Page | 1

AMITY INSTITUTE OF BIOTECHNOLOGY

ENZYMOLOGY
ASSIGNMENT-I
ENZYMES USED IN DETERGENTS

DATE OF SUBMISSION- 22.O4.08

SUBMITTED TO: SUBMITTED BY:

DR. S.M. BHATT MAYANK JAIN
FACULTY,ENZYMOLOGY ROLL NO.-77
AIB SECTION-U
AIB

CONTENTS: PAGE NO.

HISTORY................................................................................. 3
INTRODUCTION................................................................... 3
DETERGENT ENZYMES
PROTEASES....................................................................... 5
AMYLASES......................................................................... 7
LIPASE................................................................................ 8
CELLULASE...................................................................... 9
 Page | 2

MISCELLANEOUS DETERGENTS
PEROXIDASES................................................................. 10
PULLULANASE................................................................ 10
ENZYME FORMULATION................................................... 11
PRODUCTION OF ENZYME BASED DETERGENT........ 13
ENZYME STABILITY............................................................. 14
APPLICATION OF ENZYME BASED DETERGENT........ 16
BENEFIT OF USING ENZYME IN DETERGENT.............. 19
CONCLUSION .......................................................................... 20
BIBLIOGRAPHY....................................................................... 22

History
The original idea of using enzyme as detergents was described in 1913 by Dr
Otto Rohm, who patented the use of crude pancreatic extracts in laundry pre-
soak compositions to improve the removal of biological stains. In the same year,
the first enzymatic detergent named Burnus was launched, but was not popular
because of its own limitations. Subsequently, Bio- 40 - a detergent containing a
bacterial protease was produced in Switzerland and launched in the market in
1959 and it gradually became popular. In the period from 1965 to 1970, use and
sale of detergent enzymes grew very fast. In 1970, the use was distorted due to
dust production by formulations leading to allergies to some workers. This
problem was overcome in 1975 by encapsulating the granules of enzyme. From
1980s to the 1990s, several changes took place in the detergent industry like
development of softening through the wash, development of concentrated
heavy-duty power detergents, development of concentrated or structured or non-
aqueous liquid detergent.

Introduction
Enzymes have been used to improve the cleaning efficiency of detergents for
more than 35 years, and are now well accepted as ingredients in powder and
liquid detergents, stain removers/laundry pre-spotters, automatic dishwashing
detergents and industrial/institutional cleaning products. Detergent enzymes
account for about 30% of the total worldwide enzyme production and represent
 Page | 3

one of the largest and most successful applications of modern industrial

biotechnology.
The largest segment within the global industrial enzyme market is the market
for technical enzymes, estimated at around uss 980 million in 2002. In the
technical enzymes category, detergent additives make up for nearly two-thirds
of the market. These enzymes are used as functional ingredients in laundry
detergents and automated dishwashing detergents. This article gives an
overview of the detergent enzymes industry and discusses its manufacturing and
downstream processing.

Enzymes used in detergents are protein catalysts that consist of long chains of
amino acids. They are similar to protein catalysts present in all living cells
where they control metabolic processes, convert food nutrients to simple
molecules, convert these molecules to energy and to new cell material. As
catalysts; enzymes speed up specific chemical reactions, in mild conditions of
temperature and pH, without being altered or consumed in the process.
Consequently, small quantities of enzyme can repeatedly catalyze the
breakdown of millions of molecules in minutes. Enzymes function optimally in
detergents at temperatures of 20 - 60C and within a pH range of pH 7.5 - 10.5.
The performance of enzymes in detergents depends on number of factors,
including the detergent’s composition, type of stains to be removed, wash
temperature, washing procedure and wash-water hardness. To help formulators
optimize enzymatic detergent washing efficiency, Specialty Enzymes provides
wash laboratory technical services. In our wash laboratory, customer, base
detergents are evaluated on standard soils in both a model wash system (Terg-O-
Tometer) and in full-scale household washing machines.
Table 1 Compositions of an enzyme detergent
Constituent Composition (%)
Sodium tripolyphosphate (water softener, loosens dirt)a 38.0
Sodium alkane sulphonate (surfactant) 25.0
Sodium perborate tetrahydrate (oxidising agent) 25.0
Soap (sodium alkane carboxylates) 3.0
Sodium sulphate (filler, water softener) 2.5
Sodium carboxymethyl cellulose (dirt-suspending agent) 1.6
Sodium metasilicate (binder, loosens dirt) 1.0
Bacillus protease (3% active) 0.8
Fluorescent brighteners 0.3
Foam-controlling agents Trace
 Page | 4

Perfume Trace
Water to 100%

Detergent Enzymes

Presently, detergent enzyme has become an integral part of detergent

formulation. A look at the market share of detergent enzyme indicates it to be
very high in comparison with other enzyme applications. Enzymes that have to
be used as detergent composite must possess the following characters:
 Stability at temperature over a broad range of 20C to 50C and even above
 The optimum pH should be in alkaline or higher alkaline range
 It should be detergent compatible
 It should have specificity towards different proteins

Major detergent enzymes include proteases, amylases, lipases, cellulases,

miscellaneous enzymes such as peroxidases and pullulanase. A recent trend
is to reduce this phosphate content for environmental reasons. It may be
replaced by sodium carbonate plus extra protease.

Proteases

Proteases were introduced in the market in 1959 in the detergent Bio-40,

produced by Schnyder Ltd in Switzerland. Most powder and liquid laundry
detergents in the market, today, contain proteases. Proteases are of two types:
 Alkaline protease from Bacillus licheniformis, having optimum pH 8, for
egg, liquid laundry product, (pH 7- 8.5), commercially known as Alcalase
-Novonordisk Optimase- Genencor Inter .
 Page | 5

 High alkaline protease from Bacillus alkalophilus and Bacillus lentus,

having an optimum pH 10. For e.g., powder laundry products, automatic
dish washing formulations, known by trade names of Savinase-Novo
Nordisk, Purafet- Genencor Inter. Proteases enhance the cleaning of
protein-based soils, such as grass and blood by catalyzing the breakdown
of the constituent proteins in these soils through hydrolysis of the amide
bonds between individual amino acids. In the case of serine
endopeptidase, it contains a catalytic triad of amino acids at the active
site;
• An aspartyl residue containing ß-COO¯
• A histidine containing the imidazole group
• A serine residue with p-OH as the functional group
The serine hydroxyl group functions as a potential nucleophile, where as both
the aspartyl and histidine functional groups behave as general base catalysts
facilitating the hydrolysis process. .

The serine group initiates the nucleophilic attack on the peptide bond to form a
tetrahedral intermediate, which undergoes an active hydrogen transfer,
facilitated by both the histidine and aspartyl residues. The net effect of the
addition of water across the bond generates the original protein. The protease
hydrolysis involves the transfer of electrons between the amino acids at the
active site and substrate. For proteases the three-dimensional arrangement of the
catalytic triad is required for the enzyme to be active. Disturbances in the
confirmation are likely to affect enzyme efficacy and therefore cleaning
performance.
 These were susceptible to oxygen bleaches and calcium sequestrates. But
now, stable protease can be obtained .

 Oxidative attack by peroxides or per acids on the methionine residue

adjacent to the catalytic serine results in nearly 90% loss of enzyme
activity. However, replacing methionine with oxidatively stable amino
acids like alanine improves stability of enzyme towards oxygen bleach
(Boguslawski et al, 1992)

 Protease substilisin requires at least one calcium ion, which maintains

three- dimensional structure of enzyme. However, calcium- sequestering
agents used in many laundry procedures to control water hardness can
remove this calcium resulting in the decreased thermal and autolytic
 Page | 6

stability. This can be corrected by the introduction of negatively charged

residues near the calcium-binding site, which increases the binding
affinity of enzyme for calcium and results in improved stability towards
calcium sequestrants (Krawczyk et at, 1997)

 Protease has limited applications towards the detergency of wool and

Constituent silk, because of the proteinaceous nature of these fibres.

 Proteases are added in an encapsulated or granulated form, which protects

them from other detergent ingredient and eliminates the problem of
autolysis or proteolysis of other enzymes. In aqueous detergent
formulations, protease inhibitors show a preventive effect of avoiding
contact of the protease molecules with each other as well as other enzyme
molecules. This effect gets nullified on dilution and enzyme molecules
are free to act on stains (Krawczyk et al, 1997)

Amylases
Amylases facilitate the removal of starch-based food soils, by catalyzing the
hydrolysis of glycosidic linkages in starch polymers. Generally, starch-
containing stains are of chocolate, gravy, spaghetti, cocoa, pudding, etc.
Amylases can be classified as: :

a-amylases: These enzymes catalyze the hydrolysis of the amylose fractions of

the starch under hydrolysis of the glycosidic bonds in the interior of the starch
chain. The first step in the reaction is called as endoreaction & leads to
oligosaccharides, where short chain water- soluble dextrins are produced.
.
ß-amylases: These enzymes acton dextrins from reducing end and forms
maltose units.
Pullulanases or isoamylases: These degrade starch directly into linear dextrins
for they also attack ci-1,6 glycosidic bonds. .
.
Amyloglycosidases: These enzymes act on the dextrin or maltose units and
forms glucose units. .

a-amylases are mostly used for detergents, although recently other carbohydrate
cleaving enzymes such as pullulanases or isoamylases have also been described
 Page | 7

for this application. a-amylases bring about the primary hydrolysis of starch into
the oligosaccharides and dextrins. Currently, these enzymes are produced from
bacteria. Bacillus subtilis. Bacillus amyloliquefaciens, and Bacillus
licheniformis. These are available under the trade names Maxamyl- Genencor
Int or Termamyl -Novo Nordisk
Lipases

Tomato-based sauces, butter, edible oils, chocolate and cosmetic stains are very
difficult to remove as they form due to greasy food stains. Body soils, sebum
and sweat on collars, cuffs and underarms, are generally composed of a mixture
of proteins starch pigments and lipids. Lipases hydrolyze the water insoluble
triglycerides components into the more water-soluble products as
monoglycerides, diglycerides, free fatty acids and glycerol. The Novo Nordisk
launched the first lipase product in 1987. They transferred the lipolase gene into
the fungus Asper6yillus oryzae for industrial production, Genencor followed in
1993 with lumafast (Pseudomonas menocina) and Gist-Brocades in 1995 with
Lupomax. .

Currently, the known sources of lipases include mammalian lipases (human

pancreases/colipases), fungal (Rhizomucor mehei, Humicola lanuginose, etc),
yeast (Candida rugosa, Candida antartica), bacterial lipase (Pseudomonas
glumae, Pseudomonas aeroginosa, Chrobacterium viscosum) (Ishida et al, 1995)
. .

Lipases possess a catalytic triad that is similar to the serine proteases of trypsin
and subtilisin type. Hence, these are also called as serine hydrolysate lysate.
Lipases can decompose a fatty stain up to 25%, which then can be removed
very easily because of the hydrophilic character (Dorrit et al, 1991). It is
generally thought that lipases get adsorbed on to the hydrophobic stain during
the washing period. And, during the drying cycle when the water content is
decreased, the enzyme is activated and can hydrolyze triglycerides in the stain.
This facilitates the removal of stain in the next wash cycle (Dorrit et al 1991).
The enzyme also has stability over a broad range of temperature 30C to 60C.
These novel alkaline lipases also retained 100% activity in the presence of
strong oxidants.

Efforts are on to manufacture enzymes that can work below the normal
temperature range of 30C to 40C to save energy. It has been observed that
energy consumption per wash in household washing machine (3 kg clothes) at
low temperature (30C) is less than 1 % of the energy used at a higher
temperature (60C) (Edvardetal, 1991). .
 Page | 8

Cellulases

These enzymes introduced in the late 1980s were described for the first time in
a Japanese patent filed by Murata. These enzymes are used in UK and US since
1991. Cellulases remove microfibrils from cotton and cotton-blended fabrics.
These microfibrils stick out from the main fibre of cotton and are formed during
use and repeated washing condition of the tissue. This makes garments and
household textiles unusable. The cellulases can be used as softening agents, to
remove soil particles and to revive colours. .
.

The overall cellulose structure has two types of region; one that has a higher
order of crystallinity is called crystalline region. The other type has less
structured order, and hence is called as amorphous regions. The activating but
not hydrolytically acting enzyme was named as C i-activity. According to this
concept, microorganisms that are able to degrade crystalline cellulose have C i-
activity. This enzyme is not present in that microorganism that attack only
substituted cellulose like carboxymethyl cellulose as they have Cx-activity.

According to a recent research, biodegradation of cellulose requires the

interaction of three different hydrolytes or at least the first two enzymes to
attack simultaneously. These include:

· Cellobiohydrolase is also called as exocellulase (C i-activity) .

.
· Endoglucanases is also called as Endocellulases (C i-activity) .
.
· ß-Glucosidase is also called as cellobiase .
.

It was observed that sebum in the interior of cotton fibres cannot be removed by
ordinary detergents satisfactorily, although they readily remove sebum on the
exterior of the fibres. Alkaline cellulase interacts selectively with cellulose in
interfiber spaces in the interior of fibre, and selectively removes the sebum soil.
The removal of the soil is by the hydrolysis of amorphous regions (Murata et al,
1991).

Cellulases can be chemically modified to have greater stability and efficiency in

alkaline medium. It can be done by treating the acid cellulases with reagents
like maleic anhydride (Bund and Singhal et al, 2002).
 Page | 9

Currently, the cellulases used in detergents are manufactured from bacteria and
fungi. Bacterial cellulases have been in use since 1987, for example, Biotex.
Some genetically engineered strains, which are widely used include
Streptomyces sp. KSM-2, Bacillus KSM-635. The fungal cellulase from
Humicola isolens DSM1800 is active under mild alkaline conditions.

Miscellaneous detergent enzymes

Peroxidases:

These are one of the newest classes of enzymes that have been included in
detergent formulations. Peroxidases are subclass of general oxidoreductases and
are very popular and commercially available for manufacturing detergents.
Novo Nordisk produces this under the brand name Guardzyme obtained from
mushroom Corprinus cinereus. It is a heme containing protein, which in the
presence of H2O2 can mediate the oxidation of fugitive dyes in solution and
inhibits the dye transfer.

Pullulanases:

In recent years, pullulanases (Pullulan 6- glucanohydrolase) a debranching

enzyme has been gaining importance due to its efficiency of starch hydrolysis
by cleaving a-1, 6 linkages. Pullulanases with other amylolytic enzymes are
used in detergents for improved stain removal and enhanced overall cleaning
performance. This enzyme was first isolated in Klebsiella pnuemoniae (Shaw et
al, 1995).
Manufacturing and downstream processing
Nearly all-detergent enzymes, which are used and marketed today, are produced
through large-scale fermentation of microorganisms. Most of enzymes "are
obtained from the bacterial or fungal strains. As low cost enzymes are needed to
support the requirements of the global detergents business, enzyme
manufacturers should consider the following points to ensure lower costs:
 P a g e | 10

 The enzyme-producing micro-organism must be capable of secreting the

enzyme extra-cellularly in the bulk fermentation broth, as the cost in
terms of both money and time to recover enzyme from the fermentation
broth is very high
 The production organism should be able to produce highest possible
yields. Strain optimisation can be accomplished either through classical
mutagenesis and screening methods or using genetic engineering · The
number of steps in the downstream processing should be kept to a
minimum to be economical and also to avoid yield losses
 The production organism should produce the desired enzyme in a highly
pure state without any contaminating side activities or proteins. This can
be done by deleting the genes, which codes for unwanted enzymes and
proteins

Enzyme formulations .
Enzymes are formulated mainly in two forms, as a liquid product or as a
granular product .

1. Liquid product formulation .

The highly concentrated liquids of the evaporator or the ultra filtration
unit can be used for the manufacturing of the liquid formulations. The
liquids, which are to be incorporated into the formulation, must be
sterilized against microbial growth. Stabilizing agents like borax, organic
boric acid derivatives, alkali salts, etc should be added along with
preservatives like urea, propel glycol, diglycol, and sorbitol. The current
trend is to formulate these liquid formulations as structured liquids with
the help of salts and polymers, so that all surfactant remains in the
structured liquid and enzymes remain in the aqueous phase. (Hermann et
al, 1997)
2. Granular enzyme products .
Highly ultra filtered and dialysed enzyme solution is subjected to
adjustment of pH, turbulences, and temperature in the suitable range,
when the enzyme crystallises out. It can be precipitated at high salt
concentrations. The following four types of granulation process are
employed:

i. Enzyme pulling: :
The enzyme (dry) is dispersed into a molten wax, non-ionic surfactant,
or polymer matrix, and then sprayed in a cooling tower to form solid,
spherical, molten water-soluble or water dispersible material with a
 P a g e | 11

melting point above 50C. This technique offers the advantages of high
throughput and ability to recycle particles that fall outside the desired
size range but has a drawback i.e., the particle has relatively poor
physical strength, leading to break- up and high dust generation in
subsequent processing. Polyethylene glycols can be used to improve
physical strength and thus lesser dust formation, but as particle break-
up cannot be completely ruled out, it is not used widely at present.

ii. Granulation by extrusion process: :

In this technique, all the ingredients like enzyme powder or liquid
concentrate with binders, such as clay sugar, starch, some anti-
clogging agents like cellulose fibres and solubility enhancers like
sodium sulphate are mixed together and an extrudable dough is
produced, which is then pressed through the perforated metal plate.
The extruded noodles are cut into small cylinders and then given a
round shape by a spheroniser. After sieving, the particles are coated
with pigments such as titanium dioxide, and protective outer layers to
achieve desired appearance and to improve granulate integrity. The
drawback of this method is the high capital investment in a multi-step
process and the sensitivity of the process variation in feedstock
moisture and composition.

iii. High shear granulation: :

In this process the enzyme is mixed with controlled amounts of water,
binders such as polyethylene glycol, ethoxylated fatty alcohols, fatty
acids, bentonite, waxes having low melting temperature and other
granulating agents so as to form a low-moisture agglomerate. This
agglomerate is then passed on to the high-shear mixer in which it is
broken up into smaller particles. The particles are then dried in a
fluidised bed and coated with a final protective layer and pigments
like titanium dioxide. .

iv. Fluid bed coating: :

In this process, on the inert support or core material like sodium
chloride, calcium alginate, urea, or saccharose beads, liquid enzyme is
sprayed and the coating material is transferred to the drying zone with
the help of heated air stream. Then once the enzyme layer has dried,
additional coatings of stabilizers, chelating agents, antioxidants and
pigments are applied. The outer coating consists of film forming
polymer such as titanium dioxide. The volume of flow for proper
fluidization is dependent on the surface area and the shape & density
of the core material. The proper fluidization should flow sufficient
core material through the spray zone to coat all the atomized liquids
 P a g e | 12

on to the core material to prevent spray drying.

.

Production of enzyme-based detergents

The manufacture of an enzyme detergent is not very different from that of the
conventional synthetic one. Modern industrial cultivation of enzymes begins
with fermentation of a vial of dried or frozen micro-organisms called a
production strain. This production strain is selected to produce large amounts of
the enzymes of interest. The production strain is first cultivated in a small flask
containing nutrients and agar. The flask is placed in an incubator which provides
the optimal temperature for the previously frozen or dried cells to germinate.
Once the flask is ready, the cells are transferred to a seed fermenter, which is a
large tank containing previously sterilized raw materials and water, known as
the medium. Seed fermentation allows the cells to reproduce and adapt to the
environment and nutrients that they will encounter later on. The cells are then
transferred to a larger tank, the main fermenter, where temperature, pH and
dissolved oxygen are carefully controlled to optimize enzyme production.
Additional nutrients may be added to enhance productivity. When main
fermentation is complete, the mixture of cells, nutrients and enzymes, referred
to as the broth, is ready for filtration and purification. The prime step for the
formulation of an enzyme-based detergent is the compatibility of the enzyme(s)
with various detergent ingredients. In general, the suitability of an enzyme
preparation mainly depends on its compatibility with the detergents at
moderately higher temperatures. An ideal enzyme for detergent preparation
should be effective at low levels (0.4–0.8%) in the detergent solution. It should
also be compatible with various detergent components along with oxidizing and
sequestering agents and possess adequate temperature stability to be active in a
wide range of cleaning temperatures. It must also have a long shelf life 11.
Moreover, the very low use concentration is due to the fact that the enzymes
added to the product are biocatalysts. In this context, the term biocatalyzation is
implied wherein the enzymes themselves are not being consumed during the
cleaning process and a single enzyme triggers numerous chemical reactions. As
a result, the disadvantages of the conventional detergents are eliminated.
The early use of enzyme powders in detergents led to dust problems in the
production process. In addition, the reduced stability of the enzymes due to
autolysis and detrimental effects by the other detergent ingredients in the
presence of moisture were encountered. These problems led to the use of
granulation techniques and enzyme prilling with enzymes being encapsulated in
an inert water soluble waxy substance. In powder detergents, the enzymes are
mixed with the finished powders as granulates or prills. Currently, wax-coated
enzyme detergent granules are being offered in colours identical to the non-
 P a g e | 13

coloured detergent granules. The coloured granules are termed as signal

granules by the detergent manufacturers which symbolize the presence of an
extra-added active ingredient in the detergent preparations.
In case of enzyme-based automatic dishwashing detergents, citrate and other
polyacrylate builders are added. Moreover, perborates and percarbonates are
also used. These peroxybleach generating systems are not too harmful for
enzymes and through the action of activators such as tetraacetylethylene
diamine (TAED), enable the acceptable bleaching action at low temperature.

Enzyme stabilization
Most early enzyme products such as detergent proteases were just powders.
Almost all of them were granulated and further protected by coatings. Another
method to prevent enzyme dust in the air is liquid formulations. Today a lot of
research work is being done in the different formulations and stabilization
techniques in many enzyme detergent production facilities.
The enzymes used in various detergent formulations are subject to proteolytic
and autolytic degradation on storage and sudden exposure to harsh operating
conditions results in rapid inactivation of enzyme activity. Loss of enzyme
activity is also encountered during storage in the factory, shipment to client(s)
and/or storage in client(s) facilities. Hence, storage stability is of prime concern
to enzyme manufactures. The rate of enzyme inactivation is largely dependent
on temperature, pH and other detergent components such as surface active
agents, sequestrants and bleaching agents. Moreover, the higher the temperature
and alkalinity, the less stable is the enzyme.
The loss of the enzyme activity is mainly due to the partial unfolding of the
polypeptide chain, since the inactivating agent breaks down the delicate balance
of noncovalent bonds which maintain the native conformation. The ideal
approach to stabilize the enzyme would be to identify the mechanism of
inactivation and then design a procedure which would prevent that mechanism.
In order to protect the enzyme against denaturation, addition of stabilizers like
calcium salts, sodium formate, borate, polyhydric alcohols and protein
preparations have proved successful. To prevent contamination of the final
commercial crude preparation during storage, addition of sodium chloride at
18–20% concentration has been suggested. These processes maintain the
enzyme activity and improve storage stability. In certain cases, for the purpose
of convenience in handling and storage, liquid enzyme preparations
are often brought to powder form by vacuum or air drying which are milder and
less expensive than lyophilization.
The stabilization of enzymes has also been made possible through use of protein
engineering to design tailor-made enzymes with specific enzyme properties and
stability and this technique is leading new insights into the process of
biocatalysis. Protein engineering is rapidly emerging today as a new science and
 P a g e | 14

is basically an art of modifying an existing protein or creating de novo, a protein

of pre-specified properties. From a commercial viewpoint, this technology is
inherently complex, costly and time consuming. Despite these inherent
drawbacks, commercial detergent enzyme producers adopt this technology for
producing novel and/or superior enzymes with stable, new and/or improved
properties like stain removing ability, improved stability due to resistance to
oxidizing agents (oxygen-based bleaching), etc..

Applications of enzyme-based detergents

Data published up to now indicate that the enzyme detergents are being mainly
explored for their application in laundry, dishwashing, textile and other such
industries. Of late, in view of their advantages and increased potentiality, some
researchers have tried to use them in the food and dairy industries. The different
applications wherein the enzyme detergents are being currently used are:
In laundry
The microbial enzymes which have found application so far in laundry are the
proteases, amylases and lipases. More recently, the cellulases have also been
employed in the detergent industry with an added dimension. The proteases
hydrolyse the proteinaceous residues of blood, egg, grass and sweat to form
soluble peptides which are subsequently easily removed by detergent suds. The
amylases degrade the residues of starchy foods like porridge, potatoes, gravies,
custard, chocolate, etc. to dextrins, while the lipases catalyse the hydrolysis of
salad oil, sauces, lipstick, etc. The cellulases in the detergents degrade mainly
the microfibrils which are generated during continuous use and repeated
washings of the garment and also help in restoring the original shine and colour
of the garment.
The washing performance of the enzyme detergent depends on many factors to
achieve better results. These are detergent composition and dosage, pH and
buffer capacity, water hardness, washing time and temperature, mechanical
handling, soiling agents, textile types to name a few. In addition, the specificity
of the enzyme is another most important parameter. As a general opinion, it is
considered that a detergent enzyme should have as wide a specificity as
possible. For example, a protease should be capable of degrading as many
proteins as possible. However, a reasonably good wash performance can be
achieved by a specific protease, in comparison to a non-specific protease . As
the hydro-lysis proceeds, small peptide fragments are formed by the action of an
unspecific protease, which are rather difficult to remove as they are not very
much soluble in detergent solutions. On the other hand, larger protein or peptide
fragments are formed on hydrolysis with a specific protease due to the
 P a g e | 15

breakdown of very few peptide bonds which can be easily removed during the
washing process. Presently, use of dual enzymes in detergent formulations is
practised, wherein the enzymatic hydrolysis and degradation can be broadened
considerably in comparison to a single enzyme approach.
Recently, workers of the Genencor International Inc., USA have developed
enzymes called endoglycosidases which deglycosylate biopolymers like
glycoproteins which are widely distributed in living organisms. They employed
rDNA technology to develop Endo-b -N-acetyl glucosaminidase H (Endo H) as
a cleaning agent. Endo H has a unique property to remove bacteria
(Staphylococci and E. coli) from glass and cloth surfaces in buffer and detergent
solutions.
At present, most of the advanced countries like Japan, United States and some
European countries almost invariably use the detergents incorporated with
enzymes. Interestingly, in Japan, all detergent brands contain enzymes. In India,
a few premium detergent brands presently available in the market like Ariel
(Procter and Gamble (India) Ltd.), Surf Ultra, Rin Biolites, Revel Plus
(Hindustan Lever Ltd.) and Zymo (Henkel) contain enzymes in their
formulations. Recently, Procter and Gamble (P&G) has introduced a new
cellulase enzyme in the detergent powder, Ariel, presently marketed in India,
that eliminates the fuzz formed during washing and tumble-drying, particularly
of the cotton fabrics. The manufacturers claim that use of this product retains
the colour and improves the texture of the fabric on repeated washings.

In dishwashing
Enzymes have been successfully used in laundry detergents for many years as
an aid to remove tough stains. However, the interest in using enzymes in
automatic dishwashing detergents (ADDs) has increased recently. Both laundry
and dishwashing detergents share similar functions such as removal of stains
from egg, milk and starch-based soilings, etc. The performance of the enzymes
in the ADDs is strongly influenced by the ADD formulation and the conditions
of the automatic dishwashing. At present, proteases and amylases are the only
two enzymes which have found major application in dishwashing detergents. In
particular, enzyme-based dishwashing detergents are less abrasive in function
and thus are suitable for use on delicate chinaware; they prevent the erosion of
designs and colours. This application was first exploited in Japan where the use
of richly decorated chinaware and wooden kitchen utensils is widespread.
Enzymatic ADDs have gained widespread usage since the last decade. In the
past 2–3 years, ADDs with enzymes were launched in several European
countries, viz. Austria, Germany, Switzerland, Denmark and the United
Kingdom. In Japan, all major ADD brands contain enzymes, whereas only one
brand in the US market currently contains enzymes. However, at present, there
are no enzymatic dishwashing detergents available in India.
 P a g e | 16

In the textile industry

Currently, in the textile industry, there is a widespread demand for faded jeans.
This involves subjecting such clothes to amylases – a process commonly
referred to as biowashing or biobleaching, an alternative to the term, enzyme-
fade. This allows elegant softness and unique shades to be given to the cloth
which overcomes the traditional methods of bleaching by sodium hypochlorite
or tumbling with pumice stones, and also offers better safety as well as
economy.
In food and dairy industries
With the better understanding of such enzymes, more and more areas of their
application are emerging, such as in dairy, food and beverage industries. The
use of enzymes in these industries in the cleaning operations helps in creating
the required hygienic conditions in such plants. Probably, the use of enzyme-
based detergents in the in-place cleaning of membranes of ultrafiltration (UF)
and reverse osmosis (RO) equipments proves promising and forms one of the
most important aspects of modern dairy and food industries. The UF and RO
membranes are put to a variety of uses including concentration, clarification
and/or sterilization of liquid foods like skim milk, whey, egg white, fruit juices
and beverages.
Despite their diverse applications, these two membrane processes have some
inherent disadvantages. The membrane filters come in contact with the feed
stock during use. Even a small degree of adsorption causes pore blockage
resulting in clogging of filters, a phenomenon called fouling, and thereby cause
a reduction in the permeate flux rate and loss in the product quality with
increase in production costs. In general, the proteins, inorganic salts and fat
residues along with bacteria constitute the common and important fouling
agents responsible for lowering the flux and affecting the product quality.
Depending on the type of application, the precise formulations are made; for
instance, proteases are used for fouled dairy filters, a -amylases and b
-glucanases in yeast and cereal, and cellulases and pectinases for wines and fruit
juices. The enzyme detergent preparations presently marketed for cleaning of
membrane systems are Terg-a-zyme (Alconox, Inc, New York, USA) and
Ultrasil 53 (Henkel KGaA, Dusseldorf, Germany). These enzyme-based
cleaners that have been marketed rely very much on the proteases to cleave and
solubilize the protein foulant. The use of alkaline proteases from Bacillus sp.
strain MK5-6 has also proved successful in our laboratory. Pilot scale evaluation
of the enzymes at plant level operations for UF membrane cleaning indicated
the enzyme preparation to be highly effective and restored 100% flux in
comparison to Terg-a-zyme, a commercial preparation which resulted in only
80% restoration of the flux. The use of proteases and lipases to degrade and
solubilize protein and fat foulants has also proved beneficial.
 P a g e | 17

Other uses
The application of enzyme-cleaners in the optical industry is important,
enabling one to give 100% safe and efficient cleaning to lenses. In India,
presently one such enzyme-based optical cleaner in the form of tablets
containing Subtilopeptidase A is being marketed by M/s Bausch and Lomb
(India) Ltd. Enzyme detergents have also found application in hospitals.
Promod 153L, a protease enzyme-based cleaner, has been used to clean surgical
instruments fouled by blood proteins.

Benefits of enzymes
The past decades with a growing number of enzyme applications in consumer
detergents have led to major improvements in terms of benefits for consumers.

Low temperature efficiency .

Enzymes catalyze the breakdown of soils and stain materials at lower
temperatures. This allowed washing at lower temperatures and using less water
throughout Europe whilst washing performance has improved. The energy-
saving in the home from the temperature reduction and consequent reduction in
environmental emissions (such as carbon dioxide) is considerable as a washing
machine operated at 40°C consumes only one third of the energy it would use at
95°C.

Weight-efficiency
Because enzymes act as catalysts (which can be used repeatedly to speed up
chemical reactions without themselves being depleted) they are very weight
efficient and cost effective. In other words, they can potentially replace a larger
usage of conventional chemicals in the detergent. From an eco-toxicological
viewpoint, enzymes can be considered as highly optimized laundry products
ingredients which contribute positively to the overall environmental profile of
detergents.

Other
Technical and consumer research has demonstrated that the formulation of P&G
detergents with enzyme has led to significant consumer benefits in terms of
performance. The benefits of enzymes are related to both the laundry process
and the wash results, and include the abilities to:
 P a g e | 18

Wash at varying pH levels, from mild to high alkalinity;

Use different wash temperatures, from 60°C to as low as the "30-40°C

range";

Retain laundering performance in the presence of chemicals such as bleach;

builder, surfactant, etc….

Soften fabrics;

Brighten their colors; Improve whiteness

Remove fatty stains at low wash temperatures;

Conclusions
Thus to conclude, cleaning forms an important aspect for the maintenance of
hygiene and safety of foods in the food processing industry. Improperly cleaned
food-contact surfaces lead to the accumulation of food particulates which favour
the formation of biofilms, i.e. attachment of microorganisms. These cause post
contamination and spoilage of foods. It is, therefore, necessary to understand the
interactions of the biotic and abiotic entities in the food-processing operations
and further effectively analyse the impacts of cleaning and sanitation from a
microbiological viewpoint. The use of enzyme-based detergents as biocleaners
can also serve as a viable option to overcome the biofilm problem in the food
industry.
Further, the technology and production of these enzymes and the enzyme-based
detergents is mostly patent-protected. As such most of the enzymes used in the
detergent industry in India are being imported. Even the large scale detergent
manufacture seems highly technical requiring specific know-how and
infrastructure. Work has been going on in the recent past in order to develop an
indigenous technology on different enzyme systems in certain well reputed
laboratories, viz. National Dairy Research Institute, Karnal, National Chemical
Laboratory, Pune and Institute of Microbial Technology, Chandigarh. Due to
their high efficiency and safety, it is assumed that the enzyme detergents will
eventually capture a bulk of the Indian detergent market.
 P a g e | 19

As in the other sectors of the chemical process industry, where enzymes are
increasingly playing a crucial role in making conventional processes more
environment-friendly, the detergents industry has also benefited from the
introduction of enzymes. The enzyme detergents are proving to be better than
the traditional detergents with respect to washing performance, but there are still
few constraints like the inability of the enzymes to withstand high alkalinity and
variable temperatures. However, these hurdles are likely to be overcome in the
near future with newer & better technologies, which would open up a wide
array of opportunities for the detergent enzymes industry.

Bibliography
 P a g e | 20

1. Kandler, J., Proceedings of the Second World Conference on Detergents,

American Oil Chemists Society, Champaign, Illinois, 1987, p. 137.
2. The Economic Times, in Kothari’s Industrial Directory of India 1996–97
(ed. Arokiaswamy, S.), Kothari Enterprises, Chennai, 1996, pp. 14–15.
3. van Tilburg, R., Innovations Biotechnol., 1984, 20, 417–422.
4. Malmos, H., Chem. Ind., 1990, March issue, pp. 183–186.
5. Anonymous, Chem. Weekly, 1994, 40, 74–75.
6. Anonymous, Report of the 83rd AOCS Annual Meeting and Exposition,
Toronto, Canada, 1992.
7. Godfrey, T. and Reichelt, J. P., Industrial Enzymology, Nature Press, New
York, 1983, pp. 1–7.
8. IB Market Forecast, Ind. Bioprocess, 1992, 14, 4–5.
9. Hodgson, J., Biotechnol., 1994, 12, 789–790.
10. Anonymous, Chem. Week, 1992, January issue, p. 34.
11. Ward, O. P., in Microbial Enzymes and Biotechnology (ed. Fogarty, W.
M.), Applied Science Publishers, London, 1985, pp. 251–317.
12. Gist-brocades International, B. V., Technical Literature, Brochure no. 93–
12.
13. Schmid, R. D., Adv. Biochem. Eng., 1979, 12, 41–118.
14. Klibanov, A. M., Adv. Appl. Microbiol., 1983, 29, 1–28.
15. Feder, J., Kochavi, D., Anderson, R. G. and Wildi, D. S., Biotechnol.
Bioeng., 1978, 20, 1865–1872.
16. Eilertson, J. H., Fog, A. D. and Gibson, K., US Patent No. 4497897,
1985.
17. Weijers, S. R. and van’t Riet, K., Biotechnol. Adv., 1992, 10, 237–249.
18. Aunstrup, K., in Economic Microbiology. Microbial Enzymes and
Bioconversions (ed. Rose, A. H.), Academic Press, New York, 1980, vol.
5, pp. 50–114.
19. Shetty, J. K., Patel, C. P. and Nicholson, M. A., European Patent Appl.,
EP 0549048, 1993.
20. Mozhaev, V. V. and Martinek, K., Enzyme Microbial Technol., 1984, 6,
50–59.
21. Svenden, A., Clausen, I. G., Patkar, S. A., Borch, K. and Thellersen, M.,
Methods Enzymol., 1997, 284, 317–339.
22. Knowles, J. R., Science, 1987, 236, 1252–1258.
23. Takagi, H., Int. J. Biochem., 1993, 25, 307–312.
24. Rubingh, D. N., Curr. Opin. Biotechnol., 1997, 8, 417–422.
 P a g e | 21

25.Lad, P. G., Abstracts of 83rd AOCS Annual Meeting and Exposition,

Toronto, Canada, 1992.
26. Dalgaard, L. H., Kochavi, D. and Thellersen, M., Inform, 1991, 2, 532–
534, 536.
27. Beaton, N. C., J. Food Protect., 1979, 42, 584–590.
28. Merin, U. and Daufin, G., Le Lait, 1990, 70, 281–291.
29.Kumar, C. G., Ph D Thesis, National Dairy Research Institute (Deemed
University), Karnal, 1997.
30. Anonymous, Biotechnol. Bull., 1989, 8,10.
31. Aaslyng, D., Gormsen, E. and Malmos, H., J. Chem. Technol.
Biotechnol., 1991, 50, 321–330.
32. Enzymes in Detergents. (n.d.). Retrieved December 3, 3003 from
www.fst.rdg.ac.uk/courses/fs560/topic1/t1a/t1a/htm
33. Examples of Industrial Enzymes. (n.d.). Retrieved October 25, 2002 from
the Biotechnology Industry Organization Web site:
www.bio.org/er/enzymes.asp
34. Microbial Enzymes for Industry. (n.d.). Retrieved October 25, 2002 from
www.pence.ualb...a/pence/english/theme/theme_e.html
35. GUPTA, R.; BEG, Q.K. and LORENZ, P. Bacterial alkaline proteases:
molecular approaches and industrial applications. Applied Microbiology
and Biotechnology , April 2002, vol. 59, no. 1, p.15-32.
36. Siezen RJ, Leunissen JAM: Subtilases: the superfamily of subtilisin-like
serine proteases. Protein Sci 1997, 6:501-523.
37. Egmont MR: Application of proteases in detergents. In Enzymes in
Detergency. Edited by Van Ee J, Misset O, Baas EJ. New York: Marcel
Dekker Inc., Surfactant Science Series 1997, 69: 61- 74.
38. Enzymes in household detergents: In Enzymes in Industry. Edited by
Aehle W. Ullmann’s Encyclopedia of Industrial Chemistry Chapter 5.2.1.
Weinheim: Wiley-VCh Verlag 2004:155-180.
39. Enzymes in automatic dishwashing: In Enzymes in Industry.Edited by
Aehle W. Ullmann’s Encyclopedia of Industrial Chemistry I Chapter
5.2.2. Weinheim: Wiley-VCh Verlag 2004:180-194.