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Chapter 2 Journey to Microbial


Chapter 4 Nutrition and Culture

of Microorganisms

Different chemical reactions and organize

many different molecules into specific

structures is known as metabolism
Catabolism breaks molecular structures down,
releasing energy in the process, and
anabolism uses energy to build larger
molecules from smaller ones.
Metabolic reactions are either catabolic,
which means energy releasing, or anabolic,
which means energy requiring.

All microbial nutrients are compounds

constructed from the chemical elements.

However, just a handful of elements
dominate living systems and are
essential: hydrogen (H), oxygen (O),
carbon (C), nitrogen (N), phosphorus
(P), sulfur (S), and selenium (Se). In
addition to these, at least 50 other
elements, although not required, are
metabolized in some way by

Besides water, which makes up 7080%

of the wet weight of a microbial cell (a

single cell of Escherichia coli weighs just
g), cells consist primarily of
macromoleculesproteins, nucleic
acids, lipids, and polysaccharides.

All cells require carbon, and most

prokaryotes require organic (carboncontaining) compounds as their source

of carbon.
Heterotrophic bacteria assimilate
organic compounds and use them to
make new cell material.
Autotrophic microorganisms build their
cellular structures from carbon dioxide
(CO2) with energy obtained from light or

A bacterial cell is about 13% nitrogen,

which is present in proteins, nucleic

constituents. The bulk of nitrogen
available in nature is in inorganic form
as ammonia (NH3), nitrate, or nitrogen
gas (N2)

Other Macronutrients: P, S, K,Mg,

Ca, Na
In addition to C, N, O, and H, many other

elements are needed by cells, but in smaller

amounts .
Phosphorus is a key element in nucleic acids
and phospholipids and is typically supplied to
a cell as phosphate (PO4)
Sulfur is present in the amino acids cysteine
and methionine and also in several vitamins,
including thiamine, biotin, and lipoic acid.
Sulfur can be supplied to cells in several
forms, including sulfide (HS2) and sulfate

Potassium (K) is required for the activity

of several enzymes, whereas

(Mg) functions to stabilize ribosomes,
membranes, and nucleic acids and is
also required for the activity of many
Calcium (Ca) is not required by all cells
but can play a role in helping to stabilize
microbial cell walls, and it plays a key
role in the heat stability of endospores.

Sodium (Na) is required by some, but not all,

microorganisms, and its requirement is

typically a reflection of the habitat. For
example,seawater contains relatively high
levels of Na, and marine
microorganisms typically require Na for
By contrast, freshwater species are usually
able to grow in the absence of Na.
K, Mg, Ca, and Na are all supplied to cells as
salts, typically as chloride or sulfate salts.

Micronutrients: Iron and Other

Trace Metals
Microorganisms require several metals

for growth
Chief among these is iron (Fe), which
plays a major role in cellular respiration.
Iron is a key component of cytochromes
and of ironsulfur proteins involved in
electron transport reactions .
Under anoxic conditions, iron is
generally in the ferrous form and
soluble. However, under oxic conditions,

Defined media are prepared by adding precise

amounts of highly purified inorganic or organic

chemicals to distilled water.
Therefore, the exact composition of a defined
medium (in both a qualitative and quantitative
sense) is known.
Major importance in any culture medium is the
carbon source because all cells need large
amounts of carbon to make new cell material

For culturing many microorganisms, knowledge of

the exact
composition of a medium is not essential. In
these instances
complex media may suffice and may even be
Complex media employ digests of microbial,
animal or plant products, such as casein (milk
protein), beef (beef extract), soybeans (tryptic soy
broth), yeast cells (yeast extract), or any of a
number of other highly nutritious yet impure

An enriched medium, often used for the

culture of otherwise difficult-to-grow

microorganisms, starts with a complex
base and is embellished with additional
nutrients such as serum, blood, or other
highly nutritious substances.

A selective medium contains compounds that

inhibit the growth of some microorganisms but

not others. For example, media are available
for the selective isolation
of pathogenic strains of E. coli from food
products, such as ground beef, that could be
contaminated with this organism.

Differential medium is one in which an

indicator, typically a reactive dye, is

added that reveals whether a particular
chemical reaction has occurred during

Solid and Liquid Culture Media

Liquid culture media are sometimes

solidified by the addition of a gelling

Solid media immobilize cells,
allowing them to grow and form
visible, isolated masses called

Solid and Liquid Culture Media

Microbial colonies are of various shapes

and sizes depending on the organism,

the culture conditions, the nutrient
supply, and several other physiological
parameters, and can contain several
billion individual cells.
Some microorganisms produce
pigments that cause the colony to be
colored. Colonies permit the
microbiologist to visualize the
composition and presumptive

Solid media are prepared in the same way as

liquid media
except that before sterilization, agar, a gelling
agent, is added to the medium, typically at a
concentration of 12%.
The agar melts during the sterilization process,
and the molten medium is then poured into sterile
glass or plastic plates and allowed to solidify
before use.

By the end of the lesson, student should be able
to :
1. define terms enzyme;
2. understand the Lock & Key model and

Induced-fit model;
3. identify the groups of enzymes; and
4. understand some factors affecting the
enzyme activities.

Enzymes are proteins that catalyze (i.e.

accelerate) and control the rates of chemical

In enzymatic reactions, the molecules at the
beginning of the process are called substrates,
and the enzyme converts them into different
molecules, the products.
Almost all processes in a biological cell need
enzymes in order to occur at significant rates.

Since enzymes are extremely selective for their

substrates and speed up only a few reactions

from among many possibilities, the set of
enzymes made in a cell determines which
metabolic pathways occur in that cell.

Enzymes are biological catalysts produced by

living cells.
Enzymes lower the amount of activation energy
They speed up the rate of biochemical reactions
in the cell but remain unchanged at the end of the
Most enzymes are globular protein molecules.

The chemicals which an enzyme acts on is called its

The enzyme combines with its substrate to form an
enzyme-substrate complex.
The complex than breaks up into product and enzyme.
A metabolic pathway is a number of reactions catalysed
by sequence of enzymes.


There are 2 main hypotheses explaining of

enzyme action.

Each enzyme is specific for one and ONLY one

substrate (one lock - one key)

active site: part of the enzyme that fits with the
Note that the active site has a specific fit for this
particular substrate and no other.
This theory has some weaknesses, but it explains
many basic things about enzyme function.

Substrate: The starting molecules for a chemical

reaction are called the substrates.

Enzyme substrate complex: The enzyme
substrate complex is transitional step when the
substrates of a chemical reaction are bound to
the enzyme.
Active site: The area on the enzyme where the
substrate or substrates attach to is called the
active site.
Enzymes are usually very large proteins and the
active site is just a small region of the enzyme

The induced-fit theory assumes that the substrate

plays a role in determining the final shape of the

enzyme and that the enzyme is partially flexible.
This explains why certain compounds can bind to
the enzyme but do not react because the enzyme
has been distorted too much.
Other molecules may be too small to induce the
proper alignment and therefore cannot react.
Only the proper substrate is capable of inducing the
proper alignment of the active site.

In the graphic, the substrate is represented by the

magenta molecule, the enzyme protein is

represented by the green and cyan colors.
The cyan colored protein is used to more sharply
define the active site.
The protein chains are flexible and fit around the

The advantages of the induced fit mechanism arise due

to the stabilizing effect of strong enzyme binding.

There are two different mechanisms of substrate
binding; uniform binding which has strong substrate
binding, and differential binding which has strong
transition state binding.
The stabilizing effect of uniform binding increases both
substrate and transition state binding affinity and
differential binding increases only transition state
binding affinity.

Both are used by enzymes and have been

evolutionarily chosen to minimize the G of the

Enzymes which are saturated, ie. have a high affinity
substrate binding, require differential binding to
reduce the G, whereas largely substrate unbound
enzymes may use either differential or uniform

How do enzymes work?

substrate: molecules upon which an enzyme

acts. The enzyme is shaped so that it can only

lock up with a specific substrate molecule.
substrate -------------> product

The diagram shows time on the horizontal axis and

the amount of energy in the chemicals involved in a

chemical reaction on the vertical axis.
The point if this diagram again is that without the
enzyme, much more activation energy is required to
get a chemical reaction to take place.

Factors Influencing Enzyme Activity

pH: the optimum (best) in most living things is

close to 7 (neutral).
High or low pH levels usually slow enzyme

Temperature: strongly influences enzyme

activity optimum (best) temperature for maximum

enzyme function is usually about 35-40 C.
Reactions proceed slowly below optimal
Above 45 C. most enzymes are denatured
(change in their shape so the enzyme active site
no longer fits with the substrate and the enzyme
can't function)

Metabolism is the sum of all biochemical

reactions occurring in living cells.

These reactions can be divided into two main


Involves the synthesis

of complex molecules
from simpler molecules
which requires energy

Involves the

breakdown of complex
molecules into simpler
molecules involving
hydrolysis or oxidation
and the release of

Energy releasing processes, ones that "generate"

energy, are termed exergonic reactions.

Reactions that require energy to initiate the
reaction are known as endergonic reactions.
All natural processes tend to proceed in such a
direction that the disorder or randomness of the
universe increases

In an exergonic reaction the change is free

energy is represented by a negative number (G), indicating free energy is released during
the reaction.

This kind of reaction is not termed a

spontaneous reaction. In order to go from the

initial state to the final state a considerable
amount of energy must be imparted to the
These kinds of reactions are associated with a
positive number (+G).

The speed V means the number of reactions per

second that are catalyzed by an enzyme.

With increasing substrate concentration [S], the
enzyme is asymptotically approaching its
maximum speed Vmax, but never actually
reaching it.
Because of that, no [S] for Vmax can be given.
Instead, the characteristic value for the enzyme
is defined by the substrate concentration at its
half-maximum speed (Vmax/2).
This KM value is also called Michaelis-Menten

Vo = Vmax
Vo = Initial reaction velocity

Vmax = Maximum velocity

Km = Michaelis constant
[S] = Substrate concentration

A non protein component of enzymes is called the

If the cofactor is organic, then it is called a
Coenzymes are relatively small molecules
compared to the protein part of the enzyme.
Many of the coenzymes are derived from vitamins.
The coenzymes make up a part of the active site,
since without the coenzyme, the enzyme will not

In the graphic on the left is the structure for

the coenzyme, NAD+, Nicotinamide

Adenine Dinucleotide.
Nicotinamide is from the niacin vitamin.
The NAD+ coenzyme is involved with
many types of oxidation reactions where
alcohols are converted to ketones or





nicotinamide adenine
dinucleotide (NAD+)

oxidation or
hydrogen transfer


flavin adenine
dinucleotide (FAD)

oxidation or
hydrogen transfer


coenzyme A (CoA)

Acetyl group carrier

vitamin B-12

coenzyme B-12

Methyl group

thiamin (B-1)


Aldehyde group

Coenzyme Q10 is a fat-soluble nutrient also known

as CoQ10, vitamin Q10, ubidecarenone, or

It is a natural product of the human body that is
primarily found in the mitochondria, which are the
cellular organelles that produce energy.
It occurs in most tissues of the human body;
however, the highest concentrations are found in
the heart, liver, kidneys, and pancreas.
Ubiquinone takes its name from a combination of
the word ubiquitous, meaning something that is
found everywhere, and quinone 10.

Quinones are substances found in all plants and

The variety found in humans has a 10-unit side chain
in its molecular structure.
Apart from the important process that provides
energy, CoQ10 also stabilizes cell membranes and
acts as an antioxidant.
In this capacity, it destroys free radicals, which are
unstable molecules that can damage normal cells.

Enzyme inhibitors are molecules that interact in some

way with the enzyme to prevent it from working in the

normal manner.
There are a variety of types of inhibitors including:
nonspecific, irreversible, reversible - competitive and
Poisons and drugs are examples of enzyme inhibitors.

A nonspecific inhibition effects all enzymes in the

same way.
Non-specific methods of inhibition include any
physical or chemical changes which ultimately
denatures the protein portion of the enzyme and
are therefore irreversible.

Temperature: Usually, the reaction rate increases with

temperature, but with enzyme reactions, a point is

reached when the reaction rate decreases with
increasing temperature.
At high temperatures the protein part of the enzyme
begins to denature, thus inhibiting the reaction.

A competitive inhibitor is any compound which

closely resembles the chemical structure and

molecular geometry of the substrate.
The inhibitor competes for the same active site as
the substrate molecule.
The inhibitor may interact with the enzyme at the
active site, but no reaction takes place.

The inhibitor is "stuck" on the enzyme and

prevents any substrate molecules from

reacting with the enzyme.
However, a competitive inhibition is usually
reversible if sufficient substrate molecules are
available to ultimately displace the inhibitor.
Therefore, the amount of enzyme inhibition
depends upon the inhibitor concentration,
substrate concentration, and the relative
affinities of the inhibitor and substrate for the
active site.

A noncompetitive inhibitor is a substance that

forms strong covalent bonds with an enzyme

and consequently may not be displaced by
the addition of excess substrate.
Therefore, noncompetitive inhibition is
A noncompetitive inhibitor may be bonded at,
near, or remote from the active site. In any
case, the basic structure of the enzyme is
modified to the degree that it ceases to work.

If the inhibition is at a place remote from the active

site, this is called allosteric inhibition.

Allosteric means "other site" or "other structure".
The interaction of an inhibitor at an allosteric site
changes the structure of the enzyme so that the active
site is also changed.

There are approximately 3000 enzymes which

have been characterised.

These are grouped into six main classes
according to the type of reaction catalysed.
At present, only a limited number are used in
enzyme electrodes or for other analytical

These enzymes catalyse oxidation and reduction

reactions involving the transfer of hydrogen

atoms or electrons.
The following are of particular importance in the
design of enzyme electrodes.
This group can be further divided into 4 main

catalyse hydrogen transfer from the substrate to

molecular oxygen producing hydrogen peroxide as a

by-product. An example of this is FAD dependent
glucose oxidase which catalyses the following reaction:
b-D-glucose + O2 = gluconolactone + H2O2

catalyse hydrogen transfer from the substrate to a

nicotinamide adenine dinucleotide cofactor

(NAD+). An example of this is lactate
dehydrogenase which catalyses the following
Lactate + NAD+ = Pyruvate + NADH + H+

catalyse oxidation of a substrate by hydrogen peroxide.
An example of this type of enzyme is horseradish

peroxidase which catalyses the oxidation of a number of

different reducing substances (dyes, amines,
hydroquinones etc.) and the concomitant reduction of
hydrogen peroxide.
The reaction below illustrates the oxidation of neutral
ferrocene to ferricinium in the presence of hydrogen
2[Fe(Cp)2] + H2O2 + 2H+= 2[Fe(Cp)2]+ + 2 H2O

catalyse substrate oxidation by molecular oxygen.
The reduced product of the reaction in this case is

water and not hydrogen peroxide.

An example of this is the oxidation of lactate to acetate
catalysed by lactate-2-monooxygenase.
lactate + O2 = acetate + CO2 + H2O

These enzymes transfer C, N, P or S containing

groups (alkyl, acyl, aldehyde, amino, phosphate

or glucosyl) from one substrate to another.
Transaminases, transketolases, transaldolases
and transmethylases belong to this group.

These enzymes catalyse cleavage reactions or the

reverse fragment condensations.

According to the type of bond cleaved, a distinction is
made between peptidases, esterases, lipases,
glycosidases, phosphatases and so on.
Examples of this class of enzyme include; cholesterol
esterase, alkaline phosphatase and glucoamylase.

These enzymes non-hydrolytically remove groups

from their substrates with the concomitant

formation of double bonds or alternatively add
new groups across double bonds.

These enzymes catalyse intramolecular

rearrangements and are subdivided into;



An example of this class of enzyme is glucose

isomerase which catalyses the isomerisation of

glucose to fructose.

Ligases split C-C, C-O, C-N, C-S and C-

halogen bonds without hydrolysis or

The reaction is usually accompanied by the
consumption of a high energy compound such
as ATP and other nucleoside triphosphates.
An example of this type of enzyme is
pyruvate carboxylase which catalyses the
following reaction:
pyruvate + HCO3- + ATP = Oxaloacetate + ADP + Pi

IEC Classification of Enzymes

Group Name
Type of Reaction Catalyzed
Oxidases or
Transfer of functional
Hydrolysis reactions
Addition to double bonds or
its reverse
Isomerization reactions
Formation of bonds with
Ligases or Synthetases
ATP cleavage

Enzymes do NOT change the equilibrium position of the

reaction, just the speed at which equilibrium is attained.

Most are globular or soluble.
Stereospecific (can recognize certain isomers only) due to
the fact that amino acids of the active site are chiral
Substrate/s bind in hydrophobic cleft (active site) between
several domains where catalysis occurs:
Van der Waals forces
Hydrophobic interactions
Electrostatic interactions

Active site has structure that is complimentary in structure to

the structure of its substrate.

Most are proteins, some are RNA.

Biological catalysts.
E + S ES EP E + P
Not changed by the reaction overall
Much higher reaction rates than uncatalyzed reactions.
Allow for biochemical reactions to occur under very mild

conditions (temperature, near-neutral pH, 1 atm pressure)

High yield of products (few side reactions or by-products)
Very specific reactions (specific for its substrate or a family of
related substrates)
Often a regulated functions:
allosteric activation or inhibition
covalent modification (phosphorylation changes)
enzyme expression controlled or cleavage of proenzyme

Describe what metabolism is?

What is the difference between anabolism and

What is a substrate?
List 6 types of enzyme and state the
characteristics each of them.