

2.2.2 IRREVERSIBLE INHIBITION

Enzymes can be irreversibly inactivated by the being of an inhibitor molecule to
the active site of an inhibitor molecule to the active site of the enzyme. The inhibitor
typically forms a covalent bond with the enzyme and thus prevents binding of the
substrate. It may also act by dedstroying some part of the active site.
Irreversible inhibitors thus reduce the effective concentration of the enzyme
without affecting the observed kinetics of the enzyme with respect to substrate.
Irreversible inhibitors are used in investigations of active site binding and kinetic
mechanism e.g. uncompetitive inhibition.
2.2.3 REVERSIBLE INHIBITION
Unlike irreversible inhibitors, reversible inhibitors can be removed from the
enzyme enabling it to recover its activity. Reversible inhibitors can be removed by
dialysis or by addition of another agent which binds with the inhibitor in solution and
effectively lowers its concentration. E.g. competitive inhibition.
Substances that reduce an enzyme’s activity in this way are known as inhibitors.
Many inhibitors are substances that structurally resemble their enzyme’s substrate
but either do not react very slowly compared to substrate. Such inhibitors are commonly
used to probe the chemical and conformational nature of an effect of a substrate-bi nding
site as part of an effect to elucidate the enzyme’s catalytic mechanism.
In addition, many enzyme inhibitors are effective chemotherpeutic agents since an
“unnatural” substrate analog can block the action of a specific enzyme. For example,
methotrexate (also called amethoptecin) chemically resembles dihydrofolate.
Methotrexate binds tightly to the enzyme dihydrofolate reductase, thereby preventing it
from carrying out its normal function defolate, an essential cofactor in the biosynthesis of
the DNA prscussorthymidylic acid.
Rapidly dividing cells, such as cancer cells, which are actively angaged in DNA
synthesis, are far more susceptible to methotrexate than are slower growing cells such as
those of most normal mammalian tissues. Hence methotrexate, when administered in
properdosage, kills cancer cells without fatally poisoning the host.

A) COMPETITIVE INHIBITION
A subtance that competes directly with a normal substrate for an enzymatic-
binding site is known as a competitive inhibitor. Such an inhibitor usually resembles the
substrate to the extent that is specially binds to the active site but differs from it so as to
be unreactive.
For ex, sucecinate dehydrogenase, a citric acid enzyme that functions to convert
succinate to formulate os competitively succinate but cannot be dehydrogenated.

Malonate has two carboxyl groups, like the substrate, succinate, and can fill the
succinate-binding site in the enzyme. However, the subsequent reaction involves the
formation of a double bond, and since malonate, unlike succinate, has only one carbon
atom between the carboxyl groups, it cannot react.
The steady-state kinetics of a single substrate single-binding site single-
intermediate enzyme-catalysed reaction in the presence of a competitive inhibitor, I, can
be studied by the following reaction scheme.
 The dissociation constant for the reaction E and I is KI. Where,

[E] [I] [E] [I]

[KI] = ; [EI] =
[EI] [KI]
In this, KI is called the inhibitor constant. Here,
Vo = K2 [ES]
EI, the enzyme-inhibitor complex , is catalytically inactive. A competitive inhibitor
thereforeacts by reducing the concentration of free enzyme available for substrate
binding.
To express vo in terms of measurable quantities; in this case, [EO], [S] and [I]. we
begin, as in the derivation of the michaclis-Menton equation, with the expression for the
conservation condition, which must now take into account the existence of EI.
[EO] = [E] + [ES] +[EI]
As in the derivation of Michaelis-Menton equation using the steady state
assumption.
The eqn (7) is of the same form as the michaclis-menten equation, the only difference
being that Km has been increased by a factor (1+[I]/KI). Therefore, for simple competitive
inhibition Vmax is unchanged, but Km is altered so that Kmapparent = Km(1+[I]/KI). Where
Kmapp is the apparent Km in the presence of competitive inhibitor.
The effect of the competitive inhibitor depends on the inhibitor concentration, the
substrate concentration and relative affinities of the substrate and inhibitor for the
enzyme.
In general,at a particular inhibitor and enzyme concentration, if the substrate
concentration is low, the inhibitor will compete favourably with the substrate (fig) for the
binding sites on the enzyme and the degree of the inhibition will be great. However, if at
this same inhibitor and enzyme concentration, the substrate concentration is high, then
the inhibitor will be much less successful in competing with the substrate for the
available binding sites and the degree of inhibition will be less marked. At very high
substrate concentrations, molecules of the substrate will greatly outnumber molecules of
inhibitor and the effect of the inhibitor will be negligible.
Hence Vmax for the reaction is unchanged. However the apparent Km, the substrate
concentration. When V0 = ½ Vmax, is early increased as a result of the inhibition.
The lineweaver – Burk equation in the presence of a competitive inhibitor will be,
reciprocal of eqn (7) results in
Lineweaver – Burk plots showing the effect of competitive inhibition are shown in the
following fig,
B) UNCOMPETITIVE INHIBITION
Uncompetitive inhibitors bind only to the enzyme – substrate complex and not to
the free enzyme. Substrate – binding could cause a conformational change to take place
in the enzyme and reveal and inhibitor binding- site, or the inhibitor could bind directly to
the enzyme- bound substrate. In neither case does the inhibitor compete with the substrate
for the same binding site, so the inhibition cannot be overcome by increasing the
substrate concentration. Both Km and Vm are altered, but a distinctive kinetic pattern
emerges under steady-state conditions.
V verses [s] plot is given as follows,
An un competitive inhibitor decreases Vmax and Km to the same extent.
The Lineweaver – Burk equation in the presence of an uncompetitive inhibitor is,
And the slope of a Lineweaver – Burk plot is equal to,
In other words, the slope of a Lineweaver – Burk plot is not altered by the
presence of an uncompetitive inhibitor, but the intercept is changed.
Uncompetitve inhibition of single – substrate enzyme reation is a rare phenomenon, one
of the few possible examples known being the inhibition of arylsulphatase by hydramine.
However, uncompetitive inhibitor patterns are seen with two – substrate reactions and
this may help in the elutiondation of the reaction mechanism.

C) NONCOMPETITIVE INHIBITON
Noncompetitive inhibitors are not substrate analogs. Inhibitors bind on sites other
than the active site and reduce enzyme affinity to the substrate. S and I bind reversibly,
randomly and independently at different sites. That is, I binds to E and to ES; S binds to
E and to EI. However, the resulting ESI complex is inactive. I might prevent the proper
positioning of the catalytic center, either by binding to the catalytic site or as a result of a
conformational change affecting the catalytic site, but does not affect substrate binding.
The reaction scheme is as follows,

Here, v = K2[ES]
Even this is a complex situation, for ES can be arrived at by alternation routes,
making it impossible for an expression of the same form as the Michaclis-Menten
equation to be derived using the general steady state assumption. However types of non-
competetive inhibition consistent with a Michaclis-Menten type equation and a linear
line-Weaver-Bark plot can occur if the equlibrium assumption is valid. In the simplest
possible model, simple linear non-competitive inhibition, the substrate does not affect
inhibitor-binding. Under these conditions the reactions E + EI and ES + I ESI
have an identical dissociation constant, KI, again called Inhibitor constant. The total
enzyme concentration is effectively reduced by the inhibitor, decreasing the value of Vmax
but not altering Km, since neither inhibitor nor substrate affect the binding of the other.
Hence
[E] [I]
= Km
[KI]
In the presence of a competitive inhibitor which will bind equally well to E or to ES, i.e
where
[E] [I] [ES] [I]
KI = =
[EI] [ESI]

2.3 TEMPERATURE EFFECTS ON ENZYME ACTIVITY

Every enzyme has a temperature range of optimum activity. Outside that temperature
range the enzyme is rendered inactive and is said to be totally inhibited. This occurs
because as the temperature changes this supplies enough energy to break some of the
intramolecular attractions between polar groups (Hydrogen bonding, dipole-dipole
attractions) as well as the Hydrophobic forces between non-polar groups within the
protein structure. When these forces are disturbed and changed, this causes a change in
the secondary and tertiary levels of protein structure, and the active site is altered in its
conformation beyond its ability to accomodate the substrate molecules it was intended to
catalyze. Most enzymes (and there are hundreds within the human organism) within the
human cells will shut down at a body temperature below a certain value which varies
according to each individual. This can happen if body temperature gets too low
(hypothermia) or too high (hyperthermia).
2.3.1 TEMPERATURE EFFECTS
Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as the
temperature is raised. A ten degree Centigrade rise in temperature will increase the
activity of most enzymes by 50 to 100%. Variations in reaction temperature as small as 1
or 2 degrees may introduce changes of 10 to 20% in the results. In the case of enzymatic
reactions, this is complicated by the fact that many enzymes are adversely affected by
high temperatures., the reaction rate increases with temperature to a maximum level, then
abruptly declines with further increase of temperature. Because most animal enzymes
rapidly become denatured at temperatures above 40·C, most enzyme determinations are
carried out somewhat below that temperature.Over a period of time, enzymes will be
deactivated at even moderate temperatures. Storage of enzymes at 5·C or below is
generally the most suitable. Some enzymes lose their activity when frozen.
All enzymes work within a range of temperature specific to the organism. Increases in
temperature generally lead to increases in reaction rates. There is a limit to the increase
because higher temperatures lead to a sharp decrease in reaction rates. This is due to the
denaturating (alteration) of protein structure resulting from the breakdown of the weak
ionic and hydrogen bonding that stabilize the three dimentional structure of the enzyme.
The optimum temperature for human enzymes is between 35-400 C. The average
temperature for humans is 370 C. Human enzymes start to denature quickly at
temperatures above 400 C. Bacteria found in the hot springs of Yellowstone National
Park have optimum temperatures of 700C. Temperature loving bacteria are called
thermophilic.Denaturating of enzymes, regardless of the cause, leads to the death of the
cell.
Changes in the pH or acidity of the environment can take place that would alter or totally
inhibit the enzyme from catalyzing a reaction. This change in the pH will affect the polar
and non-polar intra-molecular attractive and repulsive forces and alter the shape of the
enzyme and the active site as well to the point where the substrate molecule could no
longer fit, and the chemical change would be inhibited from taking place as efficiently or
not at all. In an acid solution any basic groups such as the Nitrogen groups in the protein
would be protonated. If the environment was too basic the acid groups would be de-
protonated. This would alter the electrical attractions between polar groups. Every
enzyme has an optimum pH range outside of which the enzyme is inhibited. Some
enzymes like many of the hydrolytic enzymes in the stomach such as Pepsin and
Chymotrypsin effective operate at a very low acidic pH. Other enzymes like alpha
amylase found in the saliva of the mouth operate most effectively at near neutrality. Still
other enzymes like the lipases will function most effectively at basic pH values. If the pH
drops in the blood called acidosis then enzymes in the blood will be inhibited outside
their optimal pH range. If the pH climbs to an unacceptably high value called alkalosis
then enzymes cease to function effectively. Normally, these conditions do not take place
because of the highly efficient buffers found in the blood that restrict the pH of the blood
to a very narrow range. Buffers are a substance or mixtures of substances that resist any
change in the pH. There are many buffer systems found in the body to adjust the pH so
that enzymes might continue to catalyze their reactions.
Correcting pH or temperature imbalances will usually allow the enzyme to resume its
original shape or conformation. Some substances when added to the system will
irreversably break bonds disrupting the primary structure so that the enzyme is inhibited
permanently. The enzyme is said to be irreversably denatured. Many toxic substances
will break co-valent bonds and cause the unraveling of the protein enzyme. Other toxic
substances will precipitate enzymes effectively removing them from the solution thus
preventing them from catalyzing the reaction. This is also called denaturation.
EFFECTS OF pH
Most enzymes are sensitive to pH and have specific ranges of activity. All have an
optimum pH. The pH can stop enzyme activity by denaturating (altering) the three
dimentional shape of the enzyme by breaking weak bonds such as ionic, and hydrogen.
Most enzymes function between a pH of 6 - 8; however pepsin in the stomach works best
at a pH of 2 and trypsin at a pH of 8. Bacteria that thrive at lower pH's are said to be
acidophilic (acid loving).
SALT CONCENTRATION
Most enzymes can not tolerate extremely high salt concentrations. The ions interfere with
the weak ionic bonds of proteins. As usual there are exceptions such as the halophic (salt
loving) algae and bacteria.
 ENZYME IMMOBILIZATION
Enzymes are protein molecules which serve to accelerate the chemical reactions of
living cells (often by several orders of magnitude). Without enzymes, most biochemical
reactions would be too slow to even carry out life processes. Enzymes display great
specificity and are not permanently modified by their participation in reactions. Since
they are not changed during the reactions, it is cost-effective to use them more than once.
However, if the enzymes are in solution with the reactants and/or products it is difficult to
separate them. Therefore, if they can be attached to the reactor in some way, they can be
used again after the products have been removed. The term "immobilized" means unable
to move or stationary. And that is exactly what an immobilized enzyme is: an enzyme
that is physically attached to a solid support over which a substrate is passed and
converted to product.
It is important to understand the changes in physical and chemical properties
which an enzyme would be expected to undergo upon immobilization (sometimes
referred to as insolubilization). There are a number of factors that affect the rate of the
enzyme's catalytic activities. Changes have been observed in the stability of enzymes and
in their kinetic properties due to the micro environment and the product's characteristics.
Benefits of Immobilizing an Enzyme
There are a number of advantages to attaching enzymes to a solid support and a
few of the major reasons are listed below:
• Multiple or repetitive use of a single batch of enzymes
• The ability to stop the reaction rapidly by removing the enzyme from the reaction
solution (or vice versa)
• Enzymes are usually stabilized by bounding
• Product is not contaminated with the enzyme (especially useful in the food and
pharmaceutical industries)
• Analytical purposes - long 1/2-life, predictable decay rates, elimination of reagent
preparation, etc.
When immobilizing an enzyme to a surface, it is most important to choose a method of
attachment that will prevent loss of enzyme activity by not changing the chemical nature
or reactive groups in the binding site of the enzyme. In other words, attach the enzyme
but do as little damage as possible. Considerable knowledge of the active site of the
enzyme will prove helpful in achieving this task. It is desired to avoid reaction with the
essential binding site group of the enzyme. Alternatively, an active site can be protected
during attachment as long as the protective groups can be removed later on without loss
of enzyme activity. In some cases, this protective function can be fulfilled by a substrate
or a competitive inhibitor of the enzyme.
The surface on which the enzyme is immobilized is responsible for retaining the
structure in the enzyme through hydrogen bonding or the formation of electron transition
complexes. These links will prevent vibration of the enzyme and thus increase thermal
stability. The micro environment of surface and enzyme has a charged nature that can
cause a shift in the optimum pH of the enzyme of up to 2 pH units. This may be
accompanied by a general broadening of the pH region in which the enzyme can work
effectively, allowing enzymes that normally do not have similar pH regions to work
together.
• Carrier-Binding : the binding of enzymes to water-insoluble carriers
• Cross-Linking: intermolecular cross-linking of enzymes by bi-functional or
multi-functional reagents.
• Entrapping : incorporating enzymes into the lattices of a semi-permeable gel or
enclosing the enzymes in a semi-permeable polymer membrane

Choice of Immobilization Method

When immobilizing an enzyme on a surface it is most important to choose a method of
attachment aimed at reactive groups outside the active catalytic and binding site of that
enzyme. Considerable knowledge of active sites of particular enzymes will enable
methods to be chosen that would avoid reaction with the essential groups therein.
Alternatively, these active sites can be protected during attachment as long as the
protective groups can be removed without loss of enzyme activity. In some cases, this
protective function can be fulfilled by a substrate of the enzyme or a competitive
inhibitor; this also contributes towards retention of tertiary structure of the enzyme.
The surface on which the enzyme is immobilized has several vital roles to play such as
retaining of tertiary structre in the enzyme by hydrogen bonding or the formation of
electron transition complexes. Retention of tertiary structure may also be a vital factor in
maximizingthermal stability in the immobilized state. In this respect it is wise to follow
closely the new findings in the chemical nature of soluble thermostable enzymes. The
microenvironment of surface and the immobilized enzyme has an anionic or cationic
nature of the surface that can cause a displacement in the optimum pH of the enzyme of
up to 2 pH units. This may be accompanied by a general broadening of the pH region in
which the enzyme can work effectively.
Immobilization by cross-linking the protein enzyme in order to insolubilize it or merely
immobilize it in desired location has many possibilities and is relatively cheap. Several
aldehydes and other cross-linking agents are now available for this purpose. Extension of
this approach to a process where an enzyme is an integral component of a copolymer
could permit designing for reversible polymerization.
Choice of Enzyme Reactor
In enzyme reactor, the highest specific activity, in terms of weight of enzyme and support
employed, is desirable. It is considered an added bonus if the support is employed to
fulfil another function. The most important of these functions is separation since, if this
occurs simultaniously with reaction, unfavorable equilibria may be displaced. One
approach is to use a molecular sieve as the support and, in packed reactors to pulse the
reactor bed alternatively by passage of substrate solution and water, so that bands of
unused substrate and product are progressing down the column. It so happens that these
enzymes for which this would be useful are also those which in some cases benefit in
having the enzyme immobilized on a porous support.
For an industrial reactor it is preferable to use supports that are non-biodegradable such
as glass, silica, Celite, Bentonite, alumina, or titanium oxide, if possible. Even the
linkages between enzyme and support can be non-biodegradable, as with the titanium
procedure. As far as the biodegradability of the enzyme, in some cases, when in active
use, enzymes will partially protect themselves, notably all those that effect oxidation with
production of hydrogen peroxide as a by product.
Many types of reactors have been proposed: packed beds with downward flow,
suspended particles in a fluid ben with upward flow of substrate, a simple stirred reactor,
tubular reactors, membrane reactors, and many others. In some of these the physical
nature of the surface becomes a major problem. Thus some supports that form excellent
packed beds fail to do so when coated with enzyme and particles which ideally self-
suspend in a fluid bed then aggregate during use requiring more power to pump through
substrate allied to a lowered catalytic activity from a decreased surface area. Many
problems were encountered with porous glass supports until it was realized that the
enzyme was not simply losing activity but the glass itself could dissolve. This has now
been overcome by the zirconium treatment of the glass surface. Additives to enzymes
intended to preserve enzyme activity are also rarely fully disclosed. This often causes
confusion as to the weight of protein in the enzyme product and can even interfere with
the coupling procedure or decrease its efficiency.
Properties of Immobilized Enzymes
It is important to understand the changes in physical and chemical properties which an
enzyme would be expected to undergo upon insolubilization if the best use is to be made
of the various insolubilization techniques available. Changes have been observed in the
stability of enzymes and in their kinetic properties because of the microenvironment
imposed upon them by the supporting matrix and by the products of their own action.
Stability
The stability of the enzymes might be expected to either increase or decrease on
insolubilization, depending on whether the carrier provides a microenvironment capable
of denaturing the enzymic protein or of stabilizing it. Inactivation due to autodigestion of
proteolytic enzymes should be reduced by isolating the enzyme molecules from mutual
attack by immobilizing them on a matrix. It has been found that enzymes coupled to
inorganic carriers were generally more stable than those attached to organic polymers
when stored at 4 or 23 ° centigrade. Stability to denaturing agents may also be changed
upon insolubilization.
Kinetic Properties
Changes in activity of enzymes due to the actual process of insolubilization have not been
studied very much. There is usually a decrease in specific activity of an enzyme upon
insolubilization, and this can be attributed to denaturation of the enzymic protein caused
by the coupling process. Once an enzyme has been insolubilized, however, it finds itself
in a microenvironment that may be drastically different from that existing in free
solution. The new microenvironment may be a result of the physical and chemical
character of the support matrix alone, or it may result from interactions of the matrix with
substrates or products involved in the enzymatic reaction.
The Michaelis constant has been found to decrease by more than one order of magnitude
when substrate of opposite charge to the carrier matrix was used. Again, this only
happened at low ionic strengths, and when neutral substrates were used. The electrostatic
potential was calculated by insertion of the Maxwell-Bottzmann distribution into the
Michaelis-Menton equation using the changes in Michaelis constant, and good agreement
was obtained with the value for the electrostatic potential calculated from the pH-activity
shifts.
The diffusion of substrate from the bulk solution to the micro-environment of an
immobilized enzyme can limit the rate of the enzyme reaction. The rate at which
substrate passes over the insoluble particle affects the thickness of the diffusion film,
which in turn determines the concentration of substrate in the vicinity of the enzyme and
hence the rate of reaction.
The effect of the molecular weight of the substrate can also be large. Diffusion of large
molecules will obviously be limited by steric interactions with the matrix, and this is
reflected in the fact that the relative activity of bound enymes towards high molecular
weight substrates has been generally found to be lower than towards low molecular
weight substrates. This, however, may be an advantage in some cases, since the
immobilized enzymes may be protected from attack by large inhibitor molecules.

3.1 Outline of Preparation Techniques

Methods used for the immobilization of enzymes fall into four main categories:
1. Physical adsorption onto an inert carrier,
2. Inclusion in the lattices of a polymerized gel,
3. Cross-linking of the protein with a bifunctional reagent, and
4. Covalent binding to a reactive insoluble support.
Techniques and supports for immobilization
A large number of techniques and supports are now available for the
immobilization of enzymes or cells on a variety of natural and synthetic supports. The
choice of the support as well as the technique depends on the nature of the enzyme,
nature of the substrate and its ultimate application. Therefore, it will not be possible to
suggest any universal means of immobilization. It can only be said that the search must
continue for matrices which provide facile, secure immobilization with good interaction
with substrates, and which conform in shape, size, density and so on to the use for which
they are intended. Care has to be taken to select the support materials as well as the
reagents used for immobilization, particularly when their ultimate applications are in the
food processing and pharmaceutical industries. Macromolecular, colloidal, viscous,
sticky, dense or particulate food constituents or waste streams also limit the choice of
reactor and support geometries. Commercial success has been achieved when support
materials have been chosen for their flow properties, low cost, nontoxicity, maximum
biocatalysts loading while retaining desirable flow characteristics, operational durability,
ease of availability, and ease of immobilization31. Techniques for immobilization have
been broadly classified into four categories, namely entrapment, covalent binding, cross-
linking and adsorption. A combination of one or more of these techniques has also been
investigated. It must be emphasized that in terms of economy of a process, both the
activity and the operational stability of the biocatalysts are important. They determine its
productivity, which is the activity integrated over the operational time.

Adsorption
Physical adsorption of an enzyme onto a solid is prabably the simplest way of preparing
immobilized enzymes. The method relies on non-specific physical interaction between
the enzyme protein and the surface of the matrix, brought about by mixing a concentrated
solution of enzyme with the solid.
A major advantage of adsorption as a general method of insolubilizing enzymes is that
usually no reagents and only a minimum of activation steps are required. As a result,
adsorption is cheap, easily carried out, and tends to be less disruptive to the enzymic
protein than chemical means of attachment, the binding being mainly by hydrogen bonds,
multiple salt linkages, and Van der Waal's forces. In this respect, the method bears the
greatest similarity to the situation found in biological membranes in vivo and has been
used to model such systems.
Because of the weak bonds involved, desorption of the protein resulting from changes in
temperature, pH, ionic strength or even the mere presence of substrate, is often observed.
Another disadvantage is non-specific further adsorption of other proteins or other
substances as the immobilized enzyme is used. This may alter the properties of the
immobilized enzyme or, if the substance adsorbed is a substrate for the enzyme, the rate
will probably decrease depending on the surface mobility of enzyme and substrate.
Adsorption of the enzyme may be necessary to facilitate the covalent reactions described
later. Stabilization of enzymes temporarily adsorbed onto a matrix has been achieved by
cross-linking the protein in a chemical reactiion subsequent to its physical adsorption.
This is perhaps the simplest of all the techniques and one which does not grossly alter the
activity of the bound enzyme. In case of enzymes immobilized through ionic interactions,
adsorption and desorption of the enzyme depends on the basicity of the ion exchanger.
Moreover, a dynamic equilibrium is normally observed between the adsorbed enzyme
and the support which is often affected by pH as well as the ionic strength of the
surrounding medium. This property of reversibility of binding has often been used for the
economic recovery of the support. This has been successfully adapted in industry for the
resolution of racemic mixtures of amino acids, using amino acid acylase1,3. A variety of
commercially available ion exchangers have been investigated for this purpose32. One of
the techniques, which has gained importance more recently, is the use of
polyethylenimine for imparting polycationic characteristics to many of the neutral
supports based on cellulose or inorganic materials54. Enzymes with low pI, like
invertase55, urease56, glucose oxidase57, catalase57, and other enzymes54 have been bound
through adsorption followed by cross-linking on polyethylenimine-coated supports.
Immobilization of enzymes through hydrophobic interaction has also shown
promise58,59. One of the important features of this technique, which is of great
significance, is that, unlike ionic binding, hydrophobic interactions are usually stabilized
by high ionic concentrations, thus enabling the use of high concentrations of substrates as
desired in an industrial process without the fear of desorption. Other types of strong
interactive binding techniques have also been reported for the reversible immobilization
of enzymes. A typical example is the immobilization of soybean b -amylase on phenyl-
boronate agarose, which can be reversed for the recovery of the support using sorbitol 60.
Varieties of biospecific interactions have also been investigated for the reversible
immobilization of enzymes by adsorption. Enzymes like acetyl choline esterase, ascorbic
acid oxidase, invertase, peroxidase, glucose oxidase, etc. have been immobilized by
biospecific-reversible immobilization on lectin-bound supports61,62 and invertase, using
polyclonal antiinvertase antibodies63.
Occlusion
Confining enzymes within the lattices of polymerized gels is another method for
immobilization. This allows the free diffusion of low molecular weight substrates and
reaction products. The usual method is to polymerize the hydrophilic matrix in an
aqueous solution of the enzyme and break up the polymeric mass to the desired particle
size.
As there is no bond formation between the enzyme and the polymer matrix, occlusion
provides a generally applicable method that, in theory, involoves no disruption of the
protein molecules. However, free radicals generated on the course of the polymerization
may affect the activity of entrapped enzymes. Another disadvantage is that only low
molecular weight substrates can diffuse rapidly to the enzyme, thus making the method
unsuitable for enzymes that act on macromolecular substrates, such as ribonuclease,
trypsin, dextranase, etc.
The broad distribution in pore size of synthetic gels of the polyacrylamide type inevitably
results in leakage of the entrapped enzyme, even after prolonged washing. This may be
overcome by cross-linking the entrapped protein with glutaraldehyde. Alternatively,
ultrafiltration membranes of well-defined pore size may be used to occlude the enzyme.
Covalent Binding
The most intensely studied of the insolubilization techniques is the formation of covalent
bonds between the enzyme and the support matrix. When trying to select the type of
reaction by which a given protein should be insolubilized, the choice is limited by the fact
that the binding reaction must be performed under conditions that do not cause loss of
enzymic activity, and the active site of the enzyme must be unaffected by the reagents
used.
The functional groups of proteins suitable for covalent binding under mild conditions
include (i) the alpha amino groups of the chain and the epsilon amino groups of lysine
and arginine, (ii) the alpha carboxyl group of the chain end and the beta and gamma
carboxyl groups of aspartic and glutamic acids, (iii) the phenol ring of tyrosine, (iv) the
thiol group of cysteine, (v) the hydroxyl groups of serine and threonine, (vi) the imidazile
group of histidine, and (vii) the indole group of tryptophan.
A small number of reactions have been designed to couple with functional groups on the
protein other than the amino and phenolic residues. Aminoethyl cellulose has been
coupled to the carboxylic acid residues of enzymic protein in the presence of
carbodiimide, and thiol residues of a protein have been oxidatively coupled to the thiol
groups of a cross-linked copolymer of acrylamide and N-acryloyl-cystein.
It is possible in some cases to increase the number of reactive residues of an enzyme in
order to increase the yield of insolubilized enzyme and to provide alternative reaction
sites to those essential for enzymic activity. As with cross-linking, covalent bonding
should provide stable, insolubilized enzyme derivatives that do not leach enzyme into the
surrounding solution. The wide variety of binding reactions, and insoluble carriers with
functional groups capable of covalent coupling, or being activated to give such groups,
makes this a generally applicable method of insolubilization, even if very little is known
about the protein structure or active site of the enzyme to be coupled.

Covalent binding is an extensively used technique for the immobilization of enzymes,

though it is not a good technique for the immobilization of cells. The functional groups
extensively investigated are the amino, carboxyl, and the phenolic group of tyrosine1,32.
Enzymes are covalently linked to the support through the functional groups in the
enzymes, which are not essential for the catalytic activity. It is often advisable to carry
out the immobilization in the presence of its substrate or a competitive inhibitor so as to
protect the active site. The covalent binding should also be optimized so as not to alter its
conformational flexibility. Some of these problems however, can be obviated by covalent
bonding through the carbohydrate moiety when a glycoprotein is concerned. A number of
industrially useful enzymes are glycoproteins wherein the carbohydrate moiety may not
be essential for its activity. In general, functional aldehyde group can be introduced in a
glycoprotein by oxidizing the carbohydrate moiety by periodate oxidation without
significantly affecting the enzyme activity. The enzyme could then be covalently linked
to a support containing an alky amine group through Schiffs base reaction. Enzymes like
glucose oxidase, peroxidase, invertase, etc. have been immobilized using this
technique32,38,39. Covalent binding has been extensively investigated using inorganic
supports. Enzymes covalently bound to inorganic supports have been used in the
industry40. Enzymes have also been bound to synthetic membranes, thus integrating
biconversion and downstream processing41,42. Large-scale processes using such an
approach have been demonstrated for the preparation of invert sugar using invertase43.

Carrier-Binding
The carrier-binding method is the oldest immobilization technique for enzymes. In
this method, the amount of enzyme bound to the carrier and the activity after
immobilization depend on the nature of the carrier. The following picture shows how the
enzyme is bound to the carrier:

The selection of the carrier depends on the nature of the enzyme itself, as well as the:
• Particle size
• Surface area
• Molar ratio of hydrophilic to hydrophobic groups
 • Chemical composition
In general, an increase in the ratio of hydrophilic groups and in the concentration of
bound enzymes, results in a higher activity of the immobilized enzymes. Some of the
most commonly used carriers for enzyme immobilization are polysaccharide derivatives
such as cellulose, dextran, agarose, and polyacrylamide gel.
According to the binding mode of the enzyme, the carrier-binding method can be
further sub-classified into:
• Physical Adsorption
• Ionic Binding
• Covalent Binding

Cross-Linking

Immobilization of enzymes has been achieved by intermolecular cross-linking of the

protein, either to other protein molecules or to functional groups on an insoluble support
matrix.. Cross-linking an enzyme to itself is both expensive and insufficient, as some of
the protein material will inevitably be acting mainly as a support. This will result in
relatively low enzymatic activity. Generally, cross-linking is best used in conjunction
with one of the other methods. It is used mostly as a means of stabilizing adsorbed
enzymes and also for preventing leakage from polyacrylamide gels.

Since the enzyme is covalently linked to the support matrix, very little desorption is
likely using this method. Marshall (1973), for example, reported that carbamy
phosphokinase cross-linked to alkyl amine glass with glutaraldehyde lost only 16% of its
activity after continuous use in a column at room temperature for fourteen days.
The most common reagent used for cross-linking is glutaraldehyde. Cross-linking
reactions are carried out under relatively severe conditions. These harsh conditions can
change the conformation of active center of the enzyme; and so may lead to significant
loss of activity. Immobilization of enzymes has been achieved by intermolecular cross-
linking of the protein, either to other protein molecules or to functional groups on an
insoluble support matrix.. Cross-linking an enzyme to itself is both expensive and
insufficient, as some of the protein material will inevitably be acting mainly as a support,
resulting in relatively low enzymic activity. Generally, cross-linking is best used in
conjunction with one of the other methods. Preventing leakage from polyacrylamide gels
has already been mentioned, but it is used much more widely as a means of stablilizing
adsorbed enzymes.
Since the enzyme is covalently linked to the support matrix, very little desorption is
likely using this method. Marshall (1973), for example, reported that carbamyl
phosphokinase cross-linked to alkylamine glass with glutaraldehyde lost only 16% of its
activity after continuous use in a column at room temperature for fourteen days.
Cross-linking
Biocatalysts can also be immobilized through chemical cross-linking using homo- as well
as heterobifunctional cross-linking agents1. Among these, glutaraldehyde which interacts
with the amino groups through a base reaction has been extensively used in view of its
GRAS status, low cost, high efficiency, and stability44. The enzymes or the cells have
been normally cross-linked in the presence of an inert protein like gelatine, albumin, and
collagen4,32. Studies from our laboratory have shown the use of raw hen egg white as an
economic, easily available novel proteinic support rich in lysozyme for the
immobilization of enzyme or nonviable cells either in a powder45–47, bead48 or highly
porous foam49,50 form, using glutaraldehyde as the cross-linker. The unique feature of this
support is the large concentration of lysozyme naturally present in hen egg white which
gets co-immobilized, thus imparting the bacteriolytic property to the support49,51.
Adsorption followed by cross-linking has also been used for the immobilization of
enzymes. The technique of cross-linking in the presence of an inert protein can be applied
to either enzymes or cells. The technique can also be used for the immobilization of
enzymes by cross-linking the cell homogenates52. Osmotic stabilization of cellular
organelles53 or halophilic cells15 prior to immobilization using cross-linkers has also
shown promise.

Entrapping Enzymes

The entrapment method of immobilization is based on the localization of an enzyme

within the lattice of a polymer matrix or membrane. It is done in such a way as to retain
protein while allowing penetration of substrate. It can be classified into lattice and micro
capsule types.

This method differs from the covalent binding and cross linking in that the enzyme
itself does not bind to the gel matrix or membrane. This results in a wide applicability.
The conditions used in the chemical polymerization reaction are relatively severe and
result in the loss of enzyme activity. Therefore, careful selection of the most suitable
conditions for the immobilization of various enzymes is required.
Lattice-Type entrapment involves entrapping enzymes within the interstitial spaces
of a cross-linked water-insoluble polymer. Some synthetic polymers such as
polyarylamide, polyvinylalcohol, etc... and natural polymer (starch) have been used to
immobilize enzymes using this technique.
Microcapsule-Type entrapping involves enclosing the enzymes within semi
permeable polymer membranes. The preparation of enzyme micro capsules requires
extremely well-controlled conditions and the procedures for micro capsulation of
enzymes can be classified as:
• Interfacial Polymerization Method: In this procedure, enzymes are enclosed in
semi permeable membranes of polymers. An aqueous mixture of the enzyme and
hydrophilic monomer are emulsified in a water-immiscible organic solvent. Then
the same hydrophilic monomer is added to the organic solvent by stirring.
Polymerization of the monomers then occurs at the interface between the aqueous
and organic solvent phases in the emulsion. The result is that the enzyme in the
aqueous phase is enclosed in a membrane of polymer.
• Liquid Drying: In this process, a polymer is dissolved in a water-immiscible
organic solvent which has a boiling point lower than that of water. An aqueous
solution of enzyme is dispersed in the organic phase to form a first emulsion of
water-in-oil type. The first emulsion containing aqueous micro droplets is then
dispersed in an aqueous phase containing protective colloidal substances such as
gelatin, and surfactants, and a secondary emulsion is prepared. The organic
solvent in then removed by warming in vacuum. A polymer membrane is thus
produced to give enzyme micro capsules.
• Phase Separation: One purification method for polymers involves dissolving the
polymer in an organic solvent and re-precipitating it. This is accomplished by
adding another organic solvent which is miscible with the first, but which does
not dissolve the polymer.
The form an of immobilized enzyme can be classified into four types: particles,
membranes, tubes, and filters. Most immobilized enzymes are in particle form for ease of
handling and ease of application.
• Particles - The particle form is described in the above section.
• Membranes - Enzyme membranes can be prepared by attaching enzymes to
membrane-type carriers, or by molding into membrane form. The molding is
done after the enzymes have been enclosed within semi-permeate membranes of
polymer by entrapment.
• Tubes - Enzyme tubes are produced using Nylon and polyacrylamide tubes as
carriers. The polymer tube is first treated in a series of chemical reactions and the
enzyme is bound by diazo coupling to give a tube in a final step.
• Fibers - Enzymes that have been immobilized by entrapment in fibers to form
enzyme fibers.
The solid supports used for enzyme immobilization can be inorganic or organic .
Some organic supports include: Polysaccharides, Proteins, Carbon, Polystyrenes,
Polyacrylates, Maleic Anhydride based Copolymers, Polypeptides, Vinyl and Allyl
Polymers, and Polyamides.
Entrapment has been extensively used for the immobilization of cells, but not for
enzymes. The major limitation of this technique for the immobilization of enzymes is the
possible slow leakage during continuous use in view of the small molecular size
compared to
the cells. Biocatalysts have been entrapped in natural polymers like agar, agarose and
gelatine through thermoreversal polymerization, but in alginate and carrageenan by
ionotropic gelation3,4,33. A number of synthetic polymers have also been investigated.
Notable among them are the photo-crosslinkable resins, polyurethane prepolymers34, and
acrylic polymers like polyacrylamide17,24. Among these, the most widespread matrix
made from monomeric precursors is the polyacrylamide gel. Polyacrylamide may not be
a useful support for use in food industry in view of its toxicity, but can have potentials in
the treatment of waste and in the fabrication of analytical devices containing biocatalysts.
One of the major limitations of entrapment technique is the diffusional limitation as well
as the steric hindrance, especially when the macromolecular substrates like starch and
proteins are used. Diffusional problems can be minimized by entrapment in fine fibres of
cellulose acetate or other synthetic materials32 or by using an open pore matrix35.
Recently, the development of so-called hydrogels and thermoreactive water-soluble
polymers, like the albumin-poly (ethylene glycol) hydrogel, have attracted attention in
the field of biotechnology36. In the area of health care, they offer new avenues for enzyme
immobilization. Such gels with a water content of about 96% provide a
microenvironment for the immobilized enzyme close to that of the soluble enzyme with
minimal diffusional restrictions37.
Traditional enzyme immobilization procedures involve isolation of the enzyme,
followed by use of several steps for the immobilization. Costs of enzyme purification, the
immobilization procedure, bioreactor operational stability, and bioreactor regeneration
are the major factors that determine the cost of a bioreactor process. Development of
techniques for the simultaneous isolation and immobilization of enzymes from crude
extracts has obviated these problems. Some typical examples include the immobilization
of a streptavidin-b -galactosidase fusion protein expressed in Escherichia coli and
bioselectively adsorbed from a crude cell lysate to biotin which is covalently immobilized
on controlled pore glass64, and the simultaneous purification and immobilization of D-
amino acid oxidase from Trigonopsis variabilis cell lysate adsorbed on phenyl
sepharose59. The technique can have future potentials especially in the downstream
processing and immobilization of enzyme/proteins obtained by recombinant DNA
technology.
Techniques for the adhesion of whole cells on polymeric surfaces are also currently
gaining considerable importance. The major advantage for the cells immobilized through
adhesion is reduction or elimination of the mass transfer problems associated with the
commonly used gel entrapment method. The technique of immobilization usually being
followed is the microbial colonization by recycling of the cell suspension along with
nutrients, such that a biofilm is gradually formed. This often results in the immobilization
of cells in a viable form for use in heterogeneous fermentations65. Useful techniques have
been developed also for the immobilization of nonviable cells to be used as an enzyme
source for simple chemical conversions. Notable among them include treating the cells or
the support with trivalent metal ions like Al3+ or Fe3+ or charged colloidal particles66, and
use of polycationic polymers like chitosan67. Novel techniques have been developed to
adhere cells strongly on a variety of polymeric surfaces including glass68, cotton cloth69,
cotton threads19, and other synthetic and inorganic surfaces using polyethylenimine 70.
This technique has also been recently used for the simultaneous filtration and
immobilization of cells from a flowing suspension, thus integrating downstream
processing with bioprocessing71.
Immobilized enzymes in organic solvents
In recent years, much research has centered on the conduct of enzyme reactions in
organic solvents. It is now well established that hydrolytic enzymes can catalyse
esterification and transesterification reactions in monophasic organic solvents and in
water-organic biphasic systems. Conventional immobilized enzymes are principally used
to facilitate catalysts recovery. Even though enzymes by themselves are insoluble in
organic solvents, in addition to others, the prime importance to the use of enzymes in
organic media is the necessity to avoid enzyme deactivation or denaturation. A number of
enzymes which are used in organic solvents have been immobilized using a variety of
techniques with a view to stabilizing them72. In systems containing enzymes immobilized
on solid supports and working in organic media, the support has a significant influence
on the total enzyme activity, and can also displace the reaction equilibrium (hydrolysis
towards synthesis) due to the interaction of the support with the water molecules. Thus
the choice of a suitable support material, proper water content, and the selection of the
organic solvents are crucial for the use of immobilized enzymes in organic media73,74.
Another approach that has been explored is by chemical cross-linking of enzyme crystals,
thereby stabilizing the crystalline lattice and its constituent enzyme molecules, which
result in forming highly concentrated immobilized enzyme particles that can be
lyophilized and stored indefinitely at room temperature. Cross-linked enzyme crystals
retain catalytic activity in harsh conditions, including temperature and pH extremes,
exogenous proteases, and exposure to organic or aqueous solvents75,76. Lyophilized cross-
linked enzyme crystals can be reconstituted easily in these solvents as active
monodisperse suspensions. Cross-linked enzyme crystals have shown promise in a
variety of applications like the synthesis of aspartame, using thermolysin 76, and for the
resolution of chiral esters77. The techniques of immobilization can also be extended to
obtain organic solvent-soluble enzymes by treating them with hydrophobic molecules
like the lipids78.
IMMOBILIZATION OF ENZYMES FOR THE FABRICATION OF
BIOSENSORS
Most of the techniques described above have been used for the immobilization of
biocatalyst for biosensor applications79. The choice of the support and the technique for
the preparation of membranes has been dictated by the low diffusional resistance of the
membrane coupled with its ability to incorporate optimal amount of enzyme per unit area.
In this respect, stable membranes have been prepared by binding glucose oxidase to
cheese cloth in the fabrication of a glucose biosensor80,81. Enzymes entrapped inside the
reversed micelle have also shown promise in the fabrication of biosensors 82. Cross-linked
enzyme crystals (CLCs) described above provide their own supports and so achieve
enzyme concentration close to the theoretical packing limit in excess of even highly
concentrated enzyme solutions. In view of this, CLCs are particularly attractive in
biosensor applications where the largest possible signal per unit volume is often critical75.
Sensors based on small transducer or thinner enzyme immobilized membranes (miniature
biosensors) are also emerging. The development of molecular devices incorporating a
sophisticated and highly organized biological information processing function, is a long-
term goal of bioelectronics. For this purpose, it is necessary in the future to develop
suitable methods for microimmobilizing the proteins/enzymes into an organized
array/pattern, as well as designing molecular structures capable of performing the
required function. A typical example is the microimmobilization of proteins into
organized patterns on a silicone wafer based on a specific binding reaction between
strepatavidin and biotin combined with photolithography techniques83. Immobilized
enzymes have also been used for various other analytical purposes. A recent development
has been in obtaining a stable dry immobilized enzyme, like acetylcholineesterase, on
polystyrene microtitration plates for mass screening of its inhibitors in water and
biological fluids84.
APPLICATIONS OF IMMOBILIZED ENZYMES
The first industrial use of an immobilized enzyme is amino acid acylase by Tanabe
Seiyaku Company, Japan, for the resolution of recemic mixtures of chemically
synthesized amino acids. Amino acid acylase catalyses the deacetylation of the L form of
the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid, that can be easily
separated, racemized and recycled. Some of the immobilized preparations used for this
purpose include enzyme immobilized by ionic binding to DEAE-sephadex and the
enzyme entrapped as microdroplets of its aqueous solution into fibres of cellulose
triacetate by means of fibre wet spinning developed by Snam Progetti. Rohm GmbH have
immobilized this enzyme on macroporous beads made of flexiglass-like material93,94.
By far, the most important application of immobilized enzymes in industry is for the
conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase95.
Some of the commercial preparations have been listed in Table 2. It is evident that most
of the commercial preparations use either the adsorption or the cross-linking technique.
Application of glucose isomerase technology has gained considerable importance,
especially in nontropical countries that have abundant starch raw material. Unlike these
countries, in tropical countries like India, where sugarcane cultivation is abundant, the
high fructose syrups can be obtained by a simpler process of hydrolysis of sucrose using
invertase. Compared to sucrose, invert sugar has a higher humectancy, higher solubility
and osmotic pressure. Historically, invertase is perhaps the first reported enzyme in an
immobilized form96. A large number of immobilized invertase systems have been
patented97. The possible use of whole cells of yeast as a source of invertase was
demonstrated by D’Souza and Nadkarni as early as 1978 (ref. 98). A systematic study has
been carried out in our laboratory for the preparation of invert sugar using immobilized
invertase or the whole cells of yeast17–19,38,45,55,68,69,71,98. These comprehensive studies
carried out on various aspects in our laboratory of utilizing immobilized whole-yeast
have resulted in an industrial process for the production of invert sugar.
L-aspartic acid is widely used in medicines and as a food additive. The enzyme aspartase
catalyses a one-step stereospecific addition of ammonia to the double bond of fumaric
acid. The enzymes have been immobilized using the whole cells of Escherichia coli. This
is considered as the first industrial application of an immobilized microbial cell. The
initial process made use of polyacrylamide entrapment which was later substituted with
the carragenan treated with glutaraldehyde and hexamethylenediamine. Kyowa Hakko
Kogyo Co. uses Duolite A7, a phenolformaldehyde resin, for adsorbing aspartase used in
their continuous process99. Other firms include Mitsubishi Petrochemical Co.100 and
Purification Engineering Inc101. Some of the firms, specially in Japan like Tanabe Seiyaku
and Kyowa Hakko, have used the immobilized fumarase for the production of malic acid
(for pharmaceutical use)94. These processes make use of immobilized nonviable cells of
Brevibacterium ammoniagenes or B. flavus as a source of fumarase. Malic acid is
becoming of greater market interest as food acidulant in competition with citric acid.
Studies from our laboratory have shown the possibility of using immobilized
mitochondria as a source of fumarase6.
One of the major applications of immobilized biocatalysts in dairy industry is in the
preparation of lactose-hydrolysed milk and whey, using b -galactosidase. A large
population of lactose intolerants can consume lactose-hydrolysed milk. This is of great
significance in a country like India where lactose intolerance is quite prevalent102. Lactose
hydrolysis also enhances the sweetness and solubility of the sugars, and can find future
potentials in preparation of a variety of dairy products. Lactose-hydrolysed whey may be
used as a component of whey-based beverages, leavening agents, feed stuffs, or may be
fermented to produce ethanol and yeast, thus converting an inexpensive byproduct into a
highly nutritious, good quality food ingredient99. The first company to commercially
hydrolyse lactose in milk by immobilized lactase was Centrale del Latte of Milan, Italy,
utilizing the Snamprogetti technology. The process makes use of a neutral lactase from
yeast entrapped in synthetic fibres103. Specialist Dairy Ingredients, a joint venture
between the Milk Marketing Board of England and Wales and Corning, had set up an
immobilized b -galctosidase plant in North Wales for the production of lactose-
hydrolysed whey. Unlike the milk, the acidic b -galactosidase of fungal origin has been
used for this purpose31. Some of the commercial b -galactosidase systems have been
summarized in Table 3. An immobilized preparation obtained by cross-linking β-
galactosidase in hen egg white (lyophilized dry powder) has been used in our laboratory
for the hydrolysis of lactose47. A major problem in the large-scale continuous processing
of milk using immobilized enzyme is the microbial contamination which has necessitated
the introduction of intermittent sanitation steps. A co-immobilizate obtained by binding
of glucose oxidase on the microbial cell wall using Con A has been used to minimize the
bacterial contamination during the continuous hydrolysis of lactose by the initiation of
the natural lacto-peroxidase system in milk88. A novel technique for the removal of
lactose by heterogeneous fermentation of the milk using immobilized viable cells of K.
fragilis has also been developed10.
One of the major applications of immobilized enzymes in pharmaceutical industry is the
production of 6-aminopenicillanic acid (6-APA) by the deacylation of the side chain in
either penicillin G or V, using penicillin acylase (penicillin amidase)104. More than 50%
of 6-APA produced today is enzymatically using the immobilized route. One of the major
reasons for its success is in obtaining a purer product, thereby minimizing the purification
costs. The first setting up of industrial process for the production of 6-APA was in 1970s
simultaneously by Squibb (USA), Astra (Sweden) and Riga Biochemical Plant (USSR).
Currently, most of the pharmaceutical giants make use of this technology. A number of
immobilized systems have been patented or commercially produced for penicillin acylase
which make use of a variety of techniques either using the isolated enzyme or the whole
cells100,105,106. This is also one of the major applications of the immobilized enzyme
technology in India. Similar approach has also been used for the production of 7-
aminodeacetoxy-cephalosporanic acid, an intermediate in the production of semisynthetic
cephalosporins.
Immobilized oxidoreductases are gaining considerable importance in biotechnology to
carry out synthetic transformations. Of particular significance in this regard are
oxidoreductase-mediated asymmetric synthesis of amino acids, steroids and other
pharmaceuticals and a host of speciality chemicals. They play a major role in clinical
diagnosis and other analytical applications like the biosensors. Future applications for
oxidoreductases can be in areas as diverse as polymer synthesis, pollution control, and
oxygenation of hydrocarbons107. Immobilized glucose oxidase can find application in the
production of gluconic acid, removal of oxygen from beverages, and in the removal of
glucose from eggs prior to dehydration in order to prevent Maillard reaction. Studies
carried out in this direction in our laboratory have shown that glucose can be removed
from egg, using glucose oxidase and catalase which are co-immobilized either on
polycationic cotton cloth57 or in hen egg white foam matrix50. Alternatively, glucose can
also be removed by rapid heterogeneous fermentation of egg melange, using immobilized
yeast108. Immobilized D-amino acid oxidase has been investigated for the production of
keto acid analogues of the amino acids, which find application in the management of
chronic uremia. Keto acids can be obtained using either L- or D-amino acid oxidases. The
use of D-amino acid oxidase has the advantage of simultaneous separation of natural L-
isomer from DL-recemates along with the conversion of D-isomer to the corresponding
keto acid which can then be transamina-ted in the body to give the L-amino acid. Of the
several microorganisms screened, the triangular yeast T. variabilis was found to be the
most potent source of D-amino acid oxidase with the ability to deaminate most of the D-
amino acids109. The permeabilized cells entrapped either in radiation polymerized
acrylamide24 Ca-alginate23 or gelatin25 have shown promise in the preparation of a -keto
acids. Another interesting enzyme that can be used profitably in immobilized form is
catalase for the destruction of hydrogen peroxide employed in the cold sterilization of
milk. A few reports are available on its immobilization using yeast cells11,22.
Lipase catalyses a series of different reactions. Although they were designed by nature
to cleave the ester bonds of triacylglycerols (hydrolysis), lipase are also able to catalyse
the reverse reaction under microaqueous conditions, viz. formation of ester bonds
between alcohol and carboxylic acid moieties. These two basic processes can be
combined in a sequential fashion to give rise to a set of reactions generally termed as
interesterification. Immobilized lipases have been investigated for both these processes.
Lipases possess a variety of industrial potentials starting from use in detergents; leather
treatment controlled hydrolysis of milk fat for acceleration of cheese ripening; hydrolysis,
glycerolysis and alcoholysis of bulk fats and oils; production of optically pure
compounds, flavours, etc. Lipases are spontaneously soluble in aqueous phase but their
natural substrates (lipids) are not. Although use of proper organic solvents as an
emulsifier helps in overcoming the problem of intimate contact between the substrate and
enzyme, the practical use of lipases in such psuedohomogeneous reactions poses
technological difficulties. Varieties of approaches to solve these, using immobilized
lipases, have recently been reviewed110.
Significant research has also been carried out on the immobilization and use of
glucoamylase. This is an example of an immobilized enzyme that probably is not
competitive with the free enzyme and hence has not found large-scale industrial
application111. This is mainly because soluble enzyme is cheap and has been used for over
two decades in a very optimized process without technical problems. Immobilization has
 also not found to significantly enhance the thermostability of amylase 111. Immobilized
renin or other proteases might allow for the continuous coagulation of milk for cheese
manufacture112. One of the major limitations in the use of enzymes which act on
macromolecular substrates or particulate or colloidal substrates like starch or cellulose
pectin or proteins has been the low retention of their realistic activities with natural
substrates due to the steric hindrance. Efforts have been made to minimize these
problems by attaching enzymes through spacer arms113. In this direction, application of
tris (hydroxymethyl) phosphine as a coupling agent114 may have future potentials for the
immobilization of enzymes which act on macromolecular substrates. Other problem,
when particulate materials are used as the substrates for an enzyme, is difficulty in the
separation of the immobilized enzyme from the final mixture. Efforts have been made in
this direction to magnetize the bicatalyst either by directly binding the enzyme on
magnetic materials (magnetite or stainless steel powder) or by co-entrapping magnetic
material so that they can be recovered using an external magnet98,115. Magnetized
biocatalysts also help in the fabrication of magnetofluidized bed reactor116.
A variety of biologically active peptides are gaining importance in various fields
including in pharmaceuti-cal industries and in food industries as sweeteners, flavourings,
antioxidants and nutritional supplements. Proteases have emerged over the last two
decades as powerful catalysts for the synthesis and modification of peptides. The field of
immobilized proteases may have a future role in this area 117,118. One of the important large
scale applications will be in the synthesis of peptide sweetener using immobilized
enzymes like the thermolysin119. Proteolytic enzymes, such as subtilisin, a-chymotrypsin,
papain, ficin or bromelain, which have been immobilized by covalent binding, adsorption
or cross-linking to polymeric supports are used (Bayer AG) to resolve A N-acyl-DL-
phenylglycine ester racemate, yielding N-acyl-D-esters or N-acyl-D-amides and N-acyl-
L-acids100. Immobilized aminopeptidases have been used to separate DL-
phenylgycinamide racemates100. SNAM-Progetti SpA-UK have used the immobilized
hydropyrimidine hydrolase to prepare D-carmamyl amino acids and the corresponding D-
amino acids from various substituted hydantoins100.
IMPORTANT APPLICATIONS OF IMMOBILIZED ENZYMES-
ENZYME SUBSTRATE REACTOR PRODUCT
USED*
Aminoacylase (immobilized N-acyl-DL- amino acids PBR L-amino acids
on anion exchange resins)
Aspartate ammonialyase Fumaric acid + NH4+ PBR L-aspartic acid
Cyanidase Cyanide present in industrial Formic acid
waste, food or feed
Glucoamylase Dextrins produced by α- D-glucose
amylase
Glucose isomerase D-glucose in glucose syrup PBR High fructose
(immobilized with corn syrup
glutaraldehyde by cross
linking)
Invertase Sucrose PBR Invert sugar
Lactase (immobilized in Milk and whey STR Lactose-free
cellulose triacetate fibers) milk and whey
Lipase Vegetable oils, e.g., palm oil Cocoa butter
substitute
Nitrile hydratase (immobilized Acrylonitrile Acrylamide
cells)
Penicillin amylases Penicillin G and penicillin V STR, PBR Penicillins
Raffinase (immobilized cells) Raffinose in beet juice, STR Raffinose-free
soybean milk solutions
PRODUCTION OF ENZYMES

Enzyme technology broadly involves production, isolation, purification and use of enzyme for the
ultimate benefit of humankind. The first enzyme produced industrial was takadiastase (a fungal
analyse) in 1896, in united states. It was as a pharamaceutical agent to cure digestive disorders.
Commercial enzymes can be produced from a wide range of biological sources. At
present, a great majority (80%) of them are microbial sources. The different organisms and their
relative contribution for the production of commercial enzymes are given below
Fungi – 60%
Bacteria – 24%
Yeast – 4%
Streptomyces – 2%
Higher animals – 6%
Higher plants – 4%
Enzymes from animal and plant sources
In the early days, animal and plat sources largely contributed to enzymes. Even now for
certain enzymes they are the major sources.
A selected list of plant ( Table 1) and animal (Table 2) enzymes with their sources aqnd
applications are given.
Table 1. Commercially produced enzymes from plant sources their applications.

Enzyme Source(s) Application(s)

β -Amylase Barely, soy bean Baking, preparation of maltose syrup.
Bromelain Pineapple Baking
Esterase Wheat Ester hydrolysis
Ficin Fig Meat tebneriser
Papin Ppaya Meat tenderizer, tanning, baking
Peroxidase Horse radish Diagnostic
Urease Jack bean Diagnostic

Table 2
Commercially produced enzymes from animal sources and their applications
Enzymes Sources Applications
Amylase, esterase Lamb,calf Digestive aids
Pepsin, trypsin Bovine Preparation of cheese
Lipase, rennin Poreine
(chymosin),phospholipase, phytase
Lysozme Hen eggs Cell wall breakage in bacteria
Human urine Urokinase For dissolution of blood clots
 Animal organs and tissues are very good sources foe enzymes such as lipases, esterase
and proteases. The enzyme lysozyme is mostly obtained from hen eggs. Some plants excellent
sources for certain enzymes-papain (papaya), bromelain (pincapple).
Limitation
There are several dracobacks associated with the manufacture of enzymes fraom animal
and plant sources. The quantities are limited and there is a wide variation in their distribution.
The most important limitations are the difficulties in isolating, purifying the enzume and the cost
factor. For these reasons microbial production of enzymes is preferred.

4.1.1 ENZYMES FROM MICROBIAL SOURCES

Microorganisms are the most signicant and convenient sources of commercial enzyme.
They can be made to produce abundant quantitic of enzymes under suitable growth conditions.
Microorganisms can be cultivateo by using inexoansive media and production can take place in a
short period. In addition it is easy to manipulate microorganisms in genetic engineering
techniques to increase the production of desired enzymes. Recovery, isolation and purification
process are easy with microbial enzymes than that with animal or plant sources.
In fact, most enzymes of industrial applications have been successfully produced by
microorganisms. Various fungi, bacteria and yeasts are employed for this purpose. A selected list
of enzymes, microbial sources and the applications are given in Table-3.

4.1.2 THE TECHNOLOGY OF ENZYME PRODUCTION GENERAL

CONSIDERATIONS
In general, the techniques employed for microbial production of enzymes are comparable
to the methods used for manufacture of other industrial products. The sailent features are briefly
described.
1. Selection of organisms
2. Formulation of medium
3. Production process
4. Recovery and purification of enzymes.
An outline of the flow chart for enzyme production by microorganisms is depicted in fig.1.
Selection of organism .
The most important criteria for selecting the microorganism are that the organism should
produce the maximum quantities of desired enzyme in a short time while the amounts of other
metabolite produced are minimal. Once the oraganism is selected, strain improvement for
optimizing the enzyme production can be done by appropriate methods (mutagens, OV rays).
From the organism chosen, inoculum can be prepared in a liquid.
Formulation of Medium
The culture medium chosen should contain all the nutrients to support adequate growth of
microorganisms that will ultimately result in good quantities of enzyme production. The
ingredients of the medium should be readily available at low cost and are nutritionally safe.
Some of the commonly used substrates for the medium are starch hydrolysate, molasses,
corn steep liquor yeast extract whey and soy bean meal. The pH of the medium should be kept
optimal for good microbial growth and enzyme production.
Production process
Industrial production of enzymes is mostly carried out by submerged liquid conditions,
and to a lesser extent by solid-substrate fermentation.
In submerged culture technique, the fields are more and the chances of infection are less. Hence,
this is a preferred method. However solid substrate fermentation is historically important and still
in use for the production of fungal enzymes. E.g. amylases, celluloses, proteases.
The medium can be sterilized by employing batch or continuous sterilization techniques.
The fermentation is started by inoculating the medium. The growth conditions (pH, temperature,
O2 supply, nutrient addition) are maintained at optimal levels. The froth formation can be
minimized by adding antifoan agents.
The production of enzymes is mostly carried ouet by batch fermentation, and to a lesser
extent by continuous process. The bioreactor system must be maintained sterile throughout the
fermentation process the duration of fermentation is variable around 2-7 days, in most production
processes. Besides the desired enzyme of several other metabolitesare also produced. The
enzyme(s) have to be recovered and purified.
Purification of Enzymes
After fermentation, the cells are separated from the growth medium by filtration or by
centrifugation. Depending on nature of enzyme namely, intracellular or extracellular either the
cells or the fermentation broth is further processed to separate and purity the enzyme.
Table.3. A selected list of industry (microblally) produced enzymes, their sources and
APPLICATIONS
Enzyme Source(s) Application(s)
α -Amylase Aspergillus oryzae Production of beer and alcohol,
Aspergillus niger Preparation of glucose syrups,
Bacillus subbtilus As a digestive aid
Bacillus licheniforns Removal of starch sizes
Amyloglucosidase Aspergillus niger Stacrh hydrolysis
Rhizopus nivous
Cellulose Aspergillus niger Alcohol and glucose production
Tricoderma koningi
Glucoamylase Aspergillus niger Production of beer and alcohol
Bacillus Starch hydrolysis
amyloliquefaciens
Glucose isomerase Arthrobacter sp Bacillus Manufacture of high fructose syrups
sp
Glucose oxidase Aspergillus niger Antioxidant in prepared foods

Invertase Saccharomyces cerevisiae Surcose inversion

Prepartion of artificial honey,
Confectionaries
Keratinase Streptomyces fradiae Removal of hair from hide
Lactase Kluyveromyus sp Lactose Hydrolysis
Saccharomyces fragilis Removal of lactose from whey
Lipase Candida lipolytica Preparation of cheese
Aspergillus niger Flavour production
Pectinase Aspergillus sp Clarification of fruit juices and wines
Sclerotina libertina Alcohol production, coffee concentration
Penicillin acylase Escherichia coli Production of 6-aminopenicillanic acid
Penicillanase Bacillus subtills Removal of Penicillin
Protease, acid Aspergillus niger Digestive aid,Substitute for calf rennet
Protease, neutral Bacillus Fish and meat tenderizer
amyloliquefaciens
Protease, alkaline Aspergillus oryzae Meat tenderizer
Streptomyces griseus Detergent additive
Bacillus sp Beer stabilizer
Pollulanase Klebsiella aerogens Hydrolysis of starch
Takadiastase Aspergillus oryzae Suplement to bread,Digestive aid
Extra cellular enzyme purification is easy from the broth. The recovery of intracellular enzyme is
more complicated and involves the disruption of cells and removal of cell debris and nucleic
acids. In some cases, enzyme may be both intracellular and extracellular, which requires
processing of both broth and cells. Intracellular enzymes may be released to medium by
increasing the permeability of cell membranes.

4.2 RECOVERY AND PURIFICATION OF ENZYMES

The desired enzyme produced may be excreted into the culture medium (extracellular
enzymes) or my be present within the cells (intracellular enzymes). Depending on the
requirement, the commercial enzyme may be crude or highly purified. Further, it may be in the
solid or liquid form. The steps involved in downstream processing i.e recovery and purification
steps employed will depend on the nature of the enzyme and the degree of purity desired.
The extraction and purification of a biotechnological product from fermentation is
referred to as downstream processing (DSP) or product recovery.
In fig.1 an outline of the major steps in downstream processing is given, and they are described in
some detail as follows
The four main stages of down stream processing is,
1. Solid-liquid separation
2. Concentration
3. Purification
4. Formulation
4.2.1 Release of Intracellular Products
In general, recovery of an extra cellular enzyme which is present in the broth is relatively
simpler compared to an intracellular enzyme. For the release of intracellular enzymes, special
techniques are needed for cell disruption. The method of disruption varies with the type of cells
and the nature of intracellular products. Since there is a wide veriation in the property of cell
disruption or breakage for instance, Gram-negative bacteria and filamentous fungi can be more
easily broken compound to Gram positive bacteria and yeasts.

4.2.2 CELL DISRUPTION

The cell disruption methods can be broadly classified into mechanical and non
mechanical methods
Mechanical methods
Mechanical methods can be applied to a liquid or solid medium. First consider some
methods applied to a liquid medium.
Ultrasonication:
Ultrasonication vibrators (sonicators) are used to disrupt the cell wall and membrane of
bacterial cells. Wave density is usually around 20 kHz/S. Rods are broken more readily than coai
and gram-negative cells more easily than gram-positive cells.
An electronic generator is used to generate ultrasonic waves and a transducer converts
these waves into mechanical oscillations by a titanium probe immersed in cell suspension.
Intracellular enzymes are released into the broth upon cell disruption.
High pressure Homogenization
This technique involves forcing of cell suspension at high pressure through a very narrow
orifice to come out to atmospheric pressure. The sudden release of high pressure creates a liquid
shear that can break the cells.

SOLID SHEAR
Grinding with glass beads
The cells mixed with glass beads are subjected to a very high speed in a reaction vessel.
The cells break as they are forced against the wall of the vessel by the beads. Several factors
influence the cell breakage size and quantity of the glass beads. Concentration and age of cells,
temperature and age of cells, temperature and agitator speed. Under optical conditions, one can
expect a maximal breakage of about 80% of the cells.
A diagrammatic representation of a cell disrupter employing glass bead is shown in figure 3.
It contains a cylindrical body with an inlet, outlet and central motor – driven shaft. To
this shaft are fitted radial agitators. The cylinders are fitted with glass beads. The cells
suspension is added through the inlet and the disrupted cells come out through the outlet. The
body of the cell disrupter is kept cool while the operation is on. Eg. Dyno – mill, Ball mill,
French press, etc.

NON – MECHANICAL – PHYSICAL METHODS:

Osmotic Shock:
Osmotic shock and rupture with ice crystals are commonly used methods. By slowly
freezing and then thawing a cell paste, the cell wall and membrane may be broken, releasing
enzymes into the media. Changes in osmotic pressure of the medium may result in the release of
certain enzymes, particularly periplasmic proteins in gram – negative cells.

Heat Shock:
Breakage of cells by subjecting them to heat is relatively easy and cheap. But this
technique can be used only for a very few heat – stable intracellular products.

CHEMICAL METHODS:
Treatment with alkalies, organic solvents and detergents can lyse the cells to release the
contents.

ALKALIES:
Alkalie treatment has been used for the extraction of some bacterial proteins. However,
the alkali stability of the desired product is very crucial for the success of this method, e.g.
recombinant growth hormone can be efficiently released from E.Coli by treatment with sodium
hydroxide at pH 11.

ORGANIC SOLVENTS:
Several water miscible organic solvents can be used to disrupt the cells. Eg. Methanol,
ethanol, isopropanol, butanol. The organic solvent toluene is frequently used. It is believed that
toluene dissolves membrane phospholipids and creates membrane pores for release of
intracellular contents.
Detergents:
Detergents that are ionic in nature, cationic – cetyl trimethyl ammonium bromide or
aninonic – sodium lauryl sulfate can denature membrane proteins and lyse the cells.
Non – ionic detergents Triton X – low or Tween are also used some extent.

ENZYMATIC METHODS OF CELL DISRUPTION:

Cell disruption by enzymatic methods has ceratin advantages i.e. lysis of cells occurs
under mild conditions in a selective manner. This is quire advantageous for product recovery.
Lysozyme is the most frequently used enzyme and is commercially available (produced from hen
egg white). It hydrolyses β - 1, 4 – glycosidic bonds of the mucopeptide inbacterial cell
walls. The Gram – positive bacteria (with high content of cell wall mucopeptides) are more
susceptible for the action of lysozyme.
 For Gram – negative bacteria, lysozyme in association with EDTA can break the cells. As
the cell wall gets digested by lysozyme, the osmotic effects break the periplasmic membrane to
release the intracellular contents.
Certain other enzymes are also used, although less frequently for cell disruption. For the
lysis of yeast cell walls, glucanase and mannanase in combination with proteases are used.

SOLID – LIQUID SEPARATION:

The first step in product recovery is the separation of whole cells (cell biomass) and other
insoluble ingredients from the culture broth. Several methods are in use for solid – liquid
separation. These include filtration and centrifugation.
Filtration:
Filtration is the most commonly used technique for separation the biomass and culture
filtrate.
The efficiency of filtration depends on many factors – the size of the organism, presence
of other organisms, viscosity of the medium and temperature. Several filters such as depth filters,
absolute filters, rotary drum vacuum filters and membrane filters are in use.
Depth filters:
They are composed of a filament matrix such as glass wool, asbestos or filter paper. The
particles are trapped within the matrix and the fluid passes out. Filamentous fungi can be
removed by using depth filters.
Absolute filters:
These filters are with specific pore sizes that are smaller than the particles to be removed.
Bacteria from culture medium can be removed by absolute filters.
Rotary Drum Vacuum filters:
These filters are frequently used for separation of broth containing 10 – 40% solids (by
volume) and particles in the size of 0.5 – 10 µ m. Rotary drum vacuum filters have been
successfully used for filtration of yeast cells and filamentous fungi.
The equipment is simple with low power consumption and is easy to operate (Fig.4).
The filtration unit consists of a rotating drum partially immersed in tank of broth. As the
drum rotates, it picks up the biomass which gets deposited as a cake on the drum surface. This
filter cake can be easily removed.
Membrane Filters:
In this type of filtration, membranes with specific pore sizes can be used. However,
clogging of filters is a major limitation. These are two types of membrane filtrations – static and
cross – flow filtration (Fig. 5).
In cross – flow filtration, the culture broth is pumped in a crosswise fashion across the
membrane. This reduces the clogging process and hence better than the static filtration.

CENTRIFUGATION:
The technique of centrifugation is based on the principle of density differences between
the particles to be separated and the medium. Thus centrifugation is mostly used for separating
solid particles from liquid phase.
The different types of centrifuges are depicted in fig.6. and briefly described Lereunder.
Tubular bowl centrifuge: (Fig 6.a.)
This is a simple and a small centrifuge, commonly used in pilot plants. Tubular bowl
centrifuge can be operated at a high centrifugal speed and can be run in both batch or continuous
mode. The solids are removed manually.
Disc Centrifuge: (Fig. 6.b.)
It consists of several discs that separate the bowl into settling zones. The feed slurry is
fed through a central tube, the clarified fluid moves upwards while the solids settle at the lower
surface.
Multi chamber Centrifuge:
This is basically a modification of tubular bowl type of centrifuge. It consists of several
chambers connected in such a way that the feed flows in a zig – zag fashion. There is a variation
in the centrifugal fore in different chambers. The force is much higher in the periphery chambers,
as a results smallest particles settle down in the outer most chamber.

Concentration:
The filtrate that is free from suspended paticles (Cells, cell debris, etc) usually contains
80 – 98% of water. The desired product is a very minor constituent. The water has to be removed
to achieve the product concentration. The commonly used techniques for concentrating biological
products are evaporation, liquid – liquid extraction, membrane filtration, precipitation and
adsorption. The actual product adopted on the nature of desired product (quality and quantity to
be retained as far as possible) and the cost factor.
Evaporation:
Water in the broth filtrate can be removed by a simple evaporation process. The
evaporators in general have a heating device for supply of steam and unit for the separation of
concentrated product and vapour, a condenser for condensing vapour, accessories and control
equipment. The capacity of the equipment is variable that may range from small laboratory scale
to industrial scale. Some of the important types of evaporators in common uses are as follows:
Eg. Plate evaporators, falling film evaporators, forced film evaporators, centrifugal forced film
evaporators.
LIQUID – LIQUID EXTRACTION:
The concentration of biological products can be achieved by transferring the desired
product (solute) from one liquid phase to another liquid phase, a phenomenon referred to liquid –
liquid extraction. Besides concentration, this technique is also useful for partial purification of a
product. The efficiency of extraction is dependent on the partition coefficient i.e. the relative
distribution of a substance between the two liquid phases.
The process of liquid – liquid extraction may be broadly categorized as ‘extraction of low
molecular – weight products’ and extraction of ‘high molecular weight products’.
EXTRACTION OF LOW MOLECULAR WEIGHT PRODUCTS:
By using organic solvents, the lipophilic compounds can be conveniently extracted.
EXTRACTION OF HIGH MOLECULAR WEIGHT PRODUCTS:
Proteins are the most predominant high molecular weight products produced in
fermentations industries. Organic solvents cannot be used for protein extraction, as they lose
their biological activities. They are extracted by using an aqueous two – phase systems.
Aqueous two – phase systems (ATPs):
They can be prepared by mixing a polymer (e.g. polyethyle glycol) and a salt solution
(ammonium sulphate) or two different polymers. Water is the main component in ATPs, but the
two phases are not miscible.
Cells and other solids remain in one phase while the proteins are transferred to other
phase. The distribution of the desired product is based on its surface and ionic character and the
nature of phases. The separation takes much longer time by ATPs.

PRECIPITATION:
Precipitation is the most commonly used technique in industry for the concentration of
macromolecules such as proteins and polysaccharides. Further, precipitation technique can also
be employed for the removal of creation unwanted by products, e.g. nuclei acids, pigments,
neutral salts, organic solvents, high molecular weight polymers (ionic or non – ionic), besides
alteration in temperature and pH are used in precipitation.
Isoelectric point:
 Enzymes and other proteins are highly charged molecules and can be precipitated with
appropriate change neutralizing chemicals. Once their changes are broken they form aggregates
and settle down as precipitate. When an acid or base is added, the enzyme protein can be brought
to its isoelectric pH. At this pH, there is no net charge on enzyme molecules and electrostatic
repulsion between them is low so that they tend to aggregate. Therefore adjusting the pH to the
isoelectric point of a protein causes its precipitation.
Salting Out:
‘Salting Out’ of proteins is achieved by increasing the ionic strength of a protein
containing solution by adding salts such as (NH4)2SO4 or Na2SO4. The added ions interact with
water more strongly, causing protein molecules to precipitate. The solubility of proteins in a
solution as a function of the ionic strength of the solution is given by,
log (S/S0) = – K'S (I)
Where, S – Solubility of protein in solution (g/l)
S0 – Solubility of protein when Z = 0, Z – ionic strength of solution
K'S – Salting – out constant, which is a function of temperature and pH.
Organic solvents:
Ethanol, acetone and propanol are the commonly used organic solvents for protein
precipitation.
Organic solvents addition at low temperature (T< - 5°C) cause the precipitation of
proteins by the reducing the dielectric constant of the solution. Thesolubility of protein as a
function of the dielectric constant of a solution is given by
log (S/S0) = – K' /DS2
Where, DS is the dielectric constant of a solution results in stronger electrostatic forces between
the protein molecules and facilitates protein precipitation. The addition of solvents reduces
protein – water molecule interactions and therefore decreases protein solubility.
NON – IONIC POLYMERS: Polyethylene glycol (PEG) is a high molecular weight non – ionic
polymer that can precipitate proteins. It reduces the quantity of water available for protein
salvation and precipitates proteins.
IONIC POLYMERS: The charged polymers such as polyacrylic acid and polyethyleneimine are
used. They form complexes with oppositely charged protein molecules that causes charge
neutralization and precipitation.
Increases in temperature:
The heat sensitive proteins can be precipitated by increasing the temperatures.
PRECIPITATION BY LIGANDS:
Ligands with specific binding sites for proteins have been successfully used for selective
precipitation.
FURTHER PURIFICATION PROCEDURE:
Further purification of enzymes after extracting the crude enzyme extract by the above
methods, involves Dialysis, Chromotography and electrophoresis.
Dialysis:
Dialysis is the process that is used to remove small molecules from enzyme. For this
enzyme precipitate obtained in previous step is dissolved in a small quantity of buffer solution in
which the enzyme was originally extracted.
The solution can be taken in a dialysis bag (may be a semi permeable membrane such as
a cellulose membrane with pores). The bag is suspened in either distilled water of a buffer of
known molarity and ionic composition. Some other salts or chemical smay have to be added
sometimes in the outer solution to prevent the loss of enzyme activity during dialysis (Fig. 7.).

Molecules having dimensions significantly greater than the pore diameter are retained
inside the dialysis bag, whereas smaller molecules and ions traverse the pores of such a
membrane and emerge in the dialysate outside the bag.
CHROMATOGRAPHY:
Chromatographic separation of proteins is the most common method of enzyme
purification.
It is basically an analytical technique dealing with the separation of closely related
compounds from a mixture. Chromatography usually consists of a ‘stationary phase’ and ‘mobile
phase’. The stationary phase is the porous solid matrix packed in a column onto which the
mixture of compounds to be separated is loaded? The compounds are elated by a mobile phase.
The eluate from the column can be monitored continuously (E.g. protein elution can be monitored
by ultraviolet adsorption at 280 nm and collected in fractions of definite volumes.
The different types of chromatography techniques used for separation (mainly proteins)
along with, the principles are given in Table.1.
Table 1: Chromatographic techniques along with the principles for separation of proteins

Chromatography Principle
Gel – filtration (size exclusion) Size and Shape
Ion – exchange Net charge
Affinity Biological affinity and molecular recognition

A large number of matrices are commercially available for purification of proteins e.g.
agarose, cellulose, polyacrylaminde, porous silica,m cross – linked dextan polystryrene. Some of
the important features of selected Cheomalographic techniques are briefly described.

GEL – FILTRATION CHROMATOGRAPHY:

In this chromatography, various proteins are separated on the basis of differences in their
molecular sizes. This type of chromatography is also known as molecular exclusion
chromatography or molecular sieve chromatography (Fiog. 8)
A column made up of glass or steel is taken and packed with a gel (E.g. Sephadex,
Sepharose and Biogel) or Porous bead made of an insoluble but highly hydrated polymer such as
dextran or agarose or polyaccylamind. (The beads are typically 100µ m (0.1 mm) in diameter).
A solution mixture containing molecules of different sizes (e.g. different proteins) is applied to
the top of the column and eluted. The smaller molecules enter the gel beads through their pores
and get trapped. On the other hand, the larger molecules cannot pass through the pores and
therefore come out first with the mobile liquid.
The elution volume is logarithmically proportional to the molecular weights.

ION EXCHANGE CHROMATOGRPHY:

It involves the separation of molecules based on their surface charges. Ion exchangers are
of two types:
• Cation exchangers – which have negatively charged groups like carboxymethyl and
sulfonate, phosphocellulose.
• Anion exchangers – With positively charged groups like diethyl amino ethyl (DEAE),
quaternary ammonium, groups such as triethyl amino ethyl (TEAE) and QAE.
The matrix material for the column is formed from beads of some inactive material, often
a carbohydrate such as cellulose or dextrans.
In ion – exchange chromatography, the pH of the medium is very crucial, since the net
charge caries with pH. In other words, the pH determines the effective charge on hboth the target
molecules and the ion – exchanger. (Fig. 9)
If an protein has a net positive charge at pH 7, it will usually bind two a column of beads
containing carboxylate groups, whereas a negatively charged protein will not.
 A positively charged protein bound to such a column ear than be eluted (released) by
increasing the concentration of sodium chloride or another salt in the elulting buffer because
sodium ions compete with positively charged groups on the protein for finding to the column.
Proteins that have a low density of net positive charge will emerge first, followed by those having
a higher charge density.
Positively charged proteins (Cationic proteins) can be separated on negatively charged
carboxymethyl – cellulose (CM – Cellulose) columns. Conversely, negatively charged proteins
(anionic proteins) can be separated by chromatography on positively charged diethylamine ethyl
– cellulose (DEAE – Cellulose) columns.

AFFINITY CHROMATOGRAPHY:
In this method, enzymes are purified according to their speficicity for a particular
substrate or cofactor.Affinity chromatography is based on the highly specific interaction between
solute molecules and ligands attached on polymeric or ceramic beads in a packed column.
The concept of affinity chromatography is described in Fig.10.
The matrix is usually agarrose. However pollyaisylamide, hydroxyethyl methaerylate,
cellulose and porous glass can also be used as the matrix bead. Spacer arms between the matrix
and ligand are usually llnear aliphatic hydrocarbons. The use of space arms between the matrix
and ligand may reduce the steric hindrance generated by the matrix.
Coupling between the matrix and ligand depends on the functional groups present on the matrix
and ligand. Chemically reactive groups on the support matrix usually are – OH, - NH2 or –
COOH groups. If the reactive group on the matrix is an – OH group (polysaccharides, glass
hydrooxyalkyl methacrylate), then cyanogens bromide (CnBr) is used as a coupling agent. The
cyanogens bromide – activated agarose reacts with primary amine groups present in proteins that
act as ligands.
After the desired solutes are fbound to theligand, elution is achieved by changing the pH or ionic
strength in the column. Ligand – solute molecule interaction in affinity chromatography are very
specifi. That is an enzyme inhibitor or substare may be used as a lighand in separating specific
enzyme from a mixture.

ELECTROPHORESIS:
Electroporesi is a technique in which enzyme molecules are separated by difference in
their net charge in the presence of an externally applied electric field. This technique is routinely
used in enzyme purification and isozyme separation in the laboratories, although it has found only
limited application at large scale. Since the technique is time consuming and is a bit expensive.
Various types of instrumental approaches have been used to separate and purity charged
molecules using electrophoresis. However, the most common method for purifying enzymes is
though electrophoresis on polyacrylamide gel. Polycrylamide is a polymer of acrylamide and
methylene bisacarylamide and when prepared as a gel it is transparent, thermostable, non – ionic
and extremely regular in structure.
The gel may be taken either in the form of a column or a slab, although the later is
preferred over the former (F.g 11). The protein mixture is loaded in the gel and the components
are separated under a direct current of constant voltage. The migration rate of various components
of the mixture is dependent upon their charge and molecular weight.
Sodium doderyl sulphate polyacrylamide gel electrophoresis (ADS – PAGE):

This form of polyacrylamide gel electrophoresis is the most widely used method for
analyzing protein mixtures qualitatively. It is particularly useful for monitoring protein
purification and because the method is based on the separation of proteins according to size, the
method can also be used to determine the relative molecular mass of proteins.
 SDS, (CH3 – (CH2)10 – CH2OSO3– Na+) is an anionic detergent, samples to be run on SDS
– PAGE are firstly boiled for 5 min in sample buffer containing β - mereaptoethanol and SDS.
The mercaptoethanol reduces any disulphide bridges present that are holding together the protein
tertiary structure and the SDS binds strongly to and denatures the protein. Each protein in the
mixture is therefore fully denatured by this treatment and opens up into a red – shaped structure
with x series of negatively charged as molecules along the polypeptide chain.
Once the samples are loaded a current is passed through the gel. The negatively charted
protein – SDS complexes now continue to move towards the anode and because they have the
same charge per unit length, they travel into the separating gel under the applied electric field
with the same mobility. However, as they pass through the separating gel the proteins separate,
owing to the molecular siwving properties of the gel. Quite simply, the smaller the protein the
more easily it can pass through the pores of the gel, whereas large proteins are successively
retarded by frictional resistance due to the sieving effect of the gels.
The sample buffer also contains an ionisable tracting due, usually bromophenol blue, that
allows the electrophoretic run to be monitored. When the dyue reaches to bottom of the gel, the
current is turned off and the gel is removed from between the glass plates andn shjaken in an
appropriate stain solution (usually Coomassie Brilliant Blue) for a few hours and then washed in
destain solution overnight. The destain solution removes unbound background due from the gel
leaving stained proteins visible as blue bands on a clear background.

DRYING AND PACKING:

The concentrations form of the enzyme can be obtained by dying. This can be done by
film evaporator or freezer dryers (lyophilizers).

FREEZE DRYING:
Freeze drying or lylophilization is the most preferred method for dying and formulation
of an enzyme. This is mainly because freeze – drying usually does not cause loss of biological
activity of the enzyme.
Lyophilization is based on the principle of sublimation of a liquid from a frozen state. In
the actual techniques the liquid containing the product in frozen and then dried in a freeze – dryer
under vacuum. The vacuum can now be released and the product containing vials can be sealed.
The dried enzyme can be packed and marketed. For certain enzymes, stability can be
achieved by keeping them in ammonium sulphate suspensions.
All the enzymes used in foods or medical treatments must be of high grade purity and
must meet the required specifications by the regulatory bodies. These enzymes should be totally
free from toxic materials, harmful micro organisms and should not cause allergic reactions.

MONITORING OF PURIFICATIONS OF AN ENZYME:

Successful enzyme purification is recognized by high specific activity of the final

purified reaction, may fold purification and high yield.

Yield (in fraction ) =

Units of enzyme in fraction
Units of enzyme in original preparation
The amount of enzyme present in a particular fraction is usually expressed in terms of
units, which are based upon the rate of the reaction that the enzyme catalyses. One International
unit of an enzyme that will convert 1µ mole of substarate to product in 1 min under defined
conditions (such as temperature at 30°C and the optimum pH).
A good indicator of enzyme purification is the fold purification. It is determined by
dividing the specific activity of enzyme in a particular fraction by the original specific activity of
enzyme in a particular fraction by the original specific activity of enzyme in a particular fraction
by the original specific activity (of crude homogenate).
Specific activity of fraction
Fold purification =
Original specific activity
The specific activity relates to the total activity in a fraction to the toal amount of protein
present in that fraction.
Total Units of enzyme in fraction
Specific activity =
Total amount of protein in fraction

A steady increase in the specific activity of fractions from the crude homogenate
indicates the successful approach in enzyme purification.
CENTRIFUGATION
Centrifugation is used to separate particles of size between 100 and 0.1µ m from liquid
by centrifugal forces. The sub-cellular location of many enzymes may be revealed by
microscopy, provided suitable fixation and staining procedures are followed.
The sedimentation characteristics of the various sub-cellular organelles are different, so it
is possible to separate them by centrifugation of a tissue homogenate, and then to investigate
which enzymes are associated with each cell fraction.
There are two types of centrifugation, they are as follows,
A. Differential centrifugation
B. Density-gradient centrifugation

A) Differential Centrifugation
The simplest and most cuidely used method for separating the various sub-cellular organelles
from each other is different centrifugation.
A tissue homogenate is prepared in a medium of low density (e.g. 0.25 M sucrose) and
centrifuged in a series of stages, the centrifugal field of each step being higher than for the
previous one. At the end of each stage the sedimented pellet, consisting of particles of similar
sedimentation characteristics is removed. (fig .1)
A simplified scheme for the fractionation a rat liver homogenate is shown in fig. 2

Liver homogenate

Centrifuge 1000 g, 10 minutes

Supernatant

Pellet Centrifuge 10,000 g, 10 minutes

Nuclei unbroken cells

Supernatant
Pellet
 miltochondrialysosomes Centrifuge 100,000 g, 90
minutes

Pellet supernatant
Microsomes Free ribosomes
enzymes of cytosol

Fig 2. Simplified scheme for the fractionation by differential centrifugation of a 10% rat liver
homogenate in 0.25M sucrose at 0° C. Note that microsomes are not organelles but particles
produced during homogenization. Largely from endoplasmic reticulam, and consisting a specific
sedimentation fraction.

B) DENSITY-GRADIENT CENTRIFUGATION
Centrifugation techniques where the density of the suspending medium is not uniform
throughout. There are two methods of density gradient centrifugation, the rate zonal technique
and the isopycnic (isodensity or equal density) technique, and both can be used when the
quantitative seperation ofall the components of a mixture of particle is required.
RATE ZONAL CENTRIFUGATION
Particle seperation by the rate zonal technique is based upon differences in size, shape
and density of the particles, the density and viscosity of the medium and the applied centrifugal
field. (fig.3)
Seperation of similar types of similar types of particles by the rate, zonal technique is
based mainly upon differences in their size. Subcellular organelles, therefore, such as
mitochondria, lysosomes and peroxisomes, whoch have different densities but are similar in size,
do not separate efficiently using this method.
The technique involves carefully layering a sample solution on top of a preformed liquid
density gradient, the highest density of which does not exceed that of the densest particles to be
separated.
The sample is then centrifuged, until the desired degree of seperation is effected, i.e. for
sufficient time for the particles to travel through the gradient to form discrete zones or bonds
(fig.3) which are spaced according to the relativevelocities of the particles.
ISOPYNIC CENTRIFUGATION
Isopynic centrifugation depends solely upon the density of the particle and not its shape
or size and is independent of time, the size of the particle affecting only the rate at which it
reaches it s isopycnic position in the gradient.
The technique is used to separate particles of similar size but of differing density.
The subcellular organelles such as Gol;gi apparatus, mitochondria, and peroxisomes can be
effectively separated.The sample is initially mixed with the gradient medium to give a solution of
uniform density, the gradient self-forming, by sedimentation equilibrium, during centrifugation.
(fig.4).

CHARACTERIZATION OF ENZYMES
Molecular Weight Determination of an Enzymes
The information regarding the complete three-dimensional structure of an enzyme at
atomic or near atomic resolution provides a basis for understanding the properties of the enzyme,
especially its catalystic activity. The determination of this detailed strucure is a daunting task for
even the smallest enzyme and has been found to require several years of work. Assuming that a
supply of purified enzyme is available, the primary stage of the work is the determination of
relative molecule lass Mr.
The term relative molecular mass (Mr) is now used in place of molecular weight. Mr is a
dimensionless number and is the ratio of the molecular mass of a molecule to 1/12 the mass of
one atom of 12C. The latter value is known as a Dalton. Molecular masses are often quoted in
Daltons or kilodaltons.
The determination of Mr.
Enzymes are macromolecules with Mr values ranging from about 10,000 to several
million. Therefore methods for determining Mr such as mass spectrometry or freezing point
depression which are applicable to small molecules are not suitable for enzymes. The majority of
present-day determinations of Mr values of enzymes are performed by use of one or more of the
following techniques,

1. Gel filteration
2. Ultracentrifugation
3. Sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS-PAGE).
4. GEL FILTERATION

Which separate molecules on the basis of size provides one way of doing this. A packed column
is calibrated by applying proteins of known molecular weight to the top of the column and
determining the volume of buffer required for the elution of each protein; as each protein leaves
the column there should be an absorbance peak at 280nm in the column eluate, thus providing a
simple way of monitoring the elution.
From the data obtained, a graph may be drawn of elution volume against molecular
weight. The protein of unknown molecular weight is then passed through the column and its
elution volume determined exactly as for the marker proteins. Hence, by reference to the
calibration graph, the molecular weight of the protein may be estimated.

ULTRA CENTRIFUGATION
This technique is also widely used for the determination of molecular weights of proteins.
In this, the ultracentrifuge is operated at high speeds to generate centrifugal forces that are
sufficiently intense to sediment the macromolecules. The sedimentation of an enzyme can be
monitored by suitable optical means, and from the measurements the sedimentation coefficient
‘S’ can be calculated.
The sedimentation coefficient cannot by itself be used to calculate the Mr of the enzyme,
since the rate of sedimentation will depend on other factors such as the shpe of the
macromolecule. However, if we have other information, such as the value of the diffusion
coefficient (D) of the macromolecule, its partial specific volume (v) and the density of the
solution (ρ), the Mr can be calculated from the formula,
This is known as the Svedberg equation.
Where, R – gas constant, T – absolute temperature and D – diffusion constant of the molecule.

RTS
Mr =
D(1 – ύρ)

SDS – Polyacrylamide gel electrophoresis:

 The mobility of a charged molecule in an electric field is a function of various factors
such as the size and shape of the molecule and the charge it carries and it would therefore be
expected that electrophoresis would not normally give any reliable estimates of Mr.
Hedrick and Smith have shown that the Mr of a protein can be estimated by measuring its
mobility as a function of acryl amide concentration. However, this method is only reliable if the
standard proteins for calibration have the same shape, degree of hydration and partial specific
volume as the unknown protein.
In the case of proteins we use the detergent sodium dodechyl sulphate, SDS which has
the structure CH3(CH2)11OSO3– Na+. A reducing agen6t such as 2 – merraptoethanol is also added
to break disulphide bonds. Addition of the detergent has two principal effects.
1. Nearly all proteins bind SDS in a more or less constant ratio, 1.4 g SDS per gram of
protein. Since the negative charge carried by the SDS overwhelms any charge carried by
the protein, the protein – SDS complex has a constant charge/ mass ratio.
2. The three dimensional structure of the protein is Dost and the protein – SDS complex is
rod – shaped with a length proportional to the Mr of the protein.

Since the charge and hydrodynamie properties of the protein – SDS complex are both simple
functions of the Mr, the mobility of electrophoresis is a function of Mr alone. The larger
molecules have lower mobilities means that the hydrodynamic effects (i.e., sieving) predominate
over the charge effects.
Relative Mobility →

1.0 –

0.5 –

A graph of the logarithm of the unknown protein can be determined by reference to the
standard line. Different ranges of Mr can be examined by the use of gels of different
polyacrylamide concentration or by the use of gradienet gels.
0.0 – | | |
4.0 4.5 5.0
Uses of Mr information: log (Mr) →
The Mr of an enzyme is a fundamental piece of information because it cane be used in a
variety of ways such as in consideration of composition, catalytic activity and ligand binding.
Measurements of Mr made in the absence of presence of denaturing agents will show whether or
not the enzyme is composed of subunits and may indicate the number of such subunits.

IMMONOCHEMICAL METHODS OF ENZYMATIC ASSAY

These are currently two main types of enzyme – Immunoassay (EIA), they are

• Enzyme – Linked Immunosorbent assay (ELISA)

• Enzyme Multiplied Immnoassay Technique (EMIT)
Enzyme – Linked Immunosorbent Assay (ELISA):

‘ELISA’ is an immunological technique used for the quantitative determination of the

concentration of certain antigens or antibodies.
The ‘ELSIA’ technique was first introduced in 1970 by Engvall and Perlmann.
An antibody (AB0 reacts with the concerned Antigen (Ag) in a highly specific manner
(i.e. an antibody reacts only with that determinant or region of an antigen for which it is specific)
to produce an Ag – Ab complex.

ELISA PROCEDURE:
A generalized procedure and the basic principle of ELISA is as follows:

Primary Reaction:
• The antigen (Ab) of interest is immobilized on the surface of a test tube, petriplate or
micro titter well.
• Now, Antibody (Ag) specific to the Ag (Ab) is added and allowed to react with the
absorbed antigen. Unreacted molecules of the Ab(Ag) are washed away, leaving only the
Ag- Ab complex.

Secondary Reaction:
In the secondary reaction on anti – immunoglobulin (anti – Ig: an antibody that reacts
with the antibody0 is added into the vessel and allowed to react with the Ag – Ab complex
already formed; the anti – Ig binds to the antibody component of the Ag – Ab complex.
The anti – Ig is linked to an appropriate enzyme molecule (i.e. labeled with an enxyme
molecule) in such a way that its anti – Ig activity is not impaired (eg. Alkaline phosphatase,
horseradish peroxidase and β - galactosidase).
An enzyme conjugated with an antibody reacted with a colourless substrate to generate a
coloured reaction product. This substrate are known as chromogenic substrates.
The unreacted anti – Ig is washed away and finally substrate of the enzyme is added
alongwith the necessary reagents to develop colour due to the enzyme activity.
The intensity of colour is proportional to the enzyme concentration; therefore colour
intensity is used to determine the quantity of antigen or antibody or simply to detect their
presence. The sensitivity of ELISA isin the range of nanograms (10-4g)/ml.
For an easy and rapid assay a computerized ELISA reader may be used.

VARIOUS TYPES OF ELISA (Fig. 1):

• Direct ELISA (Sandwich ELISA)
• Indirect ELISA

DIRECT ELISA:
Antigen can be detected or quantititated by a sandwich or direct ELSIA.
In this technique, the primary antibody (Ab1) is immobilized on a microtiter well.
A sample containing antigen is added and allowed to react with the bound antibody. After
any free Ag is washed away, the presence of antigen bound to the antibody is detected by added
an enzyme – conjugated antibody specific for a different epitope on the antigen is added and
allowed to react with the bound antigen.
Any free Ab2 then is washed away and a substrate for the enzyme is added. The coloured
reaction product that foams is measured by specialized spectrophotometric plate readers, which
can measure the absorbance of a 90 – Well plate in less than a minute.
INDIRECT ELISA:
Antibody can be detected or quanititated with an indirect ELISA.
Serum or some other sample containing primary antibody (Ab1) is added to an antigen
coated microtiter well and allowed to react with the antigen attached to the well. After any free
Ab1 is washed away the presence of antibody bound to the antigen is detected by added an
enzyme – conjugated secondary anti – isotype antibody (Ab2), which binds to the primary
antibody. Afy free Ab2 is washed away and a substrate for the enzyme is added. The amount of
colored reaction product that forms is measured.

ENZYME MULTIPLIED IMUNOASSAY TECHNIQUE (EMIT):

This is a homogeneous procedure, since no separation of components is required. As with
some types of ELISA, an enzyme is attached to a specimen of antigen to act as a label. However,
in contrast to ELISA, the subsequent binding of the enzyme – labeled antigen to the antibody
results in a significant change in activity of the enzyme in most types of EMIT procedure,
enzyme activity is lost completely on binding to the antibody either as a result of steric lindranace
or conformational changes. In such an assay, specified amounts of antibody and enzyme – labeled
antigen are mixed with the sample. The antigen in the sample (Ag) then competes with the
enzyme – labeled antigen (E. Ag) for the available antibody (Ab) according to the following
reactions:
Ag + Ab Ag. Ab
E.Ag + Ab E'.Ag.Ab
The enzyme – antigen – antibody complex formed (E'.Ag.ab) has no catalytic activity, so
the total activity present is contributed by E.Ag. Hence the antigen content of the sample may be
calculated from the total enzyme activity at equilibrium; the more antigen there is in the sample,
the less E.Ag is able to bind to the antibody, so the greater will be the total enzyme activity.
EMIT has so far been used principally for the determination of relatively small (io.e. non
– protein) molecules, e.g. barbiturates. The enzymes which have been involved include lysozyme
and malate dehyrogenase.

RADIO IMMUNO ASSAY (RIA):

One of the most sensitive techniques for detecting antigen or antibody is radio immuno
assay (RIA).
The technique was first developed by two endocrinologists, S.A. Berson and Rosalyn
yalow in 1960. It combines the specificity of the immune reaction with the sensitivity of radio
isotope techniques.

PRINCIPLE:
The principle of RIA involves competitive binding of radio labeled antigen and
unlabelled antigen to a high affinity antibody.
The antigen is generally labeled with a gamma – emitting isotope such as 125I. The
labeled antigen is mixed with antibody at a concentration just saturates the antigen – binding sites
of the antibody molecule and then increasing amounts of unlabelled antigen of unknown
concentration are added.
The antibody does not distinguish labeled from unlabelled antigen and of the two kinds of
antigen compete for available binding sites on the antibody.
With increasing concentration of unlabelled antigen, more labeled antigenwill be
displaced form the binding sites.

4Ag* + 4 Ab 4Ag*Ab
4Ag + 4Ag* + 4 Ab 2Ag*Ab + 2Ag Ab + 2Ag* + 2 Ag
12Ag + 4 Ag* + 4Ab Ag*Ab + 3 Ag Ab + 3 Ag*+ 9 Ag

where, Ag* - Labeled antigen

Ag - Unlabeled antigen
Ab - Antibody

To determine the amount of labeled antigen bound, the Ag – Ab complex is precipitated

to separate it from free Ag. (Ag not bound to Ab) and the radio activity in the precipitate is
measured.
It will be realsed that the labeled antigen competes with the antigen in thesample for the
available binding sites on the antibody, so the higher the concentation of antigen in the sample,
the less radioactive antigen will be able to bind to the antibody and the greater will be the radio
active content of the free antigen fraction. In this way, the concentration of antigen in the sample
can be estimated.

Total amount of Free-labeled antigen Amount of labeled

– =
labeled antigen remaining antigen bound
A typical radio immunoassay calibration curve:
A standard curve has been plotted between bound labeled antigen (%) and known concentrations
of unlabeled antigen added.
Once a standard curve had been plotted unknown concentrations of the unlabeled antigen
can be determined from the standard curve.
Antibodies for the RIA of enzymes may be prepared in the form of antisera by
immunizing rabbits with the required enzyme; for example, the blood of a rabbit immunized in
the foot pad with human pancreatic α - amylase contains sufficient antibodies within a few
weeks to be useable as an antiserum.
The part of the enzyme which binds to the antibody is likely to be quite distinct from the
active site, so RIA and catalytic assay of enzymes can give different information. Catalytically
inactive forms of enzymes (eg. Prienzymes) may be detected by RIA if they contain the structural
part which is recognized by the antibody. On the other hand, QRIA procedure is likely to be
specific for one particular isoenzyme (that used to produce the antibody). Hence, it can be seen
that catalytic assay and RIA procedures are not alternat6ives but can be used to complement each
other RIAs also have the advantage of being particularly sensitive (upto a thousand times more
sensitive than catalytic assays).

ENZYME ASSAYS
An assay is a measurement of a given enzyme of known characteristics in a sample. Ideally, this
means measuring a specific and characteristics biological property (or ability to catalyze a
chemical reaction) of the desired enzymes. Less ideally, we use a method, which does not in
principle, derive from the biological activity of the protein, but is a general method in which it
has a specific behaviour. Thus enzyme assay may be
(1). The former, catalytic assay and
(2). The later, stoichiometric assay
A catalytic assay is obviously more sensitive than a stoichiometric assay, which measures
amount of the enzyme by measuring a molar equivalent amount of something, such as bound
Coomassie Blue or silver stain. In a catalytic assay the amount of measured product, at least in
principle, increases indefinitely with time of incubation, yielding a greater sensitivity while a
stoichiometric assay only reaches equilibrium.
 There are two general purposes for enzyme assay:
2. To measure how much of the enzyme is present in the sample. The enzyme is the variable
measured.
3. With a constant amount of the enzyme present, how does its activity vary with conditions
such as pH, temperature, variation of substrate concentration, effect of inhibitors, etc or
in prior incubation (stability to heat, chemical modification, etc)

Assay may be either

1. Continuous: A continuous assay is one in which some change caused by the enzyme is
monitored continuously while it is happening such as absorbance of light, uptake of base
or acid in a pH – stat, or change in oxygen electrode. The quantity monitored usually
expressed as a line graph and usually one is interested in the slope of the line, the rate of
the reaction, which should be proportional to the amount of your protein present.
2. Discontinuous: In a discontinuous assay or stop – sample assay, on the other hand, the
reaction is stopped at some definite time, and do something to the reaction mixture, such
as adding acid or base or heating to develop a color, or separating radioactive products
from starting material, which makes possible the determination of amount of product
formed by that time (or amount of starting material remaining). Reactions are stopped by
adding a solution which makes further reaction impossible, such as strong acid or base or
EDTA for Mg++ dependent reaction, boiling.
3. Semi continuous: There is also semi – continuous assays, where you can make a number
of measurements on the same reaction mixture while the reaction is running, but each
measurement takes a finite amount of time – manommetric and viscometric assays, for
instance. In principle any stop – sample assay can be made semi – continuous, by taking
multiple samples from a single assay mixture, instead of setting sponutiple assays and
stopping them at different times.

Note: Enzyme activity is measured as the amount of substate lost (or product gained) per unit
time, and it should also be specimen used for assay. In 1961, the enzyme commission of the IUB
defined an ‘Enzyme Unit’ (U), later to be known as an International Unit (IT), as the amount of
enzyme causing loss of 1μmol substrate per minute under specified conditions. Later, in 1973, the
commission of Biochemical Nomenclature introduced the Katal (Kat) as the system International
(SI) units of enzyme activity; this is defined as the amount of enzyme causing loss of 1 mol
substrate per second under specified conditions. Both units are in current usage.
For a successful assay system, the reaction being catalyzed should be capable of being
accurately monitored. That is, during reaction there should be change in optical, electrical or
other properties that is directly proportional to the product formed or substrate utilized. The
product formed/substrate utilized can be directly monitored (either by stop and sample method or
by continuous method) or any of the following method. If product or substrate is not having any
detectable trait, it may be possible to study the course of reaction indirectly by coupling it to
another detectable reaction.
Mg2+
D-glucose + ATP ------------------→ D-glucose-6-phosphate + ADP
Hexxokinase
For example, the product in this reaction (D-Glu-6-P) is not easily assayable because
there is no spectrophometric absorbtion for this compound. But if this rection is coupled to the
production of D-glucono δ -lactone 6-phosphate by the reduction of NADP + to NADPH using
glucose-6phosphate dehydrogenase enzyme. The amount of NADPH can be monitored easilyas it
has an absorption spectrum at 340nm, whereas NADP+ is not having. Thus D-glucose 6-
Phosphate can be indirectly measured in a couple assay.
D-glucose + ATP D-glucose-6-phosphate + ADP

NADP+
NADPH+H+
D-glucono δ -lactone 6-phosphate
Another example of coupled assay is
D-alanine + O2 pyruvate + NH4+ + H2O2 (D-alumino acid oxidase)
H2O2 + chromogen colored product + H2O (peroxidase)

Cycled assays are also there which use a small amount of a compound as rate-limiting
intermediate in reactions going both ways. Strictly these are assays for the compound rather than
for an enzyme, but the amount of compound started with might be the product of an enzyme
reaction carried out on a very small scale, say one cell. An example,
Pyruvate + NADH +H+ L-lactate + NAD+ (lactate dehydrogenase)
Ppyruvate + H2O2 L-lactate +O2 (lactate oxidase)

IIn this case the amount of pyruvate is the rate limiting compound and increase in concentration
of pyruvate is directly pproportional to the rate of reaction and it can be monitored as the
absorption of NADH.
Some standard methods for enzyme assay methods which are fairly quick and easy are:
1. Colorimetry: Any substrate or product which absorb radiations in visible range can be
monitored by this method. The colorimetric assays can be extended by the use of artificial
substrate and by the production of colored derivates of the substrate or product. In some cases,
substrate or product containing certain functional group which which can also be converted to
colored derivative.
2. Spectrophotometry: This method depends on absorption of light at a specified wave length
(When the wave length range is only narrowed down by filters) by substrate or product.
Spectrophotometry typically measures compounds in the 10-3 – 10-6 M.
Two products are very commonly monitored spectrophotometrically –the coenzymes
NADH and NADPH, which have a molar extinction coefficient of 6200 L/mole.cm at 340nm.,
and p-nitrophenol, which has an extinction coefficient of 18,300 L/mole.cm. Mny hydrolytic
enzymes are assayed using p-nitrophenyl esters and glycosides, which are artificial substrates.
Many reactions are coupled to production of NADH or NADPH, or their disappearance, because
they are so convenient to observe.
If a product absorbs uniquely and the spectrophotometer is attached to a recorder this can be a

continuous assay; also, modern spectrophotometers allow us to determine the rate directly

through the instruments software.

3. Fluorimerty: Some compounds when excited by higher-energy light, UV or visible, re-emit

light lower-energy of a longer wave length. This assay is very sensitive, down to 10-9 M.
Disadvantage is fewer compounds fluoresce, and the instrumenty is more expancive. It can be
more specific, since the wave length of both the exciting and emiotted light can be selected.
NADH and NADPH can fluoresce, and can be measured moer sensitively. Methylumbelliferol
and methylumbelliferylamine are the fluorescent equivalents of p-nitrophenol; but parent
glycosides of these compounds, esters and amides donot give fluorescence, so assays for
glycosidases, esterases and amidases can use them as artificial substrates.
4. Luminometric assay: In this assay, light is produced by the chemical reaction without prior
irradiation. The best known in assay of ATP by the firely tail luciferin-luciferase reaction, for
which ATP is a cofactor, but now of some importance is measurement of Ca ++ by the jellyfish
protein, which undergoes a light-producing oxidative reaction when it binds Ca++.
Luciferin +ATP +O2 Oxyluciferin + AMP + PPi + CO2 + Light
5. Titration: This assay is with an instrument called a pH-stat, which is essentially a pH meter
controlling an automatic buret, so that if the pH drops below a set value base is added to restore
it, and the amount of base is added is recorded. The same can be done for acid added to counter a
rising pH. Thus reactions producing acid, such as the hydrolysis of acetyltyrosine ethyl ester, a
chymotrypsin substrate, can be followed continuously, by plotting a chart recording of base
addition vs time, recording the total amount added over the period of linearity of the rate. On the
other hand, reactions producing acid or base can be measured spectrophotometrically if an
indicator is present.
6. Manometric assays: This is applicable if either product or substrate is a gas. The assay
measures uptake or release of a gas in a closed system. Enzymes which can be measured
manometrically include glutamate carboxylase, catalase, monoamine oxidase. These enzymatic
reaction results in the production of CO2 or O2. This can be monitored using Warburg manometer
or respirometers.
2H2O2 2H2O + O2 (gas)
7. Oxygen electrode and ion-selective electrode: The development of ion-selective electrode
such as those for ammonium ion, and the oxygen electrodes measuring O2 concentration in
solution are attractive methods for enzyme assays. This method is very sensitive, reproducible
can us very volume of sample.
8. Viscometric assays measuring change usually decrease in the viscosity of a solution as a
polymer is broken down. This can be useful for glycosides assay which releases reducing sugar
from a polysaccharide and to know whether it is an endoglycosidase (breaking the polymer in the
middle) or an exoglyucosidase, (chewing on the ends). The endoglycosidase will reduce the
viscosity of a solution of the polymer, the exoglycosidse won’t. The same could apply to
aminopeptidase vs a true protease (endo cleaving).
9. Radioisotopic assay: A radioactive substrate is acted on by an enzyme, then the product is to
separated from the substrate very carefully and measured by tis radioactivity. This must therefore
be a ‘stop and sample’ assay. Radioactive assays are best if the separation is quick and complete.
Although very sensitive, the use of radio isotope in an assay is restricited to applications where it
is possible to separate easily the radio labeled form of substrate or product. Exceptionally this
assay is having application if one of the product is a gas, which can be radio labeled.
HOOC CH2 CH2 CH (NH2)14 COOH → 14CO2 + HOOC CH2.CH2.NH2.14COOH
Glutamic acid Amino butyric acid
10. Immunochemical methods: Antibodies raised against a particular enzyme can be used for
highly specific assays. Enzyme Linked Immuno Sorbent Assary (ELISA) is an example. This
assay can distringuish between isoenzymes, which is of imortace in clinical diagnosis. A
monoclonal assay is available for serum prostatic acid phosphatase, which diagnose carcinoma of
prostrate.
11. Micro calorimetric methods: Most enzymatic reactions are accompanied by a minute
change in heat (enthalpy) that gives rise to a temperature change of the order of 10–2 to 10–4 °C.
Measurment of such small changes is possible using thermistors which are temperature sensitive
metal oxides. The technique requires stringent insulation of the reaction vessel. This assay may
be coupled to a secondary reaction where heat formed can be amplified to an easily detectable
level.
12. Gas chromatography and liquid chromatography: reactant and product differ in the time
they take coming through a column, whether as a volatile molecule in a flow or carrier gas or as a
solute in a flowing fluid. Gas chromatographs measure material passing thr detector by flame
ionization or by capture of an electron from a radioactive source is they contain nitrogen or
phosphorus; liquid chromatographs can detect spectrophometrically flurometrically by other
methods. The recorder can integrate the area under each peak to quantitate it. The power and
versatility is great such methods are particularly useful when substrate and product are very
similar chemically, as for instance cellobiose and glucose. Since being a continuous assay, the
drawback is the time each measurement takes.
13. Capillary electrophoresis and gel electrophoresis as for assay of a restriction endonuclease.
But the separation takes the more you’d like to find another way.
14. Biological assay which measure a more specific biological action of the protein rather than a
chemical action. Biological assays are particularly important when the chemical event carried out
is unremarkable, the importance being in its specificity – such as the many proteases which cause
specific biological effects by cleavage of one or a few bonds in specific protein substrates.
Another example, assay of growth hormone by thickness of the knees of hypophysectomized rats.
Another is the blood clotting system, the result of a cascading series of proteolytic events, the
product of each reaction being the enzyme for the next, culminating in the cleavage of fibrinogen
to fibrin and the formation of the lot. The clotting assay is timing how long it takes a clot to form;
it may be sued as an assay for any of the factors involved if a serum deficient in that factor so that
the sample added supplies what is needed to bring about clotting.

ISOELECTRIC FOCUSING (IEF) GELS:

This method is ideal for the separation of amphoteric substances such as proteins because
it is based on the separation of molecules according to their different isoelectric points. The
method has high resolution, being able to separate proteins that differ in their isoelectric pints by
as little as 0.01 of a pH unit. The most widely used system for IEF utilizes horizontal gels on
glass plates or plastic sheets. Separation is achieved by applying a potential difference across a
gel that contains a pH gradient. The pH gradient is formed by the introduction into the gel of
compounds known as ampholytes, which are complex mixtures of synthetic polyamino – poly
carboxylic acids.

– CH2 – N – (CH2)n – N – CH2 – where R = H or – (CH2)n – COOH , n = 2 or 3.

| |
(CH2)n (CH2)n
| |
NR2 COOH
‘The general formula for ampholytes’
Commercial available ampholytes include Bio – lyte and Pharmalyte. To prepare a thin layer
IEF gel, carrier ampholytes covering a suitable pH range amd riboflavin are mixed with the
acrylamide solution and the mixture is then poured over a glass ploate (typically 25 xm x 10
cm), which contain the space. The second glass plate is then placed on the top of the first to
form the gel cassette and the gel polymerized by photopolymerisation by placing the gel in
front of a bright light. The photo decompostion of the riboflavin generates a free radiacal
which initiates polymerization. This takes 2 – 3 hour.
Once the gel has set, the glass plates are prized apart to to reveal the gel stuck to one of the glass
sheets. Electrode wicks, which are thick (3 mm) strips of welted filter paper (the anode is
phosphoric acid, the cathode sodium hydroxide) are laid along the long length of each side of the
gel and a potential difference applied. Under the effect of this potential difference, the
ampholytes form a pH gradient between the anode and cathode.
The power is then turned off and samples applied by laying on the gel small squares of filter
paper soaked in the sample. A voltage is again applied for about 30 min to allow the sample to
electrophores off the paper and into the gel at which time the paper square can be removed from
the gel. Depending on which point on the pH gradient the sample has been loaded, proteins that
are initially at a pH region below their isoelectric point will be positively charged and will
initially migrate towards the cathode. As they proceed, however the surrounding pH will be
steadily increasing and therefore the positive charge on the protein will decrease correspondingly
until eventually the protein arrives at a point where the pH is equal to its isoelectric point. The
protein will now be in the Zwitterion form with no net charge, so further movement will cease.
Likewise, substances that are initially at pH regions above their isoelectric points will be
negatively charged and will migrate towards the anode until they reach their isoelectric pints and
become stationary.
Following electrophoresis, the gel must be stained to detect the proteins. The gel’s stained with
coomassie Brilliant Blue and then destained. The method is particularly useful for separationg
isoenzymes, which are different forms of the same enzyme often differing by only one or two
amino acid residues. Since the proteins are in their native form, enzymes can be detected in the
gel either by washing the unfixed and unstained gel in an appropriate substrate or by over
layering with agarose containing the substrate.

(A). Low pH (+) High pH (–)

(B). Low pH (+) Low pH (–)

A pH gradient is established in a gel before loading the sample.

(A). The sample is loaded and voltage is applied. The proteins will migrate to their isoelectric
– pH, the location at which they have no net charge.
(B). The proteins form bands that can be excised and used for further experimentation.

Further Purification Procedure:

Although a considerable degree of purification is achieved by fractional precipitation,
much other soluble protein will still be present because of overlap of solubility ranges. Precipitate
is collected by centrifugation and dissolved in a suitable volume of extractant for further
purification. Before further purification by instrumental methods generally chromatography,
extractant is adsorbed onto a suitable adsorbent. All adsorption and chromatographic methods
depend on distribution of the material being purified (here enzyme) between a stationary (usually
solid, sometimes adsorbed liquid) phase and a moving (usually liquid) phase. Distribution
coefficient [adsorbed material] /[total material].
Batch adsorption:
This is a simple technique intermediate between precipitation and chromatographic
methods. The most commonly used adsorbent is calcium phosphate. Originally in an gel form but
now usually in crystalline forms, brushite and hydroxylapatite, which can be used in columns.
Adsorbent is added to the crude extractant of enzyme and allowed enzymes to adsorb on to its
surface. After adsorption, the solid phase is collected by filtration or centrifugation like
precipitation process. Effective adsorbents, except affinity adsorbents, are likely to adsorb many
other proteins and thus require that a substantial amount be used. And since separation is only
between two phases, adsorbed and suppressant resolution is generally much less than in
chromatography. Zinc hydroxide has been used to remove pigments from enzyme preparations.
Ion exchange chromatography:
 Proteins differ from one another in the proportions of the charged amino acid (asparic and
glutamic acids, lysine arginine and histidine) than can contain. Hence proteins will differ in net
charge at a particular pH. This difference is exploited in ion exchange chromatography to
separate enzymes . In this method, enzyme of interest is bound on a solid support material
(ionexchange resin) bearing charged group of the opposite sign. Most of the enzyme purification
is done on anion exchange columns because most enzymes are negatively charged at
physiological pH. Elution of the bound enzyme is by exchanging the charged enzyme by a
corresponding cation/anion. Based on the charge exchanged, it may be a cation exchange
chromatography or anion exchange chromatography. Ion exchanger consists of a water insoluble
matrix namely dextra or cellulose to which charged groups have been charged group have been
covalently bound. Cation exchangers have acidic group with a net negative charge on the matrix
and positively charged exchangeable counter ions. Example is diethyl amino ethyl cellulose
(DEAE.)
(C2H5)2 NH CH2 CH2 – O Cellulose

Quarternary ammonium groups, such as triethylaminoethyl (TEAE) and QARE are also
used as anion exchangers. The most commonly used cation exchange group is carboxymethl
(CM). Phosphocellulose is another example of cation exchanger.
Ion exchange adsorbents are usually eluted by means of a gradient or steps of increasing KCl or
NaCl concentrations, in presence of a constant concentration of buffer. The charged form of the
buffer should be of the same charge as the ion exchange material, i.e. use Tris or another amine
with DEAE, phosphate or another acid with CM.

Chromatofocusing:
A variation of ion exchange technique, it chromatofocusing in which a linear pH gradient
is generated in the column with 2 or 4 pH units lower at the top as the column is eluted using the
acid form of an ampholyte at low ionic strength. When a protein is added to this pH gradient with
a buffer whose pH is similar to that prevailing at the top of the column, it will migrate down the
column as cation, encountering an increasing pH, until it reaches a pH corresponding to this
isoelectric ponit. Just beyond this pint it will become an anion and will be able to bind to the
positive groups of the exchanger (in this example column is anionexchanger). This process is
repeated in the column by changing the pH of the buffer and at the end protein is eluted at a pH
slightly above is isoelectric point. Proteins are claimed to to emerge in sharp, highly resolved
peaks. This technique has high capacity. Chromatofocusing gives a good resolution of quire
complex mixture of proteins, provided that there are discrete differences in their isoelectric point.
Proteins possessing very similar isoelectric points tend to be poorly resolved.

Hydrophobic chromatography:
Enzymes canstick to hydrophobic material byhydrophobic interation with nonpolar
regions of their surface (by val, phe, etc). The hydrophobic groups used in the columninclude
alkyl (octyl – Sepharose), phenyl (phenyl – Sepharose) andalkyl amino achains. The capacity is
high. Adsorption is strongest at high salt concentration, so a sample may be applied immediately
on redissolution after (NH4)2SO4 precipitation. Proteins are eluted by decreasing the salt
concentration. Resolution is not as good as in ion exchange chromatography.
Affinity Chromatography:
Affinity chromatography is a bio – specific process which exploits the formation of
specific and reversible complexes between a pair of biomolecules. One of the pair is called ligand
and is usually immobiliex on to a stationary phase while the other called counter ligand, is
adsorbed from the extract that is passing through the chromatographic column containing the
immobilized ligand. This technique enables separate closely related proteins from a mixture.
Method depend on a specific interaction the enzyme of interest and specific ligands, which may
be substrate analogue binding to its respective enzyme (affinity chromatorgraphy), a synthetic
dyes which can bind to specific protein (dye – lignad chromatography) a lectin, chinch can bind
to glycoproteins (lectin – affinity chromatography) or an antibodies binding to specific enzymes
antigen (immunoardsorbent chromatography).
Ligands and counter- lignds in affinity chromatography:
Lignad Counter Ligand Chromatography
Substrate, subnstrate,
1. Enzyme Affinity chromatography
analogue, cofactor, inhibitor
2. Glyco protein enzyme Lectin Lectin – affinity chromatography
3. Enzyme (an antigen) Antibody Immunoabsorbent chromatography
4. Enzyme Dye Dye- ligand chromatography
5. Metalloenzyme Metal ions Metal – chelate chromatography
The matrix or support is usually agarose or a cross – linked derivative, because it is very
poprous and admits large proteins to the pores, but has good strength and stability and is
reasonably derivatizable. In general any matrix useful for ion exchange or gel filtration
chromatography is also good for affinity chromatography. The attachment of ligand usually
proceeds by treating the matrix with a reactive compound like cyanogens bromide and
glutaraldehyde, which either leaves reactive groups to which ligands can be attached. The ligand
is usually attached with a spacer arm between it and the matrix to assure that theligand will be
fully accessible to the desiredprotein. An example of non-specific space is 1.6 – diaminohezane.
The protein mixture is applied to the column and the relevant enzyme is trapped by the
immobilized ligands while all other proteins pass through and are discarded. The enzyme is the
liberated from the column either by eluting with a deforming buffer at a pH which changes the
characteristics of the enzyme and nolonger allows it to bind to immobilized lignad. Another
method is using a competitive counter ligand, which displaces the immobilized ligand on the
enzyme.
Advantages of affinity chromatography included:
1. High selectivity compared to other purification techniques.
2. Extremely good purification upto several thousand folds in a single step and recoveries
greater than 90% can be expected provided conditions are carefully selected.
3. Affinity chromatography had a high concentration effect, especially when the enzyme of
interest is a minor component of a complex mixture.
4. Affinity methods can also be used to remove unwanted materials from a mixture.\

Immunoadsorbent chromatography:
Here immobilized ligand is antibodies to the desired enzyme. In principle, this technique
is the last word in specificity and tight binding, but of course there are drawbacks. First of all, in
order to prepare the antibodies it is required to purify the protein first and the antibody
preparation procedure is cumbersome. Another problem is elution of the bound enzyme antibody
will be difficult.
Dye ligand chromatography
Some reactive triazine-dyes (about 40 dyes) Cibacron F3GA, Procion Red H-E3B, etc.,
WW have very affinity for protein and can there for use in enzyme purification. These dyes will
easily attach to agarose or other matrices and thus can be used in an easy way. Elution is as with
‘true affinity columns, either with specific ligands which compete with the dye for the protein
binding site, or with high salt concetration or high pH.
Metal-binding chromatography
This can be a general approach for proteins with exposed histidines, cysteines or carboxyl groups
near each other, but in practice it is mainly for cloned proteins. To cloned protein, a short
sequence is added which facilities purification by binding to specific metals. The commonly used
method is to add a sequence of six or so histidinesd at the C-terminus. These bind well to divalent
cations of transition metals such as nickel. A column is prepared by attaching nitrilotriacetic acid
to a solid support. This binds nickel ions tightly; the resin is washed with 5mM imidazole to
remove unbond nickel. The fusion protein, perhaps denatured in 6M urea to ensure that the hexa-
His sequence is exposed, is applied to the column. The column is washed with dilute imidazole,
then more concentrated imidazole to elute the desired protein.Some variations are: mercuric ions
bound tightly to immobilized sulfhydryl groups, which can bind proteins by their exposed
sulfhydryl groups – elution is with excess free SH compound such as mercaptoethanol; and Fe+++
bound to iminodiacetic acid, which binds phosphoproteins by the phosphate groups.
Gel filtration (size-exclusion or molecular sieve chromatography)
The gel filtration material is porous, with pores the size of protein molecules. Large
molecules, too large to enter any of the pores, pass down the column through the space between
the gel particles. Very small molecules enter all the pores, and therefore spend much of their time
not moving and elute out only solely. Intermediate size molecules enter some of the pores, and
are eluted somewhere in between. Eluting buffer should be of high ionic strength to counteract
the few changes which may be present on the gel. Gel filtration is also used to separate proteins
from salts such as ammonium sulfate, using a small-pored gel such as Sephadex G-25 or BioGel
P10 which excludes all proteins; it is much faster than dialysis. Gel filtration is widely used to
separate protein detergent miscelles from excess of detergent, during purification of membrane
bound enzymes.
The gel filtration materials are the Sephadexes (cross-linked dextrans), ‘sephacry’ (cross-
linked acrylamide) and biogel (agarose).
High performance chromatographic techniques
HPLC stands for “high performance liquid chromatography” (through HP could also be
said to stand for “high pressure”). The stainless steel columns and robust packing material of
104m or less which can with stand high pressure are used and it enables the resolution in minutes.
These columns yield good separation and high resolution in short period. One advantage of fast
operation is that they can be run at room temperature without denaturing the protein, because the
protein comes off in 5 to 60 min. However, only small volumes can be purified. For protein
chromatography it was necessary to develop materials both strong enough like silica, to stand the
pressure and porous enough to have a high surface area for adsorption, or for gel filtration. HPLC
is a high resolution, but low capacity method. High performance Size Exclusion
Chromatography (HPSEC) utilizes rigid beads of porous silica with bonded hydrophilic polar
groups. High Performance Ion Exchange Chromatography (HIPEC) utilizes amines as anion
exchanges and sulphonic or carboxylic acids as cation exchanges, each bonded to a rigid support
as silica. High Performance Liquid Affinity Chromatography utilizes ligands bounds to supports
such as epoxy-silica. Proteins can also be separated by reverse phase HPLC (RP-HPLC) on
alkylsilica columns, the eluting solvents being buffered aqueous and organic mixtures.
Electrophorectic Tecniques
Electrophoresis is mainly an analytical procedure as it is suited for small amount of metal
it has also been used for purification of enzymes. The rate and direction of migration of a protein
in an electric field depends on its net charge and size of the molecule. In zone electrophoresis, the
separation occurs in a solid matrix commonly agarose gel or polycrylanide gel. This may be
vertical ‘disc gel electrophoresis’ and horizontal ‘thin slab gel electrophoresis’. Electrophoresis in
the absence of any support material is free solution electrophoresis or moving boundary
electrophoresis. The different analytical gel electrophoresis are
1. Simple or native gel electrophoresis
2. SDS-PAGE
3. Urea gel electrophoresis.
1. Native gel electrophoresis: The enzyme mixture to be separated is mixed with a buffer at a pH
where the proteins remain stable and in their native conformation. The pH range is usually 8-9
where most of the proteins carry negative charges and move towards the anode placed at the other
end. Any basic contaminant protein will remain in the cathodic buffer. In negative PAGE, the gel
is prepared by polymerzing acrylamide and a cross linker N,N-Methyl bisacrylamide (30:1)
together with ammonium persulphate as initiator of polymerization and TEMED (N,N,N,N-
Tetraethylendiamine) as catalyst. The pore size of the gel can be tailored to suit the molecular
weight of the sample proteins by altering the concentration of either the monomer acrylamide or
cross-linker. Increase in concentration of either of the two decreases the pore size and vice versa.

2. SDS-PAGE: This method involves the electrophorectic seperation of denatured protein on

polyacrylamide gel. The proteins in the solution are completely denatured by treatment with the
detergent sodium dodecyl sulphate (SDS) and β -mercaptoethnol and boiling the mixture for a
few minutes. Addition of mercaptoethnol disrupts disulphide linkage in protein. Each SDS
contains a negative charge and approximately one SDS bind to two amino acid residues giving
the polypeptide a large negative charge. Consequently the charge/size ratio is almost the same for
all polypeptide chains and separation of the polypeptides can occur only due to the molecular
size. The electrophoresis is carried out in vertical slab polyacrylamide gel using buffers such as
tris HCl-glycine and tris-tricine buffer for small molecular weight proteins.

The method has a high resolution and gives sharp zones. The molecular weight of sample
polypeptide chains can be determined by comparing their mobility with standard poly peptides
whose molecular weight is known.

3. Urea gel electrophoresis: This method is used particularly for protein that is insoluble at low
ionic strength. The proteins are solubilised by denaturing them completely with urea in the
presence of meracaptoethanol to disrupt any disulphide linkages. Urea containing starch gels are
easier to handle compared to polyacrylamide.

IMMUNOELECTROPHORESIS
Immunoelectrophoresis is an identification and quantofication by separating components
I mixtures by electrophoresis in an ager gels followed by immunoprecipition reaction one the
same gel. Imunoprecipitin reaction is a specific reaction between an antigen and its corresponding
antibody and it is observable as white precipitate in the pH range of 7 – 9.

CAPILLARY ELECTROPHORESIS:
Capillary electrophoresis is an analytical technique requiring only micro – to nagogram
samples. The components of the sample are allowed to seprate in a high voltage inside a capillary
tube filled with a suitable buffer. Column is usually made of stainless steel with a diameter of 100
micrometer and about 30 cm long.

ISOELECTRIC FOCUSING:
Isoelectric focusing is an example of moving boundary electrophoresis, in which a pH
gradient is set up between electrodes by allowing an acid (eg. Phosphoric acid) to diffuse from
anode and a base (eg. Ethanolamine) from cathode. The stabilization of this pH gradient is
achieved by using buffers called ampholytes. Ampholytes are synthetic aliphatic polyamino –
polycarboxylic acid and they have large number of positive and negatively charged functional
groups with closely spaced isoelectric points. During isoelectric focusing the ampholytes
migrates to their respective isoelectric pH and stabilizes the pH zones, usually a pH gradient is set
up with a difference of 0.02 pH units. The protein mixture when introduced and electrophotesis
gel, the components will be moving thl it its reach isoelectric pH and get precipitated there. The
zones containing each protein are very sharp as a result of this focusing and proteins whose
issoelecctric points differ by as little as 0.02 pH units can be distinguishable by this method.
Isotachophoresis:
Isotachophoresis is also moving boundary electrophoresis where migration of different
ionic species of the same signs all having the same counter ion in an applied electric field. In
principle, the mixture containing two ionic species A – and B – are separated on the basis of their
mobility in an electric field. For enzyme isolation, at mildly alkaline pH, when most would be
anions, a leading anion (eg. Phosphbbate) is added which has a faster mobility towards the anode
than any of the sample anion. A trailing or terminating anion with a slower mobility than any in
the sample is also added. The system is buffered by a counter cation, Tris. The leading and
trailing ions are applied at different sides of sample, leading near the anode. When high voltage is
applied all components will migrate toward the anode in discrete zones at the same
velocity(‘isotacho’ GK. ‘Same speed’). The sample will arranged in the order of mobilities. The
ions with higher effective mobility will move fast and those with lower effective mobility will
follow in decreasing order of their effective mobilities. The polarity of electric field is such that
with a homogeneous current density all the ions move with same speed at equilibrium and get
separated into a number of consecutive zones in immediate contact with each other and arranged
in order of their effective mobilities. Isotachophoresis is used in preparatory level.
Final Concentrating steps:
When further purification, it is considered that the relevant enzyme has been seprated
completely from other protein, mineral salt and other small molecules are removed by dialysis.

Dialysis: Dialysis is a membrane separation used for removal of low MW solutes such as
organic acids (< 500 MW) and inorganic ions (< 100 MW) from a solution. In enzyme
purification, dialysis is commonly used after salting out, to separation salt ions before further
purification steps. Cellophane or cellulose acetate membranes of different porosity are used for
dialysis. Complete removal of ions can be achieved but concentration of enzyme mixture is not
possible.
The purified enzyme preparation is likely to be quite dilute, so it might require
concentrating. Concentration of the enzyme preparation is achieved by lyephilisation or
ultrafiltration.

Lyophilisation is freeze-drying technique. The sample is frozen first and to it vacuum is applied
to sublimate all the ice crystals to vapour state. The powder obtained can be resuspended in a
required volume of buffer.

Ultrafiltration: This is the filtration of a protein solution through a membrane with pores small
enough to retain the protein of interest. It is a two – phase method – what is retained and what
passes through the filter. To hasten the process, it required either gas pressure on the solution
above the filter (for large volumes) or increase of gravitational force by centrifugation (for small
volumes). It is usually used just to concentrate a dilute protein solution, such as a crude culture
brotn and sometimes after dialysis for concentrating again until practically all small molecules
have been flushed through the membrane. It is sometimes used as a purification method with
retain the protein of interest usually large molecules, but pass through smaller ones. The bigget
problem is clogging of the pores by protein accumulating on the membrane surface. The enzymes
are purified and concentrated by this process.
The ideal way to complete purification is to crystallize the enzyme. Crystallization is achieved by
adding enough quantity of ammonium sulphate to cause precipitation of the enzyme and leaving
this in cold room for several days.
 Membrane bound enzymes may be inactive after purification unless reintroduced into
phospholpids environment.

Some general comments:

A good industrial protein purification processes should follow the five rules:

“Rule 1: Choose separation processes based on different physical, chemical or biochemical

properties”. Repeating the same process doesn’t gain much, though sometimes it makes later step
more efficient.
“Rule 2: Separate the most plentiful impurities first”. This means especially non – protein
impurities such as cell debris and small molecules.
“Rule 3: Choose those processes that will exploit the differences in the physico chemical
properties of the product and impurities in the most efficient manner”. This is done when you
know the properties of the purified protein and are designing a large-scale process, which you
want to be as efficient as possible. For purification of a recombinant protein it is also useful to
know properties of the commonest proteins of the host cell and how to remove them efficiently.
“Rule 4: Use a high – resolution step as soon as possible”. This is less obvious but eliminates as
many impurities as possible at an early stage. They mean affinity chromatography where
possible, otherwise probably ion exchange chromatography.
“Rule 5: Do the most arduous step last”. This means removal of the last few percent impurities.
High-resolution gel filtration is often the best step here.
MONITORINIG OF PURIFICATION STEPS
To monitor the efficacy of purification or to follow the progress of process, an assay of
the desired enzyme and it gives the measure of biological activity (total activity). It must be also
convenient and easy to perform because at times many fractions obtained after purification steps
are to be assayed for the desired enzyme and the fractions positive for the enzyme are pooled
together before going to next step. A detailed description of assay methods to find out biological
activity is given in the notes “enzyme assay”. The biological activity is measured as IU or units
(SI unit is katal). See the notes attached “unit of enzyme activity” for definitions. The important
criterion of purity is specific activity. Specific activity is biological activity divided by total
protein in the sample, This will show, irrespective of preconceived ideas, precisely which fraction
contain the important enzyme and will enable the degree of purification to be calculated.
Total protein in the sample can be estimated by any of the following method.
1. Kjeldahl analysis: The nitrogen content of most protein is 16% by weight. Total nitrogrn in
the biological sample is determined by kjeldahl titration method and the total protein is calculated
from that.
2. Ultraviolet absorption method: The method is relatively sensitive and applicable only to
partially or highly purified enzyme preparations. The aromatic amino acids, tyrosine and
tryptophane give an absorption maximum at a wavelength of 230. The major advantage of this
protein assay is that it is non-destructive and protein concentration of effluents from
chromatographic column can be measured continuously.
3. Biuret method: This is suitable method for measuring protein concentration of crude
homogenate and in subcellular fractions. Biuret reagent is a mixture of alkaline copper sulphate
and sodium potassium tartarate. The cupric ions form a coordination complex with four -NH
groups present in peptide bonds giving an absorption maximum at 540-560 nm wavelength.
4. Lowry (Folin-Ciocalteau) method: This is the most widely used protein assay method, which
measures protein concntration as low as 10mg cm-3 the principle is comparable to biuret method.
Protein complexes with copper in alkaline solution. This complex then reduces a
phosphomolybdate-phosphotugstic reagent to yield an intense blue colour. The intensity of the
blue-purple colour is measured at 660nm.
5. Turbidimetric method: Certain organic acids such as trichloroacetic acid and sulphosalistic
acid precipitate protein. The amount of precipitate formed can be measured by its light scattering
intensity. Scattering is measured in colorimeter or spectrophotometer.
Specific activity is used to calculate yield and purification factor. Purification factor gives
degree of purification and it is also called fold purification. Purification factor will be high for
efficient separation steps.
Specific activity after a particular step
Purification factor =
Specific activity of previous step

Yield can be directly calculated in each step as

Biological activity after a particular step

Yield =
Biological activity of initial step
If the fractions containing desired enzyme are combined, the total activity of the enzyme
in that fraction should be the same as that started with, whereas total amount of protein in this
pooled sample will be less than that originally present. The specific activity of the enzyme in the
combined fraction should therefore be greater than that in the preparation before purification and
increase in specificity will be a measure of the purification achieved. With each successive
purification step, the specific activity of the sample containing the enzyme should be greater than
before until complete purification is achieved and specific activity reaches a limiting value.
However, the finding of the same specific activity value before and after a purification
step does not necessarily mean that the enzyme preparation is completely pure, it could be simply
mean that the contaminating proteins have passed through the procedure in the same fraction as
the enzyme. Similarly crystallization cannot be taken as proof that only one protein is present, for
many mixed protein crystals have been found. Hence other criteria of purity have to be
considered.
Protocal for purification of Adenylate kinase
Minced Muscle

Step 1 Extract with 0.01 Mol dm-3 KCl

strain trough cheesecloth.
Extract
Step 2 Incubate at pH 3.5 then at pH 7.0;
Centrifuge

Supernatant

Step 3 Load on to phospho cellulose column. Elute

with pulse of AMP (5 mmol dm-3 )
Pooled fraction
containing activity
Concentrate by (NH4)2 SO4 Gel filtration –
Step 4
Pooled fractionScphadex G. 15
containing activity

Crystalline enzyme
 Crystallization at 62% saturation of
Step 5
(NH4)2SO4

Purification table for adenylate cyclase from 6Kg of pig muscle

Total Total Total Specific
Yield Purification
S.No Step Volume protein activity activity
% factor
Cm3 mg Katal Katal/Kg
I Extraction 16600 435000 0.0413 0.095 100 1.0
II Precipitation 15700 112000 0.0365 0.325 88.3 3.42
Phosphocellulose
III 1380 1716 0.0223 13.02 54.0 40.0
column
IV Gel filtration 211 462 0.0200 43.17 48.4 3.32
V Crystallization - 344 0.016 46.5 38.7 1.08
ENZYME ASSAYS
As assay is a meaurement of a given enzy,e of known characteristics in a sample. Ideally
this means measuring a specific and characteristic biological property (or ability to catalyze a
chemical reaction) of the desired enzyme. An assay must be specific for the enzyme being
purified and highly sensitive to its presence. Further more, the assay must be convenient to use
because it is to be done repeatedly especially during enzyme purification processes.

There are two general purposes for enzyme assay:

1. To measure how much of the enzyme is present in the sample. The enzyme is the variable
measured.
2. With a constant amount of the enzyme present, how does its activity vary with conditions
such as pH, temperature, variation of substrate concentration, effect of inhibitors, etc or
in prior incubation (stability to heat, chemical modification, etc). These studies help to
characterize the enzyme.

Another example of coupled assay is

D – alanine + O2 -----------------→ Pyruvate + NH4+ + H2O2 (D – amino acid odidase)
H2O2 + Chromogen ------------ → Coloured product + H2O (Peroxidase)

Cycle assays are also there which use a small amount of a compound as related liming
intermediate in reactions going both ways. Strictly these are assays for the compound rather than
or an enzyme, but the amount of compound started with might be the product of an enzyme
reaction carried out on a very small scale say one cell. An example,

Pyruvate + NADH + H+ ↔ L – lactate + NAD+ (Lactate dehydrogenase)

Pyruvate + H2O2 ↔ L – lactate + O2 (lactate oxidase)
 In this case the amount of pyruvate is the rate-limiting compound and increase in
concentration of pyruvate is directly proportional to the rate of reaction and it can be monitored
as the absorption of NADH.

Enzyme concentration in the sample can be directly measured by the following analytical
methods.

Method
Ultra filtration
Elecctrophoresis
SDS – PAGE
Capillary electrophoresis
Isoelectric focusing
N – Terminal analysis
Mass Spectroscopy
Comments
Molecular weight determination.
Impurities determination < 5% level
Enzymes with non – identical subunits
Mr determinator, excellent in impurity determination
Excellent analytical technique for Mr determination
Sensitive method
Sub unit determination drawback – determination of enzymes
with blocked N – Terminus or more than one poly peptide
Specialized technique to detect primary structure and post
translation modification.

SOURCES OF ENZYMES
Biologically active enzymes may be extracted from any living organism. A very wide range of
sources are used for commercial enzyme production from Actinoplanes to Zymomonas, from
spinach to snake venom. Of the hundred or so enzymes being used industrially, over a half are
from fungi and yeast and over a third are from bacteria with the remainder divided between
animal (8%) and plant (4%) sources (Table 2.1). A very much larger number of enzymes find use
in chemical analysis and clinical diagnosis. Non-microbial sources provide a larger proportion of
these, at the present time. Microbes are preferred to plants and animals as sources of enzymes
because:
1. they are generally cheaper to produce.
2. their enzyme contents are more predictable and controllable,
3. reliable supplies of raw material of constant composition are more easily arranged, and
4. plant and animal tissues contain more potentially harmful materials than microbes,
including phenolic compounds (from plants), endogenous enzyme inhibitors and
proteases.
Attempts are being made to overcome some of these difficulties by the use of animal and plant
cell culture.
Table 2.1. Some important industrial enzymes and their sources.
Enzymea EC Source Intra/extra Scale of Industrial use
numberb -cellularc productiond
Animal enzymes
Catalase 1.11.1.6 Liver I - Food
Chymotrypsin 3.4.21.1 Pancreas E - Leather
e
Lipase 3.1.1.3 Pancreas E - Food
Rennetf 3.4.23.4 Abomasum E + Cheese
Trypsin 3.4.21.4 Pancreas E - Leather
Plant enzymes
Actinidin 3.4.22.14 Kiwi fruit E - Food
Amylase 3.2.1.1 Malted barley E +++ Brewing
Amylase 3.2.1.2 Malted barley E +++ Brewing
Bromelain 3.4.22.4 Pineapple latex E - Brewing
g
Glucanase 3.2.1.6 Malted barley E ++ Brewing
Ficin 3.4.22.3 Fig latex E - Food
Lipoxygenase 1.13.11.12 Soybeans I - Food
Papain 3.4.22.2 Pawpaw latex E ++ Meat
Bacterial enzymes
Amylase 3.2.1.1 Bacillus E +++ Starch
Amylase 3.2.1.2 Bacillus E + Starch
Asparaginase 3.5.1.1 Escherichia coli I - Health
Glucose 5.3.1.5 Bacillus I ++ Fructose syrup
isomeraseh
Penicillin 3.5.1.11 Bacillus I - Pharmaceutical
amidase
Proteasei 3.4.21.14 Bacillus E +++ Detergent
Pullulanasej 3.2.1.41 Klebsiella E - Starch
Fungal enzymes
Amylase 3.2.1.1 Aspergillus E ++ Baking
Aminoacylase 3.5.1.14 Aspergillus I - Pharmaceutical
k
Glucoamylase 3.2.1.3 Aspergillus E +++ Starch
Catalase 1.11.1.6 Aspergillus I - Food
Cellulase 3.2.1.4 Trichoderma E - Waste
Dextranase 3.2.1.11 Penicillium E - Food
Glucose 1.1.3.4 Aspergillus I - Food
oxidase
Lactasel 3.2.1.23 Aspergillus E - Dairy
Lipasee 3.1.1.3 Rhizopus E - Food
m
Rennet 3.4.23.6 Mucor miehei E ++ Cheese
n
Pectinase 3.2.1.15 Aspergillus E ++ Drinks
Pectin lyase 4.2.2.10 Aspergillus E - Drinks
m
Protease 3.4.23.6 Aspergillus E + Baking
Raffinaseo 3.2.1.22 Mortierella I - Food
Yeast enzymes
Invertasep 3.2.1.26 Saccharomyces I/E - Confectionery
 Lactasel 3.2.1.23 Kluyveromyces I/E - Dairy
e
Lipase 3.1.1.3 Candida E - Food
o
Raffinase 3.2.1.22 Saccharomyces I - Food
In practice, the great majority of microbial enzymes come from a very limited number of genera,
of which Aspergillus species, Bacillus species and Kluyveromyces (also called Saccharomyces)
species predominate. Most of the strains used have either been employed by the food industry for
many years or have been derived from such strains by mutation and selection. There are very few
examples of the industrial use of enzymes having been developed for one task. Shining examples
of such developments are the production of high fructose syrup using glucose isomerase and the
use of pullulanase in starch hydrolysis.
Screening for novel enzymes
If a reaction is thermodynamically possible, it is likely that an enzyme exists which is
capable of catalysing it. One of the major skills of enzyme companies and suitably funded
academic laboratories is the rapid and cost-effective screening of microbial cultures for enzyme
activities. Natural samples, usually soil or compost material found near high concentrations of
likely substrates, are used as sources of cultures. It is not unusual at international congresses of
enzyme technologists to see representatives of enzyme companies collecting samples of soil to be
screened later when they return to their laboratories.
The first stage of the screening procedure for commercial enzymes is to screen ideas, i.e. to
determine the potential commercial need for a new enzyme, to estimate the size of the market and
to decide, approximately, how much potential users of the enzyme will be able to afford to pay
for it. In some cases, the determination of the potential value of an enzyme is not easy, for
instance when it might be used to produce an entirely novel substance. In others, for instance
when the novel enzyme would be used to improve an existing process, its potential value can be
costed very accurately. In either case, a cumulative cash flow must be estimated, balancing the
initial screening and investment capital costs including interest, tax liability and depreciation,
against the expected long term profits. Full account must be taken of inflation, projected variation
in feedstock price and source, publicity and other costs. In addition, the probability of potential
market competition and changes in political or legal factors must be considered. Usually the
sensitivity of the project to changes in all of these factors must be estimated, by informed
guesswork, in order to assess the risk factor involved. Financial re-appraisal must be frequently
carried out during the development process to check that it still constitutes an efficient use of
resources.
If agreement is reached, probably after discussions with potential users, that experimental work
would be commercially justifiable, the next stage involves the location of a source of the required
enzyme. Laboratory work is expensive in manpower so clearly it is worthwhile using all available
databases to search for mention of the enzyme in the academic and patents literature. Cultures
may then be sought from any sources so revealed. Some preparations of commercial enzymes are
quite rich sources of enzymes other than the enzyme which is being offered for sale, revealing
such preparations as potential inexpensive sources which are worth investigating.
If these first searches are unsuccessful, it is probably necessary to screen for new microbial
strains capable of performing the transformation required. This should not be a 'blind' screen:
there will usually be some source of microbes that could have been exposed for countless
generations to the conditions that the new enzyme should withstand or to chemicals which it is
required to modify. Hence, thermophiles are sought in hot springs, osmophiles in sugar factories,
organisms capable of metabolising wood preservatives in timber yards and so on. A classic
example of the detection of an enzyme by intelligent screening was the discovery of a
commercially useful cyanide-degrading enzyme in the microbial pathogens of plants that contain
cyanogenic glycosides.
The identification of a microbial source of an enzyme is by no means the end of the story. The
properties of the enzyme must be determined; i.e. temperature for optimum productivity,
temperature stability profile, pH optimum and stability, kinetic constants (Km, Vmax), whether
there is substrate or product inhibition, and the ability to withstand components of the expected
feedstock other than substrate. A team of scientists, engineers and accountants must then consider
the next steps. If any of these parameters is unsatisfactory, the screen must continue until
improved enzymes are located. Now that protein engineering (see Chapter 8) can be seriously
contemplated, an enzyme with sufficient potential value could be improved 'by design' to
overcome one or two shortcomings. However, this would take a long time, at the present level of
knowledge and skill, so further screening of microbes from selected sources would probably be
considered more worthwhile.
Once an enzyme with suitable properties has been located, various decisions must be made
concerning the acceptability of the organism to the regulatory authorities, the productivity of the
organism, and the way in which the enzyme is to be isolated, utilised (free or immobilised) and, if
necessary, purified. If the organism is unacceptable from a regulatory viewpoint two options
exist; to eliminate that organism altogether and continue the screening operation, or to clone the
enzyme into an acceptable organism. The latter approach is becoming increasingly attractive
especially as cloning could also be used to increase the productivity of the fermentation process.
Cloning may also be attractive when the organism originally producing the enzyme is acceptable
from the health and safety point of view but whose productivity is unacceptable (see Chapter 8).
However, cloning is not yet routine and invariably successful so there is still an excellent case to
be made for applying conventional mutation and isolation techniques for the selection of
improved strains. It should be noted that although the technology for cloning glucose isomerase
into 'routine' organisms is known, it has not yet been applied. Several of the glucose isomerase
preparations used commercially consist of whole cells, or cell fragments, of the selected strains of
species originally detected by screening.
The use of immobilised enzymes is now familiar to industry and their advantages are well
recognised so the practicality of using the new enzymes in an immobilised form will be
determined early in the screening procedure. If the enzyme is produced intracellularly, the
feasibility of using it without isolation and purification will be considered very seriously and
strains selected for their amenability to use in this way.
It should be emphasised that there will be a constant dialogue between laboratory scientists and
biochemical process engineers from the earliest stages of the screening process. Once the
biochemical engineers are satisfied that their initial criteria of productivity, activity and stability
can be met, the selected strain(s) of microbe will be grown in pilot plant conditions. It is only by
applying the type of equipment used in full scale plants that accurate costing of processes can be
achieved. Pilot studies will probably reveal imperfections, or at least areas of ignorance, that must
be corrected at the laboratory scale. If this proves possible, the pilot plant will produce samples of
the enzyme preparation to be used by customers who may well also be at the pilot plant stage in
the development of the enzyme-utilizing process. The enzyme pilot plant also produces samples
for safety and toxicological studies provided that the pilot process is exactly similar to the full
scale operation.
Screening for new enzymes is expensive so that the intellectual property generated must be
protected against copying by competitors. This is usually done by patenting the enzyme or its
production method or, most usefully, the process in which it is to be used. Patenting will be
initiated as soon as there is evidence that an innovative discovery has been made.
MEDIA FOR ENZYME PRODUCTION
Detailed description of the development and use of fermenters for the large-scale cultivation of
microorganisms for enzyme production is outside the scope of this volume but mention of media
use is appropriate because this has a bearing on the cost of the enzyme and because media
components often find their way into commercial enzyme preparations. Details of components
used in industrial scale fermentation broths for enzyme production are not readily obtained. This
is not unexpected as manufacturers have no wish to reveal information that may be of technical or
commercial value to their competitors. Also some components of media may be changed from
batch to batch as availability and cost of, for instance, carbohydrate feedstocks change. Such
changes reveal themselves in often quite profound differences in appearance from batch to batch
of a single enzyme from a single producer. The effects of changing feedstocks must be
considered in relation to downstream processing. If such variability is likely to significantly
reduce the efficiency of the standard methodology, it may be economical to use a more expensive
defined medium of easily reproducible composition.
Clearly defined media are usually out of the question for large scale use on cost grounds but may
be perfectly acceptable when enzymes are to be produced for high value uses, such as analysis or
medical therapy where very pure preparations are essential. Less-defined complex media are
composed of ingredients selected on the basis of cost and availability as well as composition.
Waste materials and by-products from the food and agricultural industries are often major
ingredients. Thus molasses, corn steep liquor, distillers solubles and wheat bran are important
components of fermentation media providing carbohydrate, minerals, nitrogen and some
vitamins. Extra carbohydrate is usually supplied as starch, sometimes refined but often simply as
ground cereal grains. Soybean meal and ammonium salts are frequently used sources of
additional nitrogen. Most of these materials will vary in quality and composition from batch to
batch causing changes in enzyme productivity.
PREPARATION OF ENZYMES
Readers of papers dealing with the preparation of enzymes for research purposes will be familiar
with tables detailing the stages of purification. Often the enzyme may be purified several
hundred-fold but the yield of the enzyme may be very poor, frequently below 10% of the activity
of the original material (Table 2.2). In contrast, industrial enzymes will be purified as little as
possible, only other enzymes and material likely to interfere with the process which the enzyme is
to catalyse, will be removed. Unnecessary purification will be avoided as each additional stage is
costly in terms of equipment, manpower and loss of enzyme activity. As a result, some
commercial enzyme preparations consist essentially of concentrated fermentation broth, plus
additives to stabilise the enzyme's activity.
Table 2.2. The effect of number of steps on the yield and costs in a typical enzyme purification
process. The realistic assumptions are made that step yields are 75%, step purifications are
three-fold and step costs are 10% of the initial costs (later purification steps are usually
intrinsically more expensive but are necessarily of smaller scale).
Relative Yield Specific Total Cost per Cost per
Step
weight (%) activity cost weight activity
1.000 100 1 1.00 1 1.00
1 0.250 75 3 1.10 4 1.47
2 0.063 56 9 1.20 19 2.13
3 0.016 42 27 1.30 83 3.08
4 0.004 32 81 1.40 358 4.92
5 0.001 24 243 1.50 1536 6.32

The content of the required enzyme should be as high as possible (e.g. 10% w/w of the protein) in
order to ease the downstream processing task. This may be achieved by developing the
fermentation conditions or, often more dramatically, by genetic engineering. It may well be
economically viable to spend some time cloning extra copies of the required gene together with a
powerful promoter back into the producing organism in order to get 'over-producers' (see Chapter
8).
It is important that the maximum activity is retained during the preparation of enzymes.
Enzyme inactivation can be caused by heat, proteolysis, sub-optimal pH, oxidation, denaturants,
irreversible inhibitors and loss of cofactors or coenzymes. Of these heat inactivation, which
together with associated pH effects, is probably the most significant. It is likely to occur during
enzyme extraction and purification if insufficient cooling is available (see Chapter 1), but the
problem is less when preparing thermophilic enzymes. Proteolysis is most likely to occur in the
early stages of extraction and purification when the proteases responsible for protein turnover in
living cells are still present. It is also the major reason for enzyme inactivation by microbial
contamination. In their native conformations, enzymes have highly structured domains which are
resistant to attack by proteases because many of the peptide bonds are mechanically inaccessible
and because many proteases are highly specific. The chances of a susceptible peptide bond in a
structured domain being available for protease attack are low. Single 'nicks' by proteases in these
circumstances may have little immediate effect on protein conformation and, therefore, activity.
The effect, however, may severely reduce the conformational stability of the enzyme to heat or
pH variation so greatly reducing its operational stability. If the domain is unfolded under these
changed conditions, the whole polypeptide chain may be available for proteolysis and the same,
specific, protease may destroy it. Clearly the best way of preventing proteolysis is to rapidly
remove, or inhibit, protease activity. Before this can be achieved it is important to keep enzyme
preparations cold to maintain their native conformation and slow any protease action that may
occur.
Some intracellular enzymes are used commercially without isolation and purification but the
majority of commercial enzymes
are either produced extracellularly
by the microbe or plant or must be
released from the cells into
solution and further processed
(Figure 2.1). Solid/liquid
separation is generally required for
the initial separation of cell mass,
the removal of cell debris after cell
breakage and the collection of
precipitates. This can be achieved
by filtration, centrifugation or
aqueous biphasic partition. In
general, filtration or aqueous
biphasic systems are used to
remove unwanted cells or cell
debris whereas centrifugation is the
preferred method for the collection
of required solid material.

Figure 2.1. Flow diagram for the

preparation of enzymes.

Centrifugation
Centrifugation separates on the
basis of the particle size and
density difference between the liquid and solid phases. Sedimentation of material in a centrifugal
field may be described by

(2.1)
where v is the rate of sedimentation, d is the particle diameter, rs is the particle density, rl is the
solution density,  is the angular velocity in radians s -1, r is the radius of rotation,  is the
kinematic viscosity, Fs is a correction factor for particle interaction during hindered settling and
 is a shape factor (=1 for spherical particles). Fs depends on the volume fraction of the solids
present; approximately equalling 1, 0.5, 0.1 and 0.05 for 1%, 3%, 12% and 20% solids volume
fraction respectively. Only material which reaches a surface during the flow through continuous
centrifuges will be removed from the centrifuge feedstock, the efficiency depending on the
residence time within the centrifuge and the distance necessary for sedimentation (D). This
residence time will equal the volumetric throughput ( ) divided by the volume of the centrifuge
(V). The maximum throughput of a centrifuge for efficient use is given by

(2.2)
The efficiency of the process is seen to depend on the solids volume fraction, the effective
clarifying surface (V/D) and the acceleration factor (2r/g, where g is the gravitational constant,
981 cm s-2; a rotor of radius 25 cm spinning at 1 rev s-1 has an acceleration factor of
approximately 1 G). Low acceleration factors of about 1 500 g may be used for harvesting cells
whereas much higher acceleration factors are needed to collect enzyme efficiently. The product of
these factors (2rV/gD) is called the sigma factor ( ) and is used to compare centrifuges and to
assist scale-up.
Laboratory centrifuges using tubes in swing-out or angle head rotors have high angular velocity
() and radius of rotation (r) but small capacity (V) and substantial sedimentation distance (D).
This type of design cannot be scaled-up safely, primarily because the mechanical stress on the
centrifuge head increases with the square of the radius, which must increase with increasing
capacity.
For large-scale use, continuous centrifuges of various types are employed (Figure 2.2). These
allow the continuous addition of feedstock, the continuous removal of supernatant and the
discontinuous, semicontinuous or continuous removal of solids. Where discontinuous or
semicontinuous removal of precipitate occurs, the precipitate is flushed out by automatic
discharge systems which cause its dilution with water or medium and may be a problem if the
precipitate is required for further treatment. Centrifugation is the generally preferred method for
the collection of enzyme-containing solids as it does not present a great hazard to most enzymes
so long as foam production, with consequent enzymic inactivation, is minimised.
Figure 2.2. Basic designs of industrial centrifuges, showing the flow of material within the bowls.
Motor drives, cooling jackets and sludge collection vessels are not shown. (a) Tubular bowl
centrifuge. This is generally operated vertically, the tubular rotor providing a long flow path
enabling clarification. The sludge collects and must be removed. (b) Continuous scroll
centrifuge. This is operated horizontally. The helical screw scrolls the solids along the bowl
surface and out of the liquid; the sludge being dewatered before discharge. The clarified liquor
overflows over an adjustable weir at the other end of the bowl. The screw conveyer rotates at a
slightly different speed to the bowl. (c) Continuous multichamber disc-stack centrifuge. The bowl
contains a number of parallel discs providing a large clarifying surface with a small
sedimentation distance. The sludge is removed through a valve.

Small particles of cell debris and precipitated protein may be sedimented using tubular bowl
centrifuges, of which Sharples centrifuges (produced by Pennwalt Ltd.) are the best known.
These semi-continuous centrifuges are long and thin enabling rapid acceleration and deceleration,
minimising the down-time required for the removal of the sedimented solids. Here the radius and
effective liquid thickness are both small allowing a high angular velocity and hence high
centrifugal force; small models can be used at acceleration factors up to 50,000 g, accumulating
0.1 Kg of wet deposit whereas large models, designed to accumulate up to 5 Kg of deposit, are
restricted to 16,000 g. The capacities of these centrifuges are only moderate.Multichamber disc-
stack centrifuges, originally designed (by Westfalia and Alpha-Laval) for cream separation,
contain multiple coned discs in a stack which are spun and on which the precipitate collects. They
may be operated either semi-continuously or, by using a centripetal pressurising pump within the
centrifuge bowl which forces the sludge out through a valve, continuously. The capacity and
radius of such devices are large and the thickness of liquid is very small, due to the large effective
surface area. The angular velocity, however, is restricted giving a maximum acceleration factor of
about 8,000 g. A different design which is rather similar in principle is the solid bowl scroll
centrifuge in which an Archimedes' screw collects the precipitate so that fluid and solids leave at
opposite ends of the apparatus. These can only be used at low acceleration (about 3,000 g) so they
are suitable only for the collection of comparatively large particles.
Although many types of centrifuge are available, the efficient precipitation of small particles of
cell debris can be difficult, sometimes near-impossible. Clearly from Equation 2.2, the efficiency
of centrifugation can be improved if the particle diameter (d) is increased. This can be done either
by coagulating or flocculating particles. Coagulation is caused by the removal of electrostatic
charges (e.g. by pH change) and allowing particles to adhere to each other. Flocculation is
achieved by adding small amounts of high-molecular-weight charged materials which bridge
oppositely-charged particles to produce a loose aggregate which may be readily removed by
centrifugation or filtration. Flocculation and coagulation are cheap and effective aids to
precipitating or otherwise harvesting whole cells, cell debris or soluble proteins but, of course, it
is essential that the agents used must not inhibit the target enzymes. It is important to note that the
choice of flocculant is determined by the pH and ionic strength of the solution and the nature of
the particles. Most flocculants have very definite optimum concentrations above which further
addition may be counter-effective. Some flocculants can be rapidly ruined by shear.

FILTRATION
Filtration separates simply on the basis of particle size. Its efficiency is limited by the
shape and compressibility of the particles, the viscosity of the liquid phase and the maximum
allowable pressures. Large-scale simple filtration employs filter cloths and filter aids in a plate
and frame press configuration, in rotary vacuum filters or centrifugal filters (Figure 2.3). The
volumetric throughput of a filter is proportional to the pressure (P) and filter area (AF) and
inversely proportional to the filter cake thickness (DF) and the dynamic viscosity

(2.3)
where k is a proportionality constant dependent on the size and nature of the particles. For very
small particles k depends on the fourth power of their diameter. Filtration of particles that are
easily compressed leads to filter blockage and the failure of Equation 2.3 to describe the system.
Under these circumstances a filter aid, such as celite, is mixed with the feedstock to improve the
mechanical stability of the filter cake. Filter aids are generally used only where the liquid phase is
required as they cause substantial problems in the recovery of solids. They also may cause loss of
enzyme activity from the solution due to physical hold-up in the filter cake. It is often difficult for
a process development manager to decide whether to attempt to recover enzyme trapped in this
way. Problems associated with the build-up of the filter cake may also be avoided by high
tangential flow of the feedstock across the surface of the filter, a process known as crossflow
microfiltration (Figure 2.4). This method dispenses with filter aids and uses special symmetric
microporous membrane assemblies capable of retaining particles down to 0.1 - 1 µm diameter (cf.
Bacillus diameter of about 2 µm).
Figure 2.3. The basic design of the rotary vacuum filter. The suspension is sucked through a
filter cloth on a rotating drum. This produces a filter cake which is removed with a blade. The
filter cake may be rinsed during its rotation. These filters are generally rather messy and difficult
to contain making them generally unsuitable for use in the production of toxic or recombinant
DNA products. There have been recent developments that improve their suitability, however,
such as the Disposable Rotary Drum Filter.

A simple and familiar filtration apparatus is the perforate bowl centrifuge or basket centrifuge, in
effect a spin drier. Cell debris is collected on a cloth with, or without, filter aid and can be
skimmed off when necessary using a suitable blade. Such centrifugal filters have a large radius
and effective liquid depth, allowing high volumes. However, safety decrees that the angular
velocity must be low and so only large particles (e.g. plant material) can be removed
satisfactorily.
Figure 2.4. Principles of (a) dead-end filtration and (b) cross-flow filtration. In dead-end
filtration the flow causes the build-up of the filter cake, which may prevent efficient operation.
This is avoided in cross-flow filtration where the flow sweeps the membrane surface clean.

AQUEOUS BIPHASIC SYSTEMS

The 'incompatibility' of certain polymers in aqueous solution was first noted by Beijerinck in
1896. In this case two phases were formed when agar was mixed with soluble starch or gelatine.
Since then, many two phase aqueous systems have been found; the most thoroughly investigated
being the aqueous dextran-polyethylene glycol system (e.g. 10% polyethylene glycol 4000/2%
dextran T500), where dextran forms the more hydrophilic, denser, lower phase and polyethylene
glycol the more hydrophobic, less dense, upper phase. Aqueous three phase systems are also
known.
Phases form when limiting concentrations of the polymers are exceeded. Both phases contain
mainly water and are enriched in one of the polymers. The limiting concentrations depend on the
type and molecular weight of the polymers and on the pH, ionic strength and temperature of the
solution. Some polymers form the upper hydrophobic phase in the presence of fairly concentrated
solutions of phosphates or sulphates (e.g. 10% polyethylene glycol 4000/12.5% potassium
phosphate buffer). A drawback to the useful dextran/polyethylene glycol system is the high cost
of the purified dextran used. This has been alleviated by the use of crude unfractionated dextran
preparations, much cheaper hydroxypropyl starch derivatives and salt-containing biphasic
systems.
Aqueous biphasic systems are of considerable value to biotechnology. They provide the
opportunity for the rapid separation of biological materials with little probability of denaturation.
The interfacial tension between the phases is very low (i.e. about 400-fold less than that between
water and an immiscible organic solvent), allowing small droplet size, large interfacial areas,
efficient mixing under very gentle stirring and rapid partition. The polymers have a stabilising
influence on most proteins. A great variety of separations have been achieved, by far the most
important being the separation of enzymes from broken crude cell material. Separation may be
achieved in a few minutes, minimising the harmful action of endogenous proteases. The systems
have also been used successfully for the separation of different types of cell membranes and
organelles, the purification of enzymes and for extractive bioconversions (see Chapter 7).
Continuous liquid two-phase separation is easier than continuous solid/liquid separation using
equipment familiar from immiscible solvent systems, for example disc-stack centrifuges and
counter-current separators. Such systems are readily amenable to scale-up and may be employed
in continuous enzyme extraction processes involving some recycling of the phases.
Cells, cell debris proteins and other material distribute themselves between the two phases in a
manner described by the partition coefficient (P) defined as:

(2.4)
where Ct and Cb represent the concentrations in the top and bottom phases respectively. The yield
and efficiency of the separation is determined by the relative amounts of material in the two
phases and therefore depends on the volume ratio (Vt/Vb). The partition coefficient is
exponentially related to the surface area (and hence molecular weight) and surface charge of the
particles in addition to the difference in the electrical potential and hydrophobicity of the phases.
It is not generally very sensitive to temperature changes. This means that proteins and larger
particles are normally partitioned into one phase whereas smaller molecules are distributed more
evenly between phases. A partition coefficient of greater than 3 is required if usable yields are to
be achieved by a single extraction process. Typical partition coefficients for proteins are 0.01-100
whereas the partition coefficients for cells and cell debris are effectively zero.
The influence of pH and salts on protein partition is complex, particularly when phosphate
buffers are present. A given protein distributes differently between the phases at different pH's
and ionic strength but the presence of phosphate ions affect the partition coefficient in an
anomalous fashion because these ions distribute themselves unequally resulting in electrostatic
potential (and pH) differences. This means that systems may be 'tuned' to enrich an enzyme in
one phase, ideally the upper phase with cell debris and unwanted enzymes in the lower phase.
An enzyme may be extracted from the upper (polyethylene glycol) phase by the addition of salts
or further polymer, generating a new biphasic system. This stage may be used to further purify
the enzyme. A powerful modification of this technique is to combine phase partitioning and
affinity partitioning. Affinity ligands (e.g. triazine dyes) may be coupled to either polymer in an
aqueous biphasic system and thus greatly increase the specificity of the extraction.

CELL BREAKAGE
Various intracellular enzymes are used in significant quantities and must be released from cells
and purified (Table 2.1). The amount of energy that must be put into the breakage of cells
depends very much on the type of organism and to some extent on the physiology of the
organism. Some types of cell are broken readily by gentle treatment such as osmotic shock (e.g.
animal cells and some gram-negative bacteria such as Azotobacter species), whilst others are
highly resistant to breakage. These include yeasts, green algae, fungal mycelia and some gram-
positive bacteria which have cell wall and membrane structures capable of resisting internal
osmotic pressures of around 20 atmospheres (2 MPa) and therefore have the strength, weight for
weight, of reinforced concrete. Consequently a variety of cell disruption techniques have been
developed involving solid or liquid shear or cell lysis.
The rate of protein released by mechanical cell disruption is usually found to be proportional to
the amount of releasable protein.

(2.5)
where P represents the protein content remaining associated with the cells, t is the time and k is a
release constant dependent on the system. Integrating from P = Pm (maximum possible protein
releasable) at time zero to P = Pt at time t gives

(2.6)

(2.7)
As the protein released from the cells (Pr) is given by
(2.8)
the following equation for cell breakage is obtained

(2.9)
It is most important in choosing cell disruption strategies to avoid damaging the enzymes. The
particular hazards to enzyme activity relevant to cell breakage are summarised in Table 2.3. The
most significant of these, in general, are heating and shear.
 Table 2.3. Hazards likely to damage enzymes during cell disruption.
All mechanical methods require a large input of energy, generating heat. Cooling is essential for
Heat most enzymes. The presence of substrates, substrate analogues or polyols may also help
stabilise the enzyme.
Shear forces are needed to disrupt cells and may damage enzymes, particularly in the presence
Shear
of heavy metal ions and/or an air interface.]
Disruption of cells will inevitably release degradative enzymes which may cause serious loss of
enzyme activity. Such action may be minimised by increased speed of processing with as much
Proteases
cooling as possible. This may be improved by the presence of an excess of alternative substrates
(e.g. inexpensive protein) or inhibitors in the extraction medium.
Buffered solutions may be necessary. The presence of substrates, substrate analogues or polyols
pH
may also help stabilise the enzyme.
Some enzymes may suffer conformational changes in the presence of detergent and/or solvents.
Polyphenolics derived from plants are potent inhibitors of enzymes. This problem may be
Chemical
overcome by the use of adsorbents, such as polyvinylpyrrolidone, and by the use of ascorbic
acid to reduce polyphenol oxidase action.
Oxidation Reducing agents (e.g. ascorbic acid, mercaptoethanol and dithiothreitol) may be necessary.
Foaming The gas-liquid phase interfaces present in foams may disrupt enzyme conformation.
Heavy metal ions (e.g. iron, copper and nickel) may be introduced by leaching from the
Heavy-metal
homogenisation apparatus. Enzymes may be protected from irreversible inactivation by the use
toxicity
of chelating reagents, such as EDTA.

Media for enzyme extraction will be selected on the basis of cost-effectiveness so will include as
few components as possible. Media will usually be buffered at a pH value which has been
determined to give the maximum stability of the enzyme to be extracted. Other components will
combat other hazards to the enzyme, primarily factors causing denaturation (Table 2.3).
Ultrasonic cell disruption
The treatment of microbial cells in suspension with inaudible ultrasound (greater than about 18
kHz) results in their inactivation and disruption. Ultrasonication utilises the rapid sinusoidal
movement of a probe within the liquid. It is characterised by high frequency (18 kHz - 1 MHz),
small displacements (less than about 50 m), moderate velocities (a few m s -1), steep transverse
velocity gradients (up to 4,000 s-1) and very high acceleration (up to about 80,000 g).
Ultrasonication produces cavitation phenomena when acoustic power inputs are sufficiently high
to allow the multiple production of microbubbles at nucleation sites in the fluid. The bubbles
grow during the rarefying phase of the sound wave, then are collapsed during the compression
phase. On collapse, a violent shock wave passes through the medium. The whole process of gas
bubble nucleation, growth and collapse due to the action of intense sound waves is called
cavitation. The collapse of the bubbles converts sonic energy into mechanical energy in the form
of shock waves equivalent to several thousand atmospheres (300 MPa) pressure. This energy
imparts motions to parts of cells which disintegrate when their kinetic energy content exceeds the
wall strength. An additional factor which increases cell breakage is the microstreaming (very high
velocity gradients causing shear stress) which occur near radially vibrating bubbles of gas caused
by the ultrasound.
Much of the energy absorbed by cell suspensions is converted to heat so effective cooling is
essential. The amount of protein released by sonication has been shown to follow Equation 2.9.
The constant (k) is independent of cell concentrations up to high levels and approximately
proportional to the input acoustic power above the threshold power necessary for cavitation.
Disintegration is independent of the sonication frequency except insofar as the cavitation
threshold frequency depends on the frequency.
Equipment for the large-scale continuous use of ultrasonics has been available for many years
and is widely used by the chemical industry but has not yet found extensive use in enzyme
production. Reasons for this may be the conformational lability of some (perhaps most) enzymes
to sonication and the damage that they may realise though oxidation by the free radicals, singlet
oxygen and hydrogen peroxide that may be concomitantly produced. Use of radical scavengers
(e.g. N2O) have been shown to reduce this inactivation. As with most cell breakage methods, very
fine cell debris particles may be produced which can hinder further processing. Sonication
remains, however, a popular, useful and simple small-scale method for cell disruption.
High pressure homogenisers
Various types of high pressure homogeniser are available for use in the food and chemicals
industries but the design which has been very extensively used for cell disruption is the Manton-
Gaulin APV type homogeniser. This consists of a positive displacement pump which draws cell
suspension (about 12% w/v) through a check valve into the pump cylinder and forces it, at high
pressures of up to 150 MPa (10 tons per square inch) and flow rates of up to 10,000 L hr -1,
through an adjustable discharge valve which has a restricted orifice (Figure 2.5). Cells are
subjected to impact, shear and a severe pressure drop across the valve but the precise mechanism
of cell disruption is not clear. The main disruptive factor is the pressure applied and consequent
pressure drop across the valve. This causes the impact and shear stress which are proportional to
the operating pressure.

Figure 2.5. A cross-section through the Manton-Gaulin homogeniser valve, showing the flow of
material. The cell suspension is pumped at high pressure through the valve impinging on it and
the impact ring. The shape of the exit nozzle from the valve seat varies between models and
appears to be a critical determinant of the homogenisation efficiency. The model depicted is the
'CD Valve' from APV Gaulin.
As narrow orifices which are vulnerable to blockage are key parts of this type of homogeniser, it
is unsuitable for the disruption of mycelial organisms but has been used extensively for the
disruption of unicellular organisms. The release of proteins can be described by Equation 2.9 but
normally a similar relationship is used where the time variable is replaced by the number of
passes (N) through the homogeniser.
 (2.10)
In the commonly-used operating range with pressures below about 75 MPa, the release constant
(k) has been found to be proportional to the pressure raised to an exponent dependent on the
organism and its growth history (e.g. k=k'P2.9 in Saccharomyces cerevesiae and k=k'P2.2 in
Escherichia coli, where P represents the operating pressure and k' is a rate constant). Different
growth media may be selected to give rise to cells of different cell wall strength. Clearly, the
higher the operating pressure, the more efficient is the disruption process. The protein release rate
constant (k) is temperature dependent, disruption being more rapid at higher temperatures. In
practice, this advantage cannot be used since the temperature rise due to adiabatic compression is
very significant so samples must be pre-cooled and cooled again between multiple passes. At an
operating pressure of 50 MPa, the temperature rise each pass is about 12 deg. C.
In addition to the fragility of the cells, the location of an enzyme within the cells can influence the
conditions of use of an homogeniser. Unbound intracellular enzymes may be released by a single
pass whereas membrane bound enzymes require several passes for reasonable yields to be
obtained. Multiple passes are undesirable because, of course, they decrease the throughput
productivity rate and because the further passage of already broken cells results in fine debris
which is excessively difficult to remove further downstream. Consequently, homogenisers will be
used at the highest pressures compatible with the reliability and safety of the equipment and the
temperature stability of the enzyme(s) released. High pressure homogenisers are acceptably good
for the disruption of unicellular organisms provided the enzymes needed are not heat labile. The
shear forces produced are not capable of damaging enzymes free in solution. The valve unit is
prone to erosion and must be precision made and well maintained.
Use of bead mills
When cell suspensions are agitated in the presence of small steel or glass beads (usually 0.2 -.1.0
mm diameter) they are broken by the high liquid shear gradients and collision with the beads. The
rate and effectiveness of enzyme release can be modified by changing the rates of agitation and
the size of the beads, as well as the dimensions of the equipment. Any type of biomass,
filamentous or unicellular, may be disrupted by bead milling but, in general, the larger sized cells
will be broken more readily than small bacteria. For the same volume of beads, a large number of
small beads will be more effective than a relatively small number of larger beads because of the
increased likelihood of collisions between beads and cells.
Bead mills are available in various sizes and configurations from the Mickle shaker which has a
maximum volume of about 40 ml to continuous process equipment capable of handling up to 200
Kg wet yeast or 20 Kg wet bacteria each hour. The bead mills that have been studied in most
detail are the Dyno-Mill and the Netsch-Molinex agitator, both of which consist of a cylindrical
vessel containing a motor-driven central shaft equipped with impellers of different types. Both
can be operated continuously, being equipped with devices which retain the beads within the
milling chamber. Glass Ballotini or stainless steel balls are used, the size range being selected for
most effective release of the enzyme required. Thus 1 mm diameter beads are satisfactory for the
rapid release of periplasmic enzymes from yeast but 0.25 mm diameter beads must be used, for a
longer period, to release membrane-bound enzymes from bacteria.
The kinetics of protein release from bead mills follows the relationship given by Equation 2.9
with respect to the time (t) that a particle spends in the mill. Unfortunately, however well
designed these mills are, when continuously operated there will be a significant amount of
backmixing which reduces the efficiency of the protein released with respect to the average
residence time ( , see the discussion concerning backmixing in reactors in Chapter 5). This is
more noticeable at low flow rates (high average residence times) and when the proportion of
protein released is high. It may be counteracted by designing the bead mill to encourage plug
flow characteristics. Under these circumstances the relationship can be shown to be
 (2.11)
where i represents the degree of backmixing (i.e. i = 0 under ideal plug flow conditions and i = 1
for ideal complete backmixing). Equation 2.11 reduces to give the simplified relationship of
Equation 2.9 at low (near zero) values of i.
In addition to bead size, the protein release rate constant (k) is a function of temperature, bead
loading, impeller rotational speed and cell loading. Impeller speeds can be increased with
advantage until bead breakage becomes significant but heat generation will also increase. At a
constant impeller speed, the efficiency of the equipment declines with throughput as the degree of
backmixing increases. There will be an optimum impeller tip speed at which the increases in
disruption are balanced by increases in backmixing.
In general, increased bead loading increases the rate of protein release but also increases the
production of heat and the power consumption. Heat production is the major problem in the use
of bead mills for enzyme release, particularly on a large (e.g. 20 litres) scale. Smaller vessels may
be cooled adequately through cooling jackets around the bead chamber but larger mills require
cooling through the agitator shaft and impellers. However, if cooling is effective there is little
damage to the enzymes released.
Use of freeze-presses
The Hughes press and the 'X' press enable frozen cell pastes to be forced under high pressure
(150 - 230 MPa, 10 - 15 tons per square inch) through narrow orifices, the disruption being
produced by phase and volume changes and by solid shear due to the ice crystals. The Hughes
press can only be used discontinuously on a small scale. The 'X' press may be used semi-
continuously and is amenable to scale-up but, although the method allows the breakage of even
the most robust organisms and the efficient recovery of heat-sensitive enzymes, freeze-pressing is
not used on a large scale for releasing enzymes from cells.
Use of lytic methods
The breakage of cells using non-mechanical methods is attractive in that it offers the prospects of
releasing enzymes under conditions that are gentle, do not subject the enzyme to heat or shear,
may be very cheap, and are quiet to the user. The methods that are available include osmotic
shock, freezing followed by thawing, cold shock, desiccation, enzymic lysis and chemical lysis.
Each method has its drawbacks but may be particularly useful under certain specific
circumstances.
Certain types of cell can be caused to lyse by osmotic shock. This would be a cheap, gentle and
convenient method of releasing enzymes but has not apparently been used on a large scale. Some
types of cell may be caused to autolyse, in particular yeasts and Bacillus species. Yeast invertase
preparations employed in the industrial manufacture of invert sugars are produced in this manner.
Autolysis is a slow process compared with mechanical methods, and microbial contamination is a
potential hazard, but it can be used on a very large scale if necessary. Where applicable,
dessication may be very useful in the preparation of enzymes on a large scale. The rate of drying
is very important in these cases, slow methods being preferred to rapid ones like lyophilisation.
Enzymic lysis using added enzymes has been used widely on the laboratory scale but is less
popular for industrial purposes. Lysozyme, from hen egg-white, is the only lytic enzyme
available on a commercial scale. It has often used to lyse Gram positive bacteria in an hour at
about 50,000 U Kg-1 (dry weight). The chief objection to its use on a large scale is its cost. Where
costs are reduced by the use of the relatively inexpensive, lysozyme-rich, dried egg white, a
major separation problem may be introduced. Yeast-lytic enzymes from Cytophaga species have
been studied in some detail and other lytic enzymes are under development. If significant markets
for lytic enzymes are identified, the scale of their production will increase and their cost is likely
to decrease.Lysis by acid, alkali, surfactants and solvents can be effective in releasing enzymes,
provided that the enzymes are sufficiently robust. Detergents, such as Triton X-100, used alone or
in combination with certain chaotropic agents, such as guanidine HCl, are effective in releasing
membrane-bound enzymes. However, such materials are costly and may be difficult to remove
from the final product.
PREPARATION OF ENZYMES FROM CLARIFIED SOLUTION
In many cases, especially when extracellular enzymes are being prepared for sale, the clarified
solution is simply concentrated, preservative materials added, and sold as a solution or as a dried
preparation. The concentration process chosen will be the cheapest which is compatible with the
retention of enzyme activity. For some enzymes rotary evaporation can be considered, followed if
necessary by spray drying. The most popular method, though, is ultrafiltration, whereby water
and low molecular weight materials are removed by passage through a membrane under pressure,
enzyme being retained. Ultrafiltration differs from conventional filtration and microfiltration with
respect to the size of particles being retained (< 50 nm diameter). It uses asymmetric microporous
membranes with a relatively dense but thin skin, containing pores, supported by a coarse strong
substructure. Membranes possessing molecular weight cut-offs from 1000 to 100,000 and usable
at pressure up to 2 MPa are available.
There are a number types of apparatus available. Stirred cells represent the simplest configuration
of ultrafiltration cell. The membrane rests on a rigid support at the base of a cylindrical vessel
which is equipped with a magnetic stirrer to combat concentration polarisation. It is not suitable
for large scale use but is useful for preliminary studies and for the concentration of laboratory
column eluates. Various large-scale units are available in which membranes are formed into wide
diameter tubes (1 - 2 cm diameter) and the tubes grouped into cartridges. These are not as
compact as capillary systems (area/volume about 25 m-1) and are very expensive but are less
liable to blockage by stray large particles in the feedstream. Cheaper thin-channel systems are
available (area/volume about 500 m-1) which use flat membrane sandwiches in filter press
arrangements of various designs chosen to produce laminar flow across the membrane and
minimise concentration polarisation. Capillary membranes represent a relatively cheap and
increasingly popular type of ultrafiltration system which uses micro-tubular membranes 0.2 - 1.1
mm diameter and provides large membrane areas within a small unit volume (area/volume about
1000 m-1). Membranes are usually mounted into modules for convenient manipulation. This
configuration of membranes can be scaled up with ease. Commercial models are available that
give ultrafiltration rates of up to 600 L hr-1.
The steady improvement in the performance, durability and reliability of membranes has been a
boon to enzyme technologists, encouraging wide use of the various ultrafiltration configurations.
Problems with membrane blockage and fouling can usually be overcome by treatment of
membranes with detergents, proteases or, with care, acids or alkalis. The initial cost of
membranes remains considerable but modern membranes are durable and cost-effective.
Ultrafiltration, done efficiently, results in little loss of enzyme activity. However, some
configurations of apparatus, particularly in which solutions are recycled, can produce sufficient
shear to damage some enzymes.
NUCLEIC ACID REMOVAL
Intracellular enzyme preparations contain nucleic acids which can give rise to increased viscosity
interfering with enzyme purification procedures, in particular ultrafiltration. Some organisms
contain sufficient nuclease activity to eliminate this problem but, otherwise, the nucleic acids
must be removed by precipitation or degraded by the addition of exogenous nucleases.
Ammonium sulphate precipitation (see later) can be effective in removing nucleic acids but will
remove some protein at the same time. Various more specific precipitants have been used, usually
positively-charged materials which form complexes with the negatively-charged phosphate
residues of the nucleic acids. These include, in order of roughly decreasing effectiveness,
polyethyleneimine, the cationic detergent cetyltrimethyl ammonium bromide, streptomycin
sulphate and protamine sulphate. All of these are expensive and possibly toxic, particularly
streptomycin sulphate. Also, they may complex undesirably with certain enzymes. They may be
necessary, however, where possible contamination of the enzyme product must be avoided, such
as in the preparation of restriction endonucleases. Otherwise, treatment with bovine pancreatic
nucleases is probably the most cost-effective method of nucleic acid removal.
CONCENTRATION BY PRECIPITATION
Precipitation of enzymes is a useful method of concentration and is ideal as an initial step in their
purification. It can be used on a large scale and is less affected by the presence of interfering
materials than any of the chromatographic methods described later.
Salting out of proteins, particularly by use of ammonium sulphate, is one of the best known and
used methods of purifying and concentrating enzymes, particularly at the laboratory scale.
Increases in the ionic strength of the solution cause a reduction in the repulsive effect of like
charges between identical molecules of a protein. It also reduces the forces holding the solvation
shell around the protein molecules. When these forces are sufficiently reduced, the protein will
precipitate; hydrophobic proteins precipitating at lower salt concentrations than hydrophilic
proteins. Ammonium sulphate is convenient and effective because of its high solubility,
cheapness, lack of toxicity to most enzymes and its stabilizing effect on some enzymes (see Table
2.4). Its large-scale use, however, is limited as it is corrosive except with stainless steel, it forms
dense solutions presenting problems to the collection of the precipitate by centrifugation, and it
may release gaseous ammonia, particularly at alkaline pH. The practice of using ammonium
sulphate precipitation is more straightforward than the theory. Reproducible results can only be
obtained provided the protein concentration, temperature and pH are kept constant. The
concentration of the salt needed to precipitate an enzyme will vary with the concentration of the
enzyme. However, fractionation of protein mixtures by the stepwise increase in the ionic strength
can be a very effective way of partly purifying enzymes.
The solubility of an enzyme can be described by the equation
(2.11)
where S is the enzyme solubility, Kintercept is the intercept constant, Ksalt is the salting out constant
and  is the ionic strength which is proportional to the concentration of a precipitating salt.
Kintercept is independent of the salt used but depends on the pH, temperature, enzyme and the other
components in the solution. Ksalt depends on both the enzyme required and the salt used but is
largely independent of other factors. This equation (2.11) may also be used to give the minimum
salt concentration necessary before enzyme will start to precipitate; the concentration change
necessary to precipitate the enzyme varying according to the magnitude of the salting out
constant.
Some enzymes do not survive ammonium sulphate precipitation. Other salts may be substituted
but the more favoured alternative is to use organic solvents such as methanol, ethanol, propan-2-
ol and acetone. These act by reducing the dielectric of the medium and consequently reducing the
solubility of proteins by favouring protein-protein rather than protein-solvent interactions.
Organic solvents are not widely used on a large scale because of their cost, their flammability,
and the tendency of proteins to undergo rapid denaturation by these solvents if the temperature is
allowed to rise much above 0°C. On safety grounds when organic solvents are used, special
flameproof laboratory areas are used and temperatures maintained below their flashpoints.
Except when enzymes are presented for sale as ammonium sulphate precipitates, the
precipitating salt or solvent must be removed. This may be done by dialysis,
ultrafiltration or by using a desalting column of, for instance, Sephadex G-25.
HEAT TREATMENT
In many cases, unwanted enzyme activities may be removed by heat treatment. Different
enzymes have differing susceptibility to heat denaturation and precipitation. Where the enzyme
required is relatively heat-stable this allows its easy and rapid purification in terms of enzymic
activity. For such enzymes heat-treatment is always considered as an option at an early stage in
their purification. This method has been particularly successfully applied to the production of
glucose isomerase, where a short incubation at a relatively high temperature is used (e.g. 60 -
85°C for 10 min). No interfering activity remains after this treatment and the heat-treated, and
hence leaky, cells may be immobilised and used directly.
CHROMATOGRAPHY
Enzyme preparations that have been clarified and concentrated are now in a suitable state for
further purification by chromatography. For enzyme purification there are three principal types of
chromatography utilising the ion-exchange, affinity and gel exclusion properties of the enzyme,
usually in that order. Ion-exchange and affinity chromatographic methods can both rapidly handle
large quantities of crude enzyme but ion-exchange materials are generally cheaper and, therefore,
preferred at an earlier stage in the purification where the scale of operation is somewhat greater.
Gel exclusion chromatography (also sometimes called 'gel filtration' or just 'gel chromatography'
although it does not separate by a filtering mechanism, larger molecules passing more rapidly
through the matrix than smaller molecules) is relatively slow and has the least capacity and
resolution. It is generally left until last as an important final purification step and also as a method
of changing the solution buffer before concentration, finishing and sale. Where sufficient
information has been gathered regarding the size and variation of charge with pH of the required
enzyme and its major contaminants, a rational purification scheme can be devised. A relatively
quick analytical method for obtaining such data utilises a two-dimensional process whereby
electrophoresis occurs in one direction and a range of pH is produced in the other; movement in
the electric field determined by the size and sign of a protein's charge, which both depend on the
pH. As the sample is applied across the range of pH, this method produces titration curves (i.e.
charge versus pH) for all proteins present.
A large effort has been applied to the development of chromatographic matrices suitable for the
separation of proteins. The main problem that has had to be overcome is that of ensuring the
matrix has sufficiently large surface area available to molecules as large as proteins (i.e. they are
macroporous) whilst remaining rigid and incompressible under rapid elution conditions. In
addition, matrices must generally be hydrophilic and inert. Although the standard bead diameters
of most of these matrices are non-uniform and fairly large (50 - 150 m), many are now
supplied as uniform-sized small beads (e.g. 4 - 6 m diameter) which allows their use in very
efficient separation processes (high performance liquid chromatography, HPLC), but at
exponentially increasing cost with decreasing bead size. Relatively high pressures are needed to
operate such columns necessitating specialised equipment and considerable additional expense.
They are used only for the small-scale production of expensive enzymes, where a high degree of
purity is required (e.g. restriction endonucleases and therapeutic enzymes).
Column manufacturers now supply equipment for monitoring and controlling chromatography
systems so that it is possible to have automated apparatus which loads the sample, collects
fractions and regenerates the column. Such equipment must, of course, have fail-safe devices to
protect both column and product.

ION-EXCHANGE CHROMATOGRAPHY
Enzymes possess a net charge in solution, dependent upon the pH and their structure and
isoelectric point. In solutions of pH below their isoelectric point they will be positively charged
and bind to cation exchangers whereas in solutions of pH above their isoelectric point they will
be negatively charged and bind to anion exchangers. The pH chosen must be sufficient to
maintain a high, but opposite, charge on both protein and ion-exchanger and the ionic strength
must be sufficient to maintain the solubility of the protein without the salt being able to
successfully compete with the protein for ion-exchange sites. The binding is predominantly
reversible and its strength is determined by the pH and ionic strength of the solution and the
structures of the enzyme and ion-exchanger. Normally the pH is kept constant and enzymes are
eluted by increasing the solution ionic strength. A very wide range of ion-exchange resins,
cellulose derivatives and large-pore gels are available for chromatographic use.
Ion-exchange materials are generally water insoluble polymers containing cationic or anionic
groups. Cation exchange matrices have anionic functional groups such as -SO3-, -OPO3- and
-COO- and anion exchange matrices usually contain the cationic tertiary and quaternary
ammonium groups, with general formulae -NHR2+ and -NR3+. Proteins become bound by
exchange with the associated counter-ions.
Ion-exchange polystyrene resins are eminently suitable for large-scale chromatographic use but
have low capacities for proteins due to their small pore size. Binding is often strong, due to the
resin hydrophobicity, and the conditions needed to elute proteins are generally severe and may be
denaturing. Nevertheless such resins are a potential means of concentrating or purifying enzymes.
Ion-exchange cellulose and large pore gels are much more generally suitable for enzyme
purification and, indeed, many were designed for that task. A variety of charged groups, anionic
or cationic, may be introduced. The practical level of substitution of cellulose is limited as
derivatisation above one mole per kilogram may lead to dissolution of the cellulose.
Consequently, proteins may be eluted from them under mild conditions. Ion-exchange cellulose
can be used in both batch and column processes but on a large scale they are used mainly
batchwise. This is because the increased speed of large-scale batchwise processing and the
avoidance of the deep-bed filtering characteristics of columns outweigh any advantage due to the
increase in resolution on columns. Careful preparation before use and regeneration after use is
essential for their effective use.
Batchwise operations involve stirring the pretreated and equilibrated ion-exchanger with the
enzyme solution in a suitable cooled vessel. Adsorption to the exchangers is usually rapid (e.g.
less than 30 minutes) but some proteins can take far longer to adsorb completely. Stirring is
essential but care must be taken not to generate fine particles (fines). Unadsorbed material may be
removed in a variety of manners. Basket centrifuges are a particularly convenient means of
hastening the removal of the initial supernatant and the elution of the adsorbed material. This is
usually done using stepwise increases in ionic strength and/or changes in pH but it is possible to
place the exchangers, plus adsorbed material, in a column and elute using a suitable gradient.
However, whilst ion-exchange cellulose are widely used for column chromatography on the
laboratory scale, their compressibility causes difficulty when attempts are made to use large scale
columns.
Some of the problems with derivatised cellulose may be overcome using more recently
introduced materials. Derivatives of cross-linked agarose (Sepharose CL-6B) and of the synthetic
polymer Trisacryl have high capacities (up to 150 mg protein ml -1) yet are not significantly
compressible. In addition, they do not change volume with pH and ionic strength which allows
them to be regenerated without removal from the chromatographic column.

AFFINITY CHROMATOGRAPHY
This is a term which now covers a variety of methods of enzyme purification, the common factor
of which is the more or less specific interaction between the enzyme and the immobilised ligand.
In its most specific form, the immobilised ligand is a substrate or competitive inhibitor of the
enzyme. Ideally it should be possible to purify an enzyme from a complex mixture in a single
step and, indeed, purification factors of up to several thousand-fold have been achieved. An
alternative, equally specific approach is to use an antibody to the enzyme as the ligand. Such
specific matrices, though, are very expensive and cannot be generally employed on a large scale.
Additionally, they often do not perform as well as might be expected due to non-specific binding
effects. In general, affinity chromatography achieves a higher purification factor (with a median
value in reported purifications of about ten fold) than ion-exchange chromatography (with a
median performance of about three fold), in spite of it generally being used at a later stage in the
purification when there is less purification possible.
A less specific approach, suitable for many enzymes, is to use analogues of coenzymes, such as
NAD+, as the ligand. This method has been used successfully but has now been superceded by the
employment of a series of water soluble dyes as ligands. These are much cheaper and, usually by
trial and error, have been found to have surprising degrees of specificity for a wide range of
enzymes. This dye-affinity chromatography was allegedly discovered by accident, certain
enzymes being found to bind to the blue-dyed dextran used, as a molecular weight standard, to
calibrate gel exclusion columns.
Another fortuitous discovery was hydrophobic interaction chromatography, found when it was
noted that certain proteins were unexpectedly retained on affinity columns containing
hydrophobic spacer arms. Hydrophobic adsorbents now available include octyl or phenyl groups.
Hydrophobic interactions are strong at high solution ionic strength so samples need not be
desalted before application to the adsorbent. Elution is achieved by changing the pH or ionic
strength or by modifying the dielectric constant of the eluent using, for instance, ethanediol. A
recent introduction is cellulose derivatised to introduce even more hydroxyl groups. This material
(Whatman HB1) is designed to interact with proteins by hydrogen bonding. Samples are applied
to the matrix in a concentrated (over 50% saturated, > 2M) solution of ammonium sulphate.
Proteins are eluted by diluting the ammonium sulphate. This introduces more water which
competes with protein for the hydrogen bonding sites. The selectivity of both of these methods is
similar to that of fractional precipitation using ammonium sulphate but their resolution may be
somewhat improved by their use in chromatographic columns rather than batchwise.
Careful choice of matrices for affinity chromatography is necessary. Particles should retain good
flow and porosity properties after attachment of the ligands and should not be capable of the non-
specific adsorption of proteins. Agarose beads fulfil these criteria and are readily available as
ligand supports (see also Chapter 3). Affinity chromatography is not used extensively in the
large-scale manufacture of enzymes, primarily because of cost. Doubtless as the relative costs of
materials are lowered, and experience in handling these materials is gained, enzyme
manufacturers will make increased use of these very powerful techniques.

GEL EXCLUSION CHROMATOGRAPHY

There is now a considerable choice of materials which can separate proteins on the basis of their
molecular size. The original cross-linked dextrans (Sephadex G- series, Pharmacia Ltd.) and
polyacrylamides (Bio-Gel P- series, BioRad Ltd.) are still, quite rightly, widely used. Both types
are available in a wide range of pore sizes and particle size distributions. However, as the pore
size increases, for use with larger enzymes, these gels become progressively less rigid and
therefore less suitable for large scale use. Consequently alternative, but generally more costly,
rigid gel materials have been developed for the fractionation of proteins of molecular weight
greater than about 75,000. These are the cross-linked derivatives of agarose (Sepharose CL and
Superose) and dextran (Sephacryl S) made by Pharmacia Ltd., the cross-linked polyacrylamide-
agarose mixtures (Ultrogel AcA) made by LKB Instruments Ltd. and the ethylene glycol-
methacrylate copolymers (Fractogel HW) made by Toyo Soda Company (TSK). These are
available in a range of forms capable of fractionating enzymes, and other materials, with
molecular weights up to 108 and at high flow rates. Although these gels are described as 'rigid', it
should be appreciated that this is a relative term. The best gels are significantly compressible so
scale-up from laboratory sized columns cannot be achieved by producing longer columns. Scale-
up is achieved by increasing the diameter of columns (up to about 1 m diameter) but retaining the
small depth. Further scale-up is done by connecting such sections in series to produce 'stacks'.
Extreme care must be taken in packing all gel columns so as to allow even, well distributed, flow
throughout the gel bed. For the same reason, the end pieces of the columns must allow even
distribution of material over the whole surface of the column. The newer materials are supplied in
a pre-swollen state which enable their rapid and efficient packing using slight pressure.
Gel exclusion chromatography invariably causes dilution of the enzyme which must then be
concentrated using one of the methods described earlier.
ENZYME BIOSENSORS

A biosensor is an analytical device which employs a biological material (enzyme, antibody,

nucleic acid, hormone, organelle or whole cell) to specifically interact with an analyte; this
interaction produces some detectable physical change which is measured and converted into an
electrical signal by a transducer. Finally, the electrical signal is amplified. interpreted and
displayed as analyte concentration in the solution / preparation. An ‘analyte’ is a compound
whose concentration is to be determined, in this case, by the biosensor.
Table 11.6 A list of some biological materials and optically or electronically active devices
commonly used in biosensors.
Biological Detection device (or transducer) Example
Material
Enzymes 1. Potentiometric Electrodes 1. Enzyme electrode for urea (based on urease)
Nucleic Acids Amperometric electrodes 2. Enzyme electrode for organophosphorus
pesticides (using acetyl cholinesterase)
2. Amperometric electrodes Glucose Biosensor (Based on glucose
oxidase)
Antibodies Wave guides (Optical biosensors)
Lectins Grating Couplers (Optical biosensors)
Cells Acoustic Wave Sensors
Organs Conductimetric sensors
Tissue Slices Thermometric Sensors
The nature of interaction between the aggregate and the biological material used in the biosensor
may be of two types.
i. The analyte may be converted into a new chemical molecule ( by enzymes; such
biosensors are called catalytic bio-sensors), and
ii. The analyte may simply bind to the biological material present on the biosensor are
known as affinity biosensors)

A Successful biosensor must have at least some of the following features.

i.It should be highly specific for the analyte,
ii. The reaction used should be as independent of factors like stirring, PH, temperature, etc., as
is manageable.
iii. The response should be linear over a useful range of analyte concentrations.
iv. The device should be tiny and bio-compatible, in case it is to be used for analyses within the
body.
v. The device should be cheap, small and easy to use, and
vi. It should be durable, i.e., should be capable of repeated use.

The biological component interacts specifically to the analyte which produces a physical change
close to the transducer surface. This physical change may be
i. Heat released or absorbed by the reaction (measured by calometric biosensors)
ii. Production of an electrical potential due to changed distribution of electrons (Potentiometric
biosensors)
iii. Movement of electrons due to redox reaction (amperometric biosensors)
iv. Light produced or absorbed during the reaction (Optical biosensors), or
v. Change in mass of the biological component as a result of the reaction (acoustic wave
biosensors).
The transducer detects and measures this change and converts it into an electrical signal. This
signal is necessarily very small, and is amplified by an amplifier before it is fed into the
microprocessor. The signal is then processed and interpreted, and is displayed in suitable units.
Thus biosensors convert a chemical information flow into an electrical information flow, which
involves the following steps.

GENERAL FEATURES
A biosensor has two distinct types of components.
i) Biological, e.g enzyme antibody, etc., and
ii) Physical, e.g transducer, amplifier etc (fig.1)

The biological component of biosensor performs two key functions.

i) It specifically recognizes the analyte, and
ii) Interacts it which it in such a manner which produces some physical change detectable by
the transducer.
These properties of the biological component impart on the biosensor its specifically sensitivity
and the ability to detect and measure the analyte.

DIAGRAM
Fig 11.10. A schematic representation of the various components of a biosensor. The biological
component may be enzyme, nucleic acid, antibody etc., the analyte must be transported from the
solution to the biological component for the reaction; ordinarily the transport is due to simple
diffusion.

1. The analyte diffuses from the solution to the surface of the biosensor.
2. The analyte reacts specifically and efficiently with the biological component of the
biosensor.
3. This reaction changes the physico-chemical properties of the transducer surface.
4. This leads to a change in the optical or electronic properties of the transducer surface.
5. The change in optical / electronic properties is measured, converted into electrical
signal which is amplified, processed and displayed.
6.
DESIGN OF ENZYME ELECTRODES
The term enzymatic electro catalysis has been used to definite the synergy between the
electrochemical and enzymatic oxido-electrochemical and the enzymatic sites are close at the
molecular level. The evolution of this concept has resulted in the deposition of chemically
modified enzymes onto electrodes, which have become known as “Biosensors” or “enzyme
electrodes”. The product of the enzymatic reaction is oxidized or reduced at this surface. Here, a
new concept must be mentioned concerning the interaction between the active site of the enzyme
and the electrode. This is the occurrence of an electron transfer directly between the active site at
the enzyme and the electrode (direct transfer) or between a conducting polymer and the active site
of the enzyme entrapped into it. In the last case, the mode of the electron transfer is:
Active site of the enzyme  Conductive polymer electrode.
Normally, the coupling of the enzymatic and electrochemical reactions when the enzyme
is covalently linked on the electrode surface, is occurring without intervention of direct electron
transfer. Typically the enzyme is an oxido reduetase which catalyses the oxidation or reduction
of the substrate into the product and at the same time the reduction or oxidation of the co
substrate or the coenzyme which react with the electrode.
Examples of enzymatic electro catalysis systems using a carbon electrode is the enzyme /
substrate / co substrate combinations of glucose oxidase / glucose / oxygen, lactic
dehydrogenase / lactate / NAD / hydrogenase / hydrogen
General Approach for the Immobilisation of Enzymes onto Electrodes.
To optimize the proximity between the enzyme and the electrode, an enzyme monolayer is
covalently linked with the electrode surface by a two step procedure involving carbodiimide as
condensation reagent. On carbon electrodes an easy way to introduce functional groups
particularly carboxylic groups is as electrochemical oxidation of the surface. The carboxylic
groups of the electrode surface are then activated by carbodiimide. After rinsing to eliminate the
excess of reagent, the electrodes are so asked in the solution of enzyme to perform the reaction
between the activated groups of the surface of the electrode and the amine groups of the enzyme

Measurement of enzymatic activity

The activity of the enzyme determines the current across the carbon electrode which
reflects the electron exchange. To have a good sensitivity, it is preferable to work at a fixed
potential imposed between the carbon electrode and a reference is such as a saturated calomel
(electrode) with a potentiostat.
A third electrode, an auxiliary platinum electrode is added to give a three electrode
configuration
In the presence of the substrate of the enzymatic reaction, a flux balance is established at the
interface of the carbon electrode between diffusion, enzymatic reaction and electrochemical
reaction. The time needed to establish this flux balance is equivalent to the time of establishment
of a diffusion – convention layer. This takes only a fraction of a second in the case of a rotating
carbon disk electrode in example of catalytic activity is the case of glucose oxidase immobilized
on a rotating disk electrode of glassy carbon the enzyme catalyses the transformation of D-
Glucose into gluconic acid with the reduction of FAD prosthetic group of the enzyme. The
FADH2 can be reoxidised by oxygen or other co substrate such as benzoquinone with the
production of electrons.

D - Glucose + Enzyme – FAD Gluconic Acid + Enzyme – FADH2

E – FAD H2 + Benzoquinone  FAD + Hydroquinone
Hydroquinone ----------> Benzoquinone + 2H++2C-
Instantaneous catalytic current is then observed as a function of the concentration of the
immobilized enzyme and glucose at the surface of the electrode.
TYPES OF BIOSENSORS
A great majority have immobilized enzymes. The performance of the biosensors is
mostly dependent on the specificity and sensitivity of the biological reaction, besides the stability
of the enzyme.
GENERAL FEATURES OF BIOSENSORS
A. biosensor has two distinct components (Fig. 21.73).
1. Biological component – enzyme, cell etc.
2. Physical component – transducer, amplifier etc.
The biological component recognizes and interacts with he analyte to produce a physical
change (a signal) that can be detected by the transducer. In practice, the biological material is
appropriately immobilized on to the transducer and the so prepared biosensors can be repeatedly
used several times (may be around 10,000 times) for a long period (many months).
Principle of a biosensor
The desired biological material (usually a specific enzyme) is immobilized by
conventional methods (physical or membrane entrapment, non-covalent material is in intimate
contact with the transducer. The analyte binds to the biological material to form a bound analyte
which in turn produces the electronic response that can be measured.
BIOLOGICAL MATERIAL + ANALYTE
In some instances, the analyte is converted to a product which may be associated with the
release of heat, gas (oxygen), electrons or hydrogen ions. The transducer can convert the product
linked changes into electrical signals which can be amplified and measured.
APPLICATIONS OF BIOSENSORS
 Biosensors have become very popular in recent years. They are widely used in various
fields. Biosensors are small in size and can be easily handled. They are specific and sensitive,
and work in a cost-effective manner. The tentative market share of biosensor applications is
given in Table 21.9 some of the important applications of biosensors are broadly described
hereunder.
APPLICATIONS IN MEDICINE AND HEALTH
Biosensors are successfully used for the quantitative estimation of several biologically
important substances in body fluids e.g. glucose, cholesterol, urea. Glucose biosensor is a boon
for diabetic patients for regular monitoring of blood glucose. Blood gas monitoring for pH, pCO 2
and pO2 and pO2 is carried out during critical care and surgical monitoring of patients.
Mutagenicity of several chemicals can be determined by using biosensors. Several toxic
compounds produced in the body can also can be detected.
APPLICATIONS IN INDUSTRY
Biosensors can be used for monitoring of fermentation products and estimation of various
ions. Thus, biosensors help for improving the fermentation conditions for a better yield.
Now a days, biosensors are employed to measure the odour and freshness of foods. For
instance, freshness of stored fish can be detected by ATPase. ATP is not found in spoiled fish
and this can be detected by using AT Pase. One pharmaceutical company has developed
immobilized cholesterol oxidase system for measurement of cholesterol concentration in foods
(e.g. butter).

Area of Application Market Share (%)

Medical and Health 60%
Industry 10%
Agriculture and veterinary 8%
Defense 7%
Environmental 6%
Research 4%
Robotics 3%
Others 2%

APPLICATIONS IN POLLUTION CONTROL

Biosensors are very helpful to monitor environmental (air, water) pollution. The
concentrations of pesticides and the biological oxygen demand (BOD) can be measured by
biosensors. Several environmental pollutants can be evaluated for their mutagenicity by
employing biosensors. For more details or biosensors to monitor environment
APPLICATIONS IN MILITARY
Biosensors have been developed to detect the toxic gases and other chemical agents used
during war. There are several limitations for the direct use of enzymes for therapeutic purposes.
These include the poor availability of the enzyme at the site of action, sensitivity to nature
inhibitors, degradation by endogenous proteases and immunogenicity of certain enzymes.
APPLICATIONS OF BIOSENSORS IN INDUSTRY HEALTH CARE AND ENVIRONMENT
Biosensors have become very popular in recent years. They are uridely used in various
fields. Biosensors are small in size and can be easily handled. They are specific and sensitive,
work in a cost effective manner, so they are put into many practical uses in medicine, industries
and environmental control some of the important applications of biosensors are broadly described
hereunder.
APPLICATIONS IN MEDICINE AND HEALTH
Biosensors are successfully used for the quantitative estimation of several biological
important substances in body fluids eg. Glucose, cholesterol, urea.
 Glucose biosensor is a boon for diabetic patients for regular monitoring of blood glucose
 Blood gas monitoring for pH, pCO2 and pO2 is carried out during critical care and
surgical monitoring of patients.
 Mutagenicity of several chemical can be determined by using biosensors. several toxic
compounds produced in the body can also be detected.
 APPLICATIONS IN INDUSTRY
Biosensors can be used for monitoring of fermentation products and estimation of various ions.
Biosensors are suitable for on-line measurements of certain compounds in continuous industrial
process. As they are very sensitive and quick in action, they have been put into many practical
uses in industries.
 A microbial biosensor made up of Immobilized cells of pseudomonas fluorescence
and O2 electrode, is used to detect glucose level in molasses.
 The total amount of assimilable sugar in fermentation media and broth can be
measured with a microbial sensor. The biosensor is made up of an immobilized layer of
Brevibacterium lacto fermentum and an O2 electrode.
 An acetic acid sensor is made combining an immobilized layer of yeast Trichosporon
brassiea and an O2 electrode. It detects acetic acid level in fermentation broths for glutamic acid
within 6-10 minutes.
Thus, biosensors help for improving the fermentation conditions for a better field.
Now a days, biosensors are employed to measure the odour and freshness of foods.
 APPLICATIONS IN POLLUTION CONTROL
Some of the important biosensors used in environment pollution monitoring are briefly
described.
 BOD BIOSENSOR
Biological oxygen demand (BOD) is a widely used test for the detection of organic
pollution. This test requires five days of incubation. A BOD biosensor using the yeast
Trichospoton cutancum with oxygen probe takes just 15 minutes for determining organic
pollution
 GAS BIO SENSORS
Microbial biosensors for the detection of gases such as sulfur dioxide (SO 2) methane and
carbon dioxide have been developed. Thiobacillus - based biosensor can detect the pollutant SO 2
while methane (CH4) can be detected by immobilized methalomonas for carbon dioxide
monitoring, a particular strain of pseudomonas is used.
 IMMUNINOASSAY BIOSENSORS
Immunoelectrodes as biosensors are useful for the detection of low concentration of
pollutants. Pesticide specific antibodies can detect the presence of low concentrations of triazines,
malathion and carbamates, by employing immuno assays.
 For instance, freshness of stored fish can be detected by Atpase. APT is not
found in spoiled fish ad this can be detected by using ATPase.
The cell number in the fermentation broth and various food items can be measured using fuel cell
type biosensor. This biosensor consists of two electrodes, each of which is made of a platinum
anode and silver peroxide cathode. The electrochemical changes caused by the microbes in the
analyze are used to measure the cell number of saccharomyces cerevisiae and lactobacillus
fermentation.
 The cell number of Bacillus subtitles in fermentation broth can be measured with a
potentiometer biosensor.
 A lactate sensor is used to count lactic acid producing bacteria in fermentation media.
 An alcohol sensor made up of an immobilized layer of Trichosporon brassieac and an O2
electrode used to measure methyl - OH and ethyl -OH in fermentation broth and beverages.
 An vitamin B, sensor is made by immobilization of lactobacillus fermentation a platinum
electrode. It measures vit B1 level in fermentation broth.
OTHER BIOSENSORS
Biosensors employing acetylcholine esterase (obtained from bovine RBC) can be used
for the detection of organ phosphorus compounds in water. In fact, portable pesticide monitors
are commercially available in some developed countries.
Biosensors for the detection of poly chlorinated biphenyls (PCBs) and chlorinated
hydrocarbons and certain other organic compounds have been developed.
Phenol oxidize enzyme (obtained from potatoes and mushrooms) containing biosensors is
used for the detection of phenol. A graphite electrode with cynobacterium and synechococurss
has been developed to measure the degree of electron transport inhibition during photosynthesis
due to certain pollutants, eg herbicides.
A selected list of environmental pollutants measured by employing biosensors is given as
follows,
Pollutant measured Biosensor - Biological component
 BOD Trichosporon Cutaneum
 SO2 Thiobacillus sp
 CH4 Methanomonas flagellae
 CO2 Pseudomonas sp
 Nitrate Azobacter Vinelandi
 NH3 and NO2 Mixed Nitrifying bacteria
 Ethanol NADH and dehydrogenase
 Phenol Phenol oxidase
 Parathion Antibody to parathion.
POTENTIAL APPLICATION OF BIOSENSOR
There are many potential application of biosensors of various types. Biosensors are
widely used in research and commercial applications. Some examples are given below:
FIELD APPLICATION
biomedical and life  Self-Monitoring –eg: Glucose Testing ;lactic Acid and Other
sciences Tests
Continuous medical monitoring –eg: Drug Delivery and Drug
Development
life sciences Proteomics, Genomics, Microbiology, Toxicology, Oncology, Other
research Medical Research Application: Veterinary Medicine
food production Measuring Ripeness, contaminant/pathogen detection, Process/quality
Control, Detection of Genetically Modified Organisms in Food

forensics DNA identification

environmental Pesticide analysis; organophosphate and other contaminants
monitoring and
remediation
enzyme biosensors For detection of
by target analyte Aspartame,Choline,Creatinine,Ethonal,Formaldehyde,Glucose,Glutamine,
Hypoxanthine ,Lactic Acid ,Nitrite,Penicillin ,Phenol,Urea
protein biosensors  Genomics
and dna bosensors  Diagnosis of cancer and other diseases
 Drug Discovery
 Food Quality Assurance
 Public Health
 Military / Homeland Defense
 Forensics
hologram -- infectious agent sensor, glucose sensor, pH sensor, alcohol sensor, and
biosensor a sensor for water in solvents. The first-ever sensor capable of detecting
live (infective) organisms.

GLUCOSE BIOSENSOR
The most widespread example of a commercial biosensor is the blood glucose biosensor,
which uses an enzyme to break blood glucose down. In so doing it transfers an electron to an
electrode and this is converted into a measure of blood glucose concentration. The high market
demand for such sensors has fueled development of associated sensor technologies.

The use of enzymes in analysis

Enzymes make excellent analytical reagents due to their specificity, selectivity and efficiency.
They are often used to determine the concentration of their substrates (as analytes) by means of
the resultant initial reaction rates. If the reaction conditions and enzyme concentrations are kept
constant, these rates of reaction (v) are proportional to the substrate concentrations ([S]) at low
substrate concentrations. When [S] < 0.1 Km, equation 1.8 simplifies to give
v = (Vmax/Km)[S] (6.1)
The rates of reaction are commonly determined from the difference in optical absorbance
between the reactants and products. An example of this is the - D-galactose dehydrogenase (EC
1.1.1.48) assay for galactose which involves the oxidation of galactose by the redox coenzyme,
nicotine-adenine dinucleotide (NAD+).
-D-galactose + NAD+ D-galactono-1,4-lactone + NADH + H+ [6.1]
A 0.1 mM solution of NADH has an absorbance at 340nm, in a 1 cm path-length cuvette, of
0.622, whereas the NAD+ from which it is derived has effectively zero absorbance at this
wavelength. The conversion (NAD+ NADH) is, therefore, accompanied by a large
increase in absorption of light at this wavelength. For the reaction to be linear with respect to the
galactose concentration, the galactose is kept within a concentration range well below the Km of
the enzyme for galactose. In contrast, the NAD+ concentration is kept within a concentration
range well above the Km of the enzyme for NAD+, in order to avoid limiting the reaction rate.
Such assays are commonly used in analytical laboratories and are, indeed, excellent where a wide
variety of analyses need to be undertaken on a relatively small number of samples. The
drawbacks to this type of analysis become apparent when a large number of repetitive assays
need to be performed. Then, they are seen to be costly in terms of expensive enzyme and
coenzyme usage, time consuming, labour intensive and in need of skilled and reproducible
operation within properly equipped analytical laboratories. For routine or on-site operation, these
disadvantages must be overcome. This is being achieved by the production of biosensors which
exploit biological systems in association with advances in micro-electronic technology.

What are biosensors?

A biosensor is an analytical device which converts a biological response into an electrical signal
(Figure 6.1). The term 'biosensor' is often used to cover sensor devices used in order to determine
the concentration of substances and other parameters of biological interest even where they do
not utilise a biological system directly. This very broad definition is used by some scientific
journals (e.g. Biosensors, Elsevier Applied Science) but will not be applied to the coverage here.
The emphasis of this Chapter concerns enzymes as the biologically responsive material, but it
should be recognised that other biological systems may be utilised by biosensors, for example,
whole cell metabolism, ligand binding and the antibody-antigen reaction. Biosensors represent a
rapidly expanding field, at the present time, with an estimated 60% annual growth rate; the major
impetus coming from the health-care industry (e.g. 6% of the western world are diabetic and
would benefit from the availability of a rapid, accurate and simple biosensor for glucose) but with
some pressure from other areas, such as food quality appraisal and environmental monitoring.
The estimated world analytical market is about £12,000,000,000 year -1 of which 30% is in the
health care area. There is clearly a vast market expansion potential as less than 0.1% of this
market is currently using biosensors. Research and development in this field is wide and
multidisciplinary, spanning biochemistry, bioreactor science, physical chemistry,
electrochemistry, electronics and software engineering. Most of this current endeavour concerns
potentiometric and amperometric biosensors and colorimetric paper enzyme strips. However, all
the main transducer types are likely to be thoroughly examined, for use in biosensors, over the
next few years.
A successful biosensor must possess at least some of the following beneficial features:
1. The biocatalyst must be highly specific for the purpose of the analyses, be stable under
normal storage conditions and, except in the case of colorimetric enzyme strips and
dipsticks (see later), show good stability over a large number of assays (i.e. much greater
than 100).
2. The reaction should be as independent of such physical parameters as stirring, pH and
temperature as is manageable. This would allow the analysis of samples with minimal
pre-treatment. If the reaction involves cofactors or coenzymes these should, preferably,
also be co-immobilised with the enzyme (see Chapter 8).
3. The response should be accurate, precise, reproducible and linear over the useful
analytical range, without dilution or concentration. It should also be free from electrical
noise.
4. If the biosensor is to be used for invasive monitoring in clinical situations, the probe must
be tiny and biocompatible, having no toxic or antigenic effects. If it is to be used in
fermenters it should be sterilisable. This is preferably performed by autoclaving but no
biosensor enzymes can presently withstand such drastic wet-heat treatment. In either
case, the biosensor should not be prone to fouling or proteolysis.
5. The complete biosensor should be cheap, small, portable and capable of being used by
semi-skilled operators.
6. There should be a market for the biosensor. There is clearly little purpose developing a
biosensor if other factors (e.g. government subsidies, the continued employment of
skilled analysts, or poor customer perception) encourage the use of traditional methods
and discourage the decentralisation of laboratory testing.
The biological response of the biosensor is determined by the biocatalytic membrane which
accomplishes the conversion of reactant to product. Immobilised enzymes possess a number of
advantageous features which makes them particularly applicable for use in such systems. They
may be re-used, which ensures that the same catalytic activity is present for a series of analyses.
This is an important factor in securing reproducible results and avoids the pitfalls associated with
the replicate pipetting of free enzyme otherwise necessary in analytical protocols. Many enzymes
are intrinsically stabilised by the immobilisation process (see Chapter 3), but even where this
does not occur there is usually considerable apparent stabilisation. It is normal to use an excess of
the enzyme within the immobilised sensor system. This gives a catalytic redundancy (i.e.  <<
1) which is sufficient to ensure an increase in the apparent stabilisation of the immobilised
enzyme (see, for example, Figures 3.11, 3.19 and 5.8). Even where there is some inactivation of
the immobilised enzyme over a period of time, this inactivation is usually steady and predictable.
Any activity decay is easily incorporated into an analytical scheme by regularly interpolating
standards between the analyses of unknown samples. For these reasons, many such immobilised
enzyme systems are re-usable up to 10,000 times over a period of several months. Clearly, this
results in a considerable saving in terms of the enzymes' cost relative to the analytical usage of
free soluble enzymes.
When the reaction, occurring at the immobilised enzyme membrane of a biosensor, is limited by
the rate of external diffusion, the reaction process will possess a number of valuable analytical
assets. In particular, it will obey the relationship shown in equation 3.27. It follows that the
biocatalyst gives a proportional change in reaction rate in response to the reactant (substrate)
concentration over a substantial linear range, several times the intrinsic K m (see Figure 3.12 line
e). This is very useful as analyte concentrations are often approximately equal to the Kms of their
appropriate enzymes which is roughly 10 times more concentrated than can be normally
determined, without dilution, by use of the free enzyme in solution. Also following from equation
3.27 is the independence of the reaction rate with respect to pH, ionic strength, temperature and
inhibitors. This simply avoids the tricky problems often encountered due to the variability of real
analytical samples (e.g, fermentation broth, blood and urine) and external conditions. Control of
biosensor response by the external diffusion of the analyte can be encouraged by the use of
permeable membranes between the enzyme and the bulk solution. The thickness of these can be
varied with associated effects on the proportionality constant between the substrate concentration
and the rate of reaction (i.e. increasing membrane thickness increases the unstirred layer ( )
which, in turn, decreases the proportionality constant, kL, in equation 3.27). Even if total
dependence on the external diffusional rate is not achieved (or achievable), any increase in the
dependence of the reaction rate on external or internal diffusion will cause a reduction in the
dependence on the pH, ionic strength, temperature and inhibitor concentrations.

Figure 6.1. Schematic diagram showing the main components of a biosensor. The biocatalyst (a)
converts the substrate to product. This reaction is determined by the transducer (b) which
converts it to an electrical signal. The output from the transducer is amplified (c), processed (d)
and displayed (e).

The key part of a biosensor is the transducer (shown as the 'black box' in Figure 6.1) which makes
use of a physical change accompanying the reaction. This may be
1. the heat output (or absorbed) by the reaction (calorimetric biosensors),
2. changes in the distribution of charges causing an electrical potential to be produced
(potentiometric biosensors),
3. movement of electrons produced in a redox reaction (amperometric biosensors),
 4. light output during the reaction or a light absorbance difference between the reactants and
products (optical biosensors), or
5. effects due to the mass of the reactants or products (piezo-electric biosensors).
There are three so-called 'generations' of biosensors; First generation biosensors where the normal
product of the reaction diffuses to the transducer and causes the electrical response, second
generation biosensors which involve specific 'mediators' between the reaction and the transducer
in order to generate improved response, and third generation biosensors where the reaction itself
causes the response and no product or mediator diffusion is directly involved.
The electrical signal from the transducer is often low and superimposed upon a relatively high
and noisy (i.e. containing a high frequency signal component of an apparently random nature, due
to electrical interference or generated within the electronic components of the transducer)
baseline. The signal processing normally involves subtracting a 'reference' baseline signal,
derived from a similar transducer without any biocatalytic membrane, from the sample signal,
amplifying the resultant signal difference and electronically filtering (smoothing) out the
unwanted signal noise. The relatively slow nature of the biosensor response considerably eases
the problem of electrical noise filtration. The analogue signal produced at this stage may be
output directly but is usually converted to a digital signal and passed to a microprocessor stage
where the data is processed, converted to concentration units and output to a display device or
data store.

Calorimetric biosensors
Many enzyme catalysed reactions are exothermic, generating heat (Table 6.1) which may be used
as a basis for measuring the rate of reaction and, hence, the analyte concentration. This represents
the most generally applicable type of biosensor. The temperature changes are usually determined
by means of thermistors at the entrance and exit of small packed bed columns containing
immobilised enzymes within a constant temperature environment (Figure 6.2). Under such
closely controlled conditions, up to 80% of the heat generated in the reaction may be registered as
a temperature change in the sample stream. This may be simply calculated from the enthalpy
change and the amount reacted. If a 1 mM reactant is completely converted to product in a
reaction generating 100 kJ mole-1 then each ml of solution generates 0.1 J of heat. At 80%
efficiency, this will cause a change in temperature of the solution amounting to approximately
0.02°C. This is about the temperature change commonly encountered and necessitates a
temperature resolution of 0.0001°C for the biosensor to be generally useful.
Table 6.1. Heat output (molar enthalpies) of enzyme catalysed reactions.
Reactant Enzyme Heat output
Cholesterol Cholesterol oxidase 53
Esters Chymotrypsin 4 - 16
Glucose Glucose oxidase 80
Hydrogen peroxide Catalase 100
Penicillin G Penicillinase 67
Peptides Trypsin 10 - 30
Starch Amylase 8
Sucrose Invertase 20
Urea Urease 61
Uric acid Uricase 49
Figure 6.2. Schematic diagram of a calorimetric biosensor. The sample stream (a) passes through
the outer insulated box (b) to the heat exchanger (c) within an aluminium block (d). From there, it
flows past the reference thermistor (e) and into the packed bed bioreactor (f, 1ml volume),
containing the biocatalyst, where the reaction occurs. The change in temperature is determined by
the thermistor (g) and the solution passed to waste (h). External electronics (l) determines the
difference in the resistance, and hence temperature, between the thermistors.

The thermistors, used to detect the temperature change, function by changing their electrical
resistance with the temperature, obeying the relationship

(6.2)
therefore:

(6.2b)
where R1 and R2 are the resistances of the thermistors at absolute temperatures T1 and T2
respectively and B is a characteristic temperature constant for the thermistor. When the
temperature change is very small, as in the present case, B(1/T 1) - (1/T2) is very much smaller
than one and this relationship may be substantially simplified using the approximation when
x<<1 that ex1 + x (x here being B(1/T1) - (1/T2),

(6.3)
As T1  T2, they both may be replaced in the denominator by T1.

(6.4)
The relative decrease in the electrical resistance ( R/R) of the thermistor is proportional to the
increase in temperature (T). A typical proportionality constant (-B/T12) is -4%°C-1. The
resistance change is converted to a proportional voltage change, using a balanced Wheatstone
bridge incorporating precision wire-wound resistors, before amplification. The expectation that
there will be a linear correlation between the response and the enzyme activity has been found to
be borne out in practice. A major problem with this biosensor is the difficulty encountered in
closely matching the characteristic temperature constants of the measurement and reference
thermistors. An equal movement of only 1°C in the background temperature of both thermistors
commonly causes an apparent change in the relative resistances of the thermistors equivalent to
0.01°C and equal to the full-scale change due to the reaction. It is clearly of great importance that
such environmental temperature changes are avoided, which accounts for inclusion of the well-
insulated aluminium block in the biosensor design (see Figure 6.2).
The sensitivity (10-4 M) and range (10-4 - 10-2 M) of thermistor biosensors are both quite low for
the majority of applications although greater sensitivity is possible using the more exothermic
reactions (e.g. catalase). The low sensitivity of the system can be increased substantially by
increasing the heat output by the reaction. In the simplest case this can be achieved by linking
together several reactions in a reaction pathway, all of which contribute to the heat output. Thus
the sensitivity of the glucose analysis using glucose oxidase can be more than doubled by the co-
immobilisation of catalase within the column reactor in order to disproportionate the hydrogen
peroxide produced. An extreme case of this amplification is shown in the following recycle
scheme for the detection of ADP.
ADP is the added analyte and excess glucose, phosphoenol pyruvate, NADH and oxygen are
present to ensure maximum reaction. Four enzymes (hexokinase, pyruvate kinase, lactate
dehydrogenase and lactate oxidase) are co-immobilised within the packed bed reactor. In spite of
the positive enthalpy of the pyruvate kinase reaction, the overall process results in a 1000 fold
increase in sensitivity, primarily due to the recycling between pyruvate and lactate. Reaction
limitation due to low oxygen solubility may be overcome by replacing it with benzoquinone,
which is reduced to hydroquinone by flavo-enzymes. Such reaction systems do, however, have
the serious disadvantage in that they increase the probability of the occurrence of interference in
the determination of the analyte of interest. Reactions involving the generation of hydrogen ions
can be made more sensitive by the inclusion of a base having a high heat of protonation. For
example, the heat output by the penicillinase reaction may be almost doubled by the use of Tris
(tris-(hydroxymethyl)aminomethane) as the buffer.In conclusion, the main advantages of the
thermistor biosensor are its general applicability and the possibility for its use on turbid or
strongly coloured solutions. The most important disadvantage is the difficulty in ensuring that the
temperature of the sample stream remains constant (± 0.01°C).
 Potentiometric biosensors
Potentiometric biosensors make use of ion-selective electrodes in order to transduce the
biological reaction into an electrical signal. In the simplest terms this consists of an immobilised
enzyme membrane surrounding the probe from a pH-meter (Figure 6.3), where the catalysed
reaction generates or absorbs hydrogen ions (Table 6.2). The reaction occurring next to the thin
sensing glass membrane causes a change in pH which may be read directly from the pH-meter's
display. Typical of the use of such electrodes is that the electrical potential is determined at very
high impedance allowing effectively zero current flow and causing no interference with the
reaction.
Figure 6.3. A simple potentiometric biosensor. A semi-permeable membrane (a) surrounds the
biocatalyst (b) entrapped next to the active glass membrane (c) of a pH probe (d). The electrical
potential (e) is generated between the internal Ag/AgCl electrode (f) bathed in dilute HCl (g) and
an external reference electrode (h).

There are three types of ion-selective electrodes which are of use in biosensors:
1. Glass electrodes for cations (e.g. normal pH electrodes) in which the sensing element is a
very thin hydrated glass membrane which generates a transverse electrical potential due
to the concentration-dependent competition between the cations for specific binding sites.
The selectivity of this membrane is determined by the composition of the glass. The
sensitivity to H+ is greater than that achievable for NH4+,
2. Glass pH electrodes coated with a gas-permeable membrane selective for CO 2, NH3 or
H2S. The diffusion of the gas through this membrane causes a change in pH of a sensing
solution between the membrane and the electrode which is then determined.
3. Solid-state electrodes where the glass membrane is replaced by a thin membrane of a
specific ion conductor made from a mixture of silver sulphide and a silver halide. The
iodide electrode is useful for the determination of I- in the peroxidase reaction (Table
6.2c) and also responds to cyanide ions.

Table 6.2. Reactions involving the release or absorption of ions that may be utilised by
potentiometric biosensors.
(a) H+ cation,
glucose oxidase H2O
D-glucose + O2 D-glucono-1,5-lactone + H2O2 D-gluconate + H+ [6.3]
 penicillinase
penicillin penicilloic acid + H+ [6.4]
urease (pH 6.0)a
+ +
H2NCONH2 + H2O + 2H 2NH4 + CO2 [6.5]
urease (pH 9.5)b
H2NCONH2 + 2H2O 2NH3 + HCO3- + H+ [6.6]
lipase
neutral lipids + H2O glycerol + fatty acids + H+ [6.7]
+
(b) NH4 cation,
L-amino acid oxidase
L-amino acid + O2 + H2O keto acid + NH4+ + H2O2 [6.8]
asparaginase `
L-asparagine + H2O L-aspartate + NH4+ [6.9]
urease (pH 7.5)
H2NCONH2 + 2H2O + H+ 2NH4++ HCO3- [6.10]
(c) I- anion,
peroxidase
H2O2 + 2H+ + 2I- I2 + 2H2O [6.11]
(d) CN-anion,
-glucosidase
amygdalin + 2H2O 2glucose + benzaldehyde + H+ + CN- [6.12]
a +
Can also be used in NH4 and CO2 (gas) potentiometric biosensors.
b
Can also be used in an NH3 (gas) potentiometric biosensor.es80ll66bp
The response of an ion-selective electrode is given by

(6.5)
where E is the measured potential (in volts), E0 is a characteristic constant for the ion-
selective/external electrode system, R is the gas constant, T is the absolute temperature (K), z is
the signed ionic charge, F is the Faraday, and [i] is the concentration of the free uncomplexed
ionic species (strictly, [i] should be the activity of the ion but at the concentrations normally
encountered in biosensors, this is effectively equal to the concentration). This means, for
example, that there is an increase in the electrical potential of 59 mv for every decade increase in
the concentration of H+ at 25°C. The logarithmic dependence of the potential on the ionic
concentration is responsible both for the wide analytical range and the low accuracy and precision
of these sensors. Their normal range of detection is 10-4 - 10-2 M, although a minority are ten-fold
more sensitive. Typical response time are between one and five minutes allowing up to 30
analyses every hour.
Biosensors which involve H+ release or utilisation necessitate the use of very weakly buffered
solutions (i.e. < 5 mM) if a significant change in potential is to be determined. The relationship
between pH change and substrate concentration is complex, including other such non-linear
effects as pH-activity variation and protein buffering. However, conditions can often be found
where there is a linear relationship between the apparent change in pH and the substrate
concentration. A recent development from ion-selective electrodes is the production of ion-
selective field effect transistors (ISFETs) and their biosensor use as enzyme-linked field effect
transistors (ENFETs, Figure 6.4). Enzyme membranes are coated on the ion-selective gates of
these electronic devices, the biosensor responding to the electrical potential change via the current
output. Thus, these are potentiometric devices although they directly produce changes in the
electric current. The main advantage of such devices is their extremely small size (<< 0.1 mm2)
which allows cheap mass-produced fabrication using integrated circuit technology. As an
example, a urea-sensitive FET (ENFET containing bound urease with a reference electrode
containing bound glycine) has been shown to show only a 15% variation in response to urea (0.05
- 10.0 mg ml-1) during its active lifetime of a month. Several analytes may be determined by
miniaturised biosensors containing arrays of ISFETs and ENFETs. The sensitivity of FETs,
however, may be affected by the composition, ionic strength and concentrations of the solutions
analysed.

Figure 6.4. Schematic diagram of the section across the width of an ENFET. The actual
dimensions of the active area is about 500 m long by 50 m wide by 300 m thick. The main
body of the biosensor is a p-type silicon chip with two n-type silicon areas; the negative source
and the positive drain. The chip is insulated by a thin layer (0.1 m thick) of silica (SiO 2) which
forms the gate of the FET. Above this gate is an equally thin layer of H +-sensitive material (e.g.
tantalum oxide), a protective ion selective membrane, the biocatalyst and the analyte solution,
which is separated from sensitive parts of the FET by an inert encapsulating polyimide
photopolymer. When a potential is applied between the electrodes, a current flows through the
FET dependent upon the positive potential detected at the ion-selective gate and its consequent
attraction of electrons into the depletion layer. This current (I) is compared with that from a
similar, but non-catalytic ISFET immersed in the same solution. (Note that the electric current is,
by convention, in the opposite direction to the flow of electrons).
Amperometric biosensors

Amperometric biosensors function by the production of a current when a potential is applied

between two electrodes. They generally have response times, dynamic ranges and sensitivities
similar to the potentiometric biosensors. The simplest amperometric biosensors in common usage
involve the Clark oxygen electrode (Figure 6.5). This consists of a platinum cathode at which
oxygen is reduced and a silver/silver chloride reference electrode. When a potential of -0.6 V,
relative to the Ag/AgCl electrode is applied to the platinum cathode, a current proportional to the
oxygen concentration is produced. Normally both electrodes are bathed in a solution of saturated
potassium chloride and separated from the bulk solution by an oxygen-permeable plastic
membrane (e.g. Teflon, polytetrafluoroethylene). The following reactions occur:
Ag anode 4Ag0 + 4Cl- 4AgCl + 4e- [6.13]
Pt cathode O2 + 4H+ + 4e- 2H2O [6.14]
The efficient reduction of oxygen at the surface of the cathode causes the oxygen concentration
there to be effectively zero. The rate of this electrochemical reduction therefore depends on the
rate of diffusion of the oxygen from the bulk solution, which is dependent on the concentration
gradient and hence the bulk oxygen concentration (see, for example, equation 3.13). It is clear
that a small, but significant, proportion of the oxygen present in the bulk is consumed by this
process; the oxygen electrode measuring the rate of a process which is far from equilibrium,
whereas ion-selective electrodes are used close to equilibrium conditions. This causes the oxygen
electrode to be much more sensitive to changes in the temperature than potentiometric sensors. A
typical application for this simple type of biosensor is the determination of glucose concentrations
by the use of an immobilised glucose oxidase membrane. The reaction (see reaction scheme
[1.1]) results in a reduction of the oxygen concentration as it diffuses through the biocatalytic
membrane to the cathode, this being detected by a reduction in the current between the electrodes
(Figure 6.6). Other oxidases may be used in a similar manner for the analysis of their substrates
(e.g. alcohol oxidase, D- and L-amino acid oxidases, cholesterol oxidase, galactose oxidase, and
urate oxidase)

Figure 6.5. Schematic diagram of a simple amperometric biosensor. A potential is applied

between the central platinum cathode and the annular silver anode. This generates a current (I)
which is carried between the electrodes by means of a saturated solution of KCl. This electrode
compartment is separated from the biocatalyst (here shown glucose oxidase, GOD) by a thin
plastic membrane, permeable only to oxygen. The analyte solution is separated from the
biocatalyst by another membrane, permeable to the substrate(s) and product(s). This biosensor is
normally about 1 cm in diameter but has been scaled down to 0.25 mm diameter using a Pt wire
cathode within a silver plated steel needle anode and utilising dip-coated membranes.
An alternative method for determining the rate of this reaction is to measure the production of
hydrogen peroxide directly by applying a potential of +0.68 V to the platinum electrode, relative
to the Ag/AgCl electrode, and causing the reactions:
Pt anode H2O2 O2 + 2H+ + 2e- [6.15]
Ag cathode 2AgCl + 2e- 2Ag0 + 2Cl-[6.16]
The major problem with these biosensors is their dependence on the dissolved oxygen
concentration. This may be overcome by the use of 'mediators' which transfer the electrons
directly to the electrode bypassing the reduction of the oxygen co-substrate. In order to be
generally applicable these mediators must possess a number of useful properties.
1. They must react rapidly with the reduced form of the enzyme.
 2. They must be sufficiently soluble, in both the oxidised and reduced forms, to be able to
rapidly diffuse between the active site of the enzyme and the electrode surface. This
solubility should, however, not be so great as to cause significant loss of the mediator
from the biosensor's microenvironment to the bulk of the solution. However soluble, the
mediator should generally be non-toxic.
3. The overpotential for the regeneration of the oxidised mediator, at the electrode, should
be low and independent of pH.
4. The reduced form of the mediator should not readily react with oxygen.
The ferrocenes represent a commonly used family of mediators (Figure 6.7a). Their reactions
may be represented as follows,

[6.17]
Electrodes have now been developed which can remove the electrons directly from the reduced
enzymes, without the necessity for such mediators. They utilise a coating of electrically
conducting organic salts, such as N-methylphenazinium cation (NMP+, Figure 6.7b) with
tetracyanoquinodimethane radical anion (TCNQ.- Figure 6.7c). Many flavo-enzymes are strongly
adsorbed by such organic conductors due to the formation of salt links, utilising the alternate
positive and negative charges, within their hydrophobic environment. Such enzyme electrodes
can be prepared by simply dipping the electrode into a solution of the enzyme and they may
remain stable for several months. These electrodes can also be used for reactions involving
NAD(P)+-dependent dehydrogenases as they also allow the electrochemical oxidation of the
reduced forms of these coenzymes. The three types of amperometric biosensor utilising product,
mediator or organic conductors represent the three generations in biosensor development (Figure
6.8). The reduction in oxidation potential, found when mediators are used, greatly reduces the
problem of interference by extraneous material.

Figure 6.7. (a) Ferrocene ( 5- bis-cyclopentadienyl iron), the parent compound of a number of
mediators. (b) TMP+, the cationic part of conducting organic crystals. (c) TCNQ .-, the anionic part
of conducting. organic crystals. It is a resonance-stabilised radical formed by the one-electron
oxidation of TCNQH2
Figure 6.8. Amperometric biosensors for flavo-oxidase enzymes illustrating the three generations
in the development of a biosensor. The biocatalyst is shown schematically by the cross-hatching.
(a) First generation electrode utilising the H2O2 produced by the reaction. (E0 = +0.68 V). (b)
Second generation electrode utilising a mediator (ferrocene) to transfer the electrons, produced by
the reaction, to the electrode. (E0 = +0.19 V). (c) Third generation electrode directly utilising the
electrons produced by the reaction. (E0 = +0.10 V). All electrode potentials (E0) are relative to the
Cl-/AgCl,Ag0 electrode. The following reaction occurs at the enzyme in all three biosensors:
Substrate(2H) + FAD-oxidase Product + FADH2-oxidase [6.18]
This is followed by the processes:
(a)
biocatalyst
FADH2-oxidase + O2 FAD-oxidase + H2O2 [6.19]
electrode
H2O2 O2 + 2H+ + 2e- [6.20]
(b)
biocatalyst
FADH2-oxidase + 2 Ferricinium+ FAD-oxidase + 2 Ferrocene + 2H+ [6.21]
electrode
2 Ferrocene 2 Ferricinium+ + 2e- [6.22]
(c)
biocatalyst/electrode
FADH2-oxidase FAD-oxidase + 2H+ + 2e- [6.23]
The current (i) produced by such amperometric biosensors is related to the rate of reaction (vA) by
the expression:
i = nFAvA (6.6)
where n represents the number of electrons transferred, A is the electrode area, and F is the
Faraday. Usually the rate of reaction is made diffusionally controlled (see equation 3.27) by use
of external membranes. Under these circumstances the electric current produced is proportional to
the analyte concentration and independent both of the enzyme and electrochemical kinetics.
Optical biosensors
There are two main areas of development in optical biosensors. These involve determining
changes in light absorption between the reactants and products of a reaction, or measuring the
light output by a luminescent process. The former usually involve the widely established, if rather
low technology, use of colorimetric test strips. These are disposable single-use cellulose pads
impregnated with enzyme and reagents. The most common use of this technology is for whole-
blood monitoring in diabetes control. In this case, the strips include glucose oxidase, horseradish
peroxidase (EC 1.11.1.7) and a chromogen (e.g. o-toluidine or 3,3',5,5'-tetramethylbenzidine).
The hydrogen peroxide, produced by the aerobic oxidation of glucose (see reaction scheme [1.1]),
oxidising the weakly coloured chromogen to a highly coloured dye.
peroxidase
chromogen(2H) + H2O2 dye + 2H2O [6.24]
The evaluation of the dyed strips is best achieved by the use of portable reflectance meters,
although direct visual comparison with a coloured chart is often used. A wide variety of test strips
involving other enzymes are commercially available at the present time.A most promising
biosensor involving luminescence uses firefly luciferase (Photinus-luciferin 4-monooxygenase
(ATP-hydrolysing), EC 1.13.12.7) to detect the presence of bacteria in food or clinical samples.
Bacteria are specifically lysed and the ATP released (roughly proportional to the number of
bacteria present) reacted with D-luciferin and oxygen in a reaction which produces yellow light in
high quantum yield.
luciferase
ATP + D-luciferin + O2 oxyluciferin + AMP + pyrophosphate + CO2 + light (562
nm) [6.25]
The light produced may be detected photometrically by use of high-voltage, and expensive,
photomultiplier tubes or low-voltage cheap photodiode systems. The sensitivity of the
photomultiplier-containing systems is, at present, somewhat greater (< 104 cells ml-1, < 10-12 M
ATP) than the simpler photon detectors which use photodiodes. Firefly luciferase is a very
expensive enzyme, only obtainable from the tails of wild fireflies. Use of immobilised luciferase
greatly reduces the cost of these analyses.

Piezo-electric biosensors
Piezo-electric crystals (e.g. quartz) vibrate under the influence of an electric field. The frequency
of this oscillation (f) depends on their thickness and cut, each crystal having a characteristic
resonant frequency. This resonant frequency changes as molecules adsorb or desorb from the
surface of the crystal, obeying the relationshipes
 (6.7)
where f is the change in resonant frequency (Hz), m is the change in mass of adsorbed
material (g), K is a constant for the particular crystal dependent on such factors as its density and
cut, and A is the adsorbing surface area (cm 2). For any piezo-electric crystal, the change in
frequency is proportional to the mass of absorbed material, up to about a 2% change. This
frequency change is easily detected by relatively unsophisticated electronic circuits. A simple use
of such a transducer is a formaldehyde biosensor, utilising a formaldehyde dehydrogenase coating
immobilised to a quartz crystal and sensitive to gaseous formaldehyde. The major drawback of
these devices is the interference from atmospheric humidity and the difficulty in using them for
the determination of material in solution. They are, however, inexpensive, small and robust, and
capable of giving a rapid response.
Immunosensors
Biosensors may be used in conjunction with enzyme-linked immunosorbent assays (ELISA). The
principles behind the ELISA technique is shown in Figure 6.9. ELISA is used to detect and
amplify an antigen-antibody reaction; the amount of enzyme-linked antigen bound to the
immobilised antibody being determined by the relative concentration of the free and conjugated
antigen and quantified by the rate of enzymic reaction. Enzymes with high turnover numbers are
used in order to achieve rapid response. The sensitivity of such assays may be further enhanced
by utilising enzyme-catalysed reactions which give intrinsically greater response; for instance,
those giving rise to highly coloured, fluorescent or bioluminescent products. Assay kits using this
technique are now available for a vast range of analyses.
Figure 6.9. Principles of a direct competitive ELISA. (i) Antibody, specific for the antigen of
interest is immobilised on the surface of a tube. A mixture of a known amount of antigen-enzyme
conjugate plus unknown concentration of sample antigen is placed in the tube and allowed to
equilibrate. (ii) After a suitable period the antigen and antigen-enzyme conjugate will be
distributed between the bound and free states dependent upon their relative concentrations. (iii)
Unbound material is washed off and discarded. The amount of antigen-enzyme conjugate that is
bound may be determined by the rate of the subsequent enzymic reaction.
Recently ELISA techniques have been combined with biosensors, to form immunosensors, in
order to increase their range, speed and sensitivity. A simple immunosensor configuration is
shown in Figure 6.10 (a), where the biosensor merely replaces the traditional colorimetric
detection system. However more advanced immunosensors are being developed (Figure 6.10 ( b))
which rely on the direct detection of antigen bound to the antibody-coated surface of the
biosensor. Piezoelectric and FET-based biosensors are particularly suited to such applications.
Figure 6.10. Principles of immunosensors. (a)(i) A tube is coated with (immobilised) antigen. An
excess of specific antibody-enzyme conjugate is placed in the tube and allowed to bind. (a)(ii)
After a suitable period any unbound material is washed off. (a)(iii) The analyte antigen solution is
passed into the tube, binding and releasing some of the antibody-enzyme conjugate dependent
upon the antigen's concentration. The amount of antibody-enzyme conjugate released is
determined by the response from the biosensor. (b)(i) A transducer is coated with (immobilised)
antibody, specific for the antigen of interest. The transducer is immersed in a solution containing
a mixture of a known amount of antigen-enzyme conjugate plus unknown concentration of
sample antigen. (b)(ii) After a suitable period the antigen and antigen-enzyme conjugate will be
distributed between the bound and free states dependent upon their relative concentrations. (b)
(iii) Unbound material is washed off and discarded. The amount of antigen-enzyme conjugate
bound is determined directly from the transduced signal.
PROBLEMS TO WORK OUT

Problems
1.Calculate the Vmax value and Km value for the given data by using various plots

[S] Vo µmol
mM
0 product/min
0.0
1 0.9
2 1.4
5 1.9
10 2.3
50 2.6
100 2.6

2. Calculate the Vmax value and Km value for the given data by using various plots