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WHAT ARE ENZYMESEnzyme are specialized organic substances, composed of polymers of amino acids, that act as catalysts to regulate the speed of the many chemical reactions involved in the metabolism of living organisms, such as digestion. The name enzyme was suggested in 1867 by the German physiologist Wilhelm Khne (1837-1900); it is derived from the Greek phrase en zym, meaning in leaven. Enzymes are classified into several broad categories, such as hydrolytic, oxidizing, and reducing, depending on the type of reaction they control. Hydrolytic enzymes accelerate reactions in which a substance is broken down into simpler compounds through reaction with water molecules. Oxidizing enzymes, known as oxidases, accelerate oxidation reactions; reducing enzymes speed up reduction reactions, in which oxygen is removed. Many other enzymes catalyze other types of reactions. Individual enzymes are named by adding ase to the name of the substrate with which they react. The enzyme that controls urea decomposition is called urease; those that control protein hydrolyses are known as proteinases. Some enzymes, such as the proteinases trypsin and pepsin, retain the names used before this nomenclature was adopted.


Enzymes are large proteins that speed up chemical reactions. In their globular structure, one or more polypeptide chains twist and fold, bringing together a small number of amino acids to form the active site, or the location on the enzyme where the substrate binds and the reaction takes place. The enzyme itself is unaffected by the reaction. When the products have been released, the enzyme is ready to bind with a new substrate.



Alcoholic fermentation is undoubtedly the oldest known enzyme reaction. This and similar phenomena were believed to be spontaneous reactions until 1857, when the French chemist Louis Pasteur proved that fermentation occurs only in the presence of living cells. Subsequently, however, the German chemist Eduard Buchner discovered (1897) that a cell-free extracts of yeast can cause alcoholic fermentation. The ancient puzzle was then solved; the yeast cell produces the enzyme, and the enzyme brings about the fermentation. As early as 1783 the Italian biologist Lazzaro Spallanzani had observed that meat could be digested by gastric juices extracted from hawks. This experiment was probably the first in which a vital reaction was performed outside the living organism.

JAMES.B.SUMNER (Crystallized Urease)

After Buchner's discovery scientists assumed that fermentations and vital reactions in general were caused by enzymes. Nevertheless, all attempts to isolate and identify their chemical nature were unsuccessful. In 1926, however, the American biochemist James B. Sumner succeeded in isolating and crystallizing urease. Four years later pepsin and trypsin were isolated and crystallized by the American biochemist John H. Northrop. Enzymes were found to be proteins, and Northrop proved that the protein was actually the enzyme and not simply a carrier for another compound.


Swedish chemist Jns Jakob Berzelius suggested in 1823, enzymes are typical catalysts: they are capable of increasing the rate of reaction without being consumed in the process.

Jns Jakob Berzelius

Some enzymes, such as pepsin and trypsin, which bring about the digestion of meat, control many different reactions, whereas others, such as urease, are extremely specific and may accelerate only one reaction. Still others release energy to make the heart beat and the lungs expand and contract. Many facilitate the conversion of sugar and foods into the various substances the body requires for tissue-building, the replacement of blood cells, and the release of chemical energy to move muscles. Pepsin, trypsin, and some other enzymes possess, in addition, the peculiar property known as autocatalysis, which permits them to cause their own formation from an inert precursor called zymogen. As a consequence, these enzymes may be reproduced in a test tube. As a class, enzymes are extraordinarily efficient. Minute quantities of an enzyme can accomplish at low temperatures what would require violent reagents and high temperatures by ordinary chemical means. About 30 g (about 1 oz) of pure crystalline pepsin, for example, would be capable of digesting nearly 2 metric tons of egg white in a few hours.

Each enzyme is selectively specific for the substance in which it causes a reaction and is most effective at a temperature peculiar to it. Although an increase in temperature may accelerate a reaction, enzymes are unstable when heated. The catalytic activity of an enzyme is determined primarily by the enzyme's amino-acid sequence and by the tertiary structurethat is, the three-dimensional folded structureof the macromolecule. Many enzymes require the presence of another ion or a molecule, called a cofactor, in order to function.


Enzymes lower the barriers that normally prevent chemical reactions from occurring (or slow them down) by decreasing the required activation energy. Thus, in the presence of enzymes, reactions proceed and/or proceed at a faster rate.





Enzymes are very specific, and it was suggested by Emil Fischer in 1894 that this was because both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This is often referred to as "the lock and key" model. However, while this model explains enzyme specificity, it fails to explain the stabilization of the transition state that enzymes achieve. The "lock and key" model has proven inaccurate and the induced fit model is the most currently accepted enzyme-substrate-coenzyme figure.


In 1958 Daniel Koshland suggested a modification to the lock and key model: since enzymes are rather flexible structures, the active site is continually reshaped by interactions with the substrate as the substrate interacts with the enzyme.

Daniel Koshland

The substrate does not simply bind to a rigid active site, the amino acid side chains which make up the active site are moulded into the precise positions that enable the enzyme to perform its catalytic function. In some cases, such as glycosidases, the substrate molecule also changes shape slightly as it enters the active site. The active site continues to change until the substrate is completely bound, at which point the final shape and charge is determined.



The most popular known application of enzymes is in the manufacture of enzymatic washing agents (detergents). Since last 40 years, the use of enzymes in detergents has been the largest of all enzyme applications. Consumers of detergents are actual users of an enzymatic product. In majority of other applications, enzymes are used as auxiliary agents at some point in the manufacturing process and are not, as a rule, present in the finished product - not at any rate in an active form.

Proteases are the most widely used enzymes in the detergent industry. They remove protein stains such as grass, blood, egg and human sweat. These organic stains have a tendency to adhere strongly to textile fibers. The proteins act as glues, preventing the waterborne detergent systems from removing some of the other components of the soiling, such as pigments and street dirt. The inefficiency of nonenzymatic detergents at removing proteins can result in permanent stains due to oxidation and denaturing caused by bleaching and drying. Blood, for example, will leave a rustcoloured spot unless it is removed before bleaching. Proteases hydrolyse proteins and break them down into more soluble polypeptides or free amino acids. As a result of the combined effect of surfactants and enzymes, stubborn stains can be removed from fibers.

Though enzymes can easily digest protein stains, oily and fatty stains have always been troublesome to remove. The trend towards lower washing temperatures has made the removal of grease spots an even bigger problem. This applies particularly to materials made up of a blend of cotton and polyester. The lipase is capable of removing fatty stains such as fats, butter, salad oil, sauces and the tough stains on collars and cuffs.

Amylases are used to remove residues of starch-based foods like potatoes, spaghetti, custards, gravies and chocolate. This type of enzyme can be used in laundry detergents as well as in dishwashing detergents.

The development of detergent enzymes has mainly focused on enzymes capable of removing stains. However, a cellulase enzyme has properties enabling it to modify the structure of cellulose fiber on cotton and cotton blends. When it is added to a detergent, it results into the following effects:

Colour brightening-When

garments made of cotton or cotton blends have been washed several times, they tend to get a 'fluffy' look and the colours become duller. This effect is due to the formation of micro fibrils that become partly detached from the main fibres. The light falling on the garment is reflected back to a greater extent giving the impression that the colour is duller. These fibrils, however, can be degraded by the cellulase enzyme, restoring a smooth surface to the fibre and restoring the garment to its original colour.


enzyme also has a significant softening effect on the fabric, probably due to the removal of the micro fibrils.

Soil removal-Some dirt particles are trapped in the network of micro fibrils and are released when the micro fibrils are removed by the cellulase enzyme.


Enzymes have been used in the pulp and paper industry to soften wood fibers, improve drainage, and present alternatives to chemical bleaching.


- Paper is made from cellulose fibers, which must be separated from a tough wood fiber called lignin. The step by step process used to separate cellulose from lignin and other wood components is known as pulping. It is a time and energy consuming

process, involving the mechanical processing of wood or the treatment of wood with harsh chemicals. In biopulping, cellulase and xylanase enzymes made by lignin-degrading fungi are used to pre-treat wood and break down the lignin fibers. Removing lignin prior to further wood pulping saves time and energy, and decreases the quantities of chemicals used.


- Enzymes can also improve water drainage during wood pulping, a process that often slows down paper production. When fine lignin fibers are degraded by enzymes, less water is absorbed, thereby reducing drainage times, lessening the energy required to dry the paper, and producing a cleaner water runoff.

Bleach Boosting

- Lignin fibers that remain in wood pulp are colored and must be bleached, usually by harsh chlorine compounds under high pressures. As an alternative, enzymes may be used to remove fine surface fibers, thereby reducing the bleaching process or eliminating it altogether.


BREAD-MAKINGBread is the most common and traditional foods around the world. But bread actually has close links with enzymes. For years, enzymes such as malt and fungal alpha-amylase have been used in bread making. Due to the changes in the baking industry and the ever-increasing demand for more natural products, enzymes have gained real importance in breadmaking.

The dough for bread, rolls, buns, etc. consists of flour, water, yeast, salt and other ingredients such as sugar and fat. Flour consists of gluten, starch, non-starch polysaccharides, lipids, etc. When the dough is made, the yeast starts to work on the fermentable sugars, transforming them into alcohol and carbon dioxide, thus rising the dough. In the beginning, the fermentation goes smoothly whether sugar has been added or not, because flour always contains a certain amount of fermentable sugar. But when this has been used up, the fermentation process will cease unless new supplies of sugar are made available to the yeast.

Amylases degrade starch and produce small dextrins for the yeast to
act. Gluten is a combination of proteins, which form a large network during dough formation. This network holds the gas in dough proofing and baking. The strength of this network is very important for the quality of all bread raised by yeast. Enzymes such as proteases, xylanases and lipases directly or indirectly improve the strength of the gluten network and so improve the quality the bread.


A small percentage of pentosans (non-starch polysaccharides) are present in flour. Pentosans have an important role in bread quality due to their water absorption capability and interaction with gluten, which is vital for the formation of the loaf structure. By hydrolysing the pentosans using some enzymes like hemicellulase, pentosanase or xylanase, the dough becomes easier to handle and the resulting bread has a bigger loaf volume and an improved crumb structure.


Another application of enzymes in baking is in the production of biscuits and crackers. The requirements of the flour are altogether different from those in bread-making; soft flour' which produces a dough with pronounced plastic properties is preferred. For this purpose, flour with relatively low protein content is desirable. The gluten protein structure should not be too strong, otherwise the dough will be too difficult to handle. Unless flour with these properties is available, it is necessary to add an agent to weaken the gluten. Reducing agents (substances which have the opposite effect to oxidizing agents) have been used for this purpose, in particular sodium bisulphite. The bisulphite has the desired effect on the gluten, but unfortunately it affects other substances in the flour, including the content of vitamin B1 (thiamine). This vitamin is completely or partially destroyed. Sodium bisulphite has been banned in certain countries and is becoming less popular due to health risks. An alternative is the application of a protein-degrading enzyme. This softens the gluten without affecting the other constituents of the dough. Several fungal and bacterial proteases can be used for this purpose. Proteases can also be used when making bread with 'hard flour' i.e. flour high in gluten protein.


Before cotton yarn or fabric can be dyed, it goes through a number of processes in a textile mill. One important step is scouring - the complete or partial removal of the non-cellulosic components of native cotton such as waxes, pectins, hemicelluloses and mineral salts as well as impurities such as machinery and size lubricants. Scouring gives a fabric with a high and even wet ability that can be bleached and dyed successfully. Today, highly alkaline chemicals such as sodium hydroxide are used for scouring. These chemicals not only remove the impurities but also attack the cellulose, leading to a reduction in strength and loss of weight of the fabric. Furthermore, the resulting wastewater has a high COD (chemical oxygen demand), BOD (biological oxygen demand) and salt content.


remove the non-cellulosic components. The enzymatic treatment leaves the cellulose structure almost intact, so it reduces weight loss and strength loss. Bio-Scouring has a number of potential advantages over traditionally prepared textiles. It reduces total water consumption by around 25%, the treated yarn/fabrics retain their strength properties, and the weight loss is much less than for processing in traditional ways. Bio-Scouring also gives softer cotton textiles.


- For fabrics made from cotton or blends, the warp threads are coated with an adhesive substance know as 'size; to prevent the threads breaking during weaving. Although many different compounds have been used to size fabrics, starch and its derivatives

have been the most common sizing agent. After weaving, the size must be removed again in order to prepare the fabric for dyeing and finishing. Enzymes like heat stable amylase, fungal amylase are used for desizing woven fabrics because of their highly efficient and specific way of desizing without harming the yarn

BIO-POLISHINGCotton and other natural fibres based on cellulose can be improved by an enzymatic treatment known as BioPolishing. This treatment gives the fabric a smoother and glossier appearance. The treatment is used to remove 'fuzz' - the tiny strands of fibre that protrude from the surface of yarn. A ball of fuzz is called a 'pill' in the textile trade. After BioPolishing, the fuzz and pilling are reduced. The other benefits of removing fuzz are a softer and smoother handle, and superior colour brightness.


What is stone washing- stone washing is a textiles manufacturing process typically utilized by the fashion industry, in order to give a newly-assembled cloth garments a worn-out appearance. Stone-washing also helps to increase the softness and flexibility of otherwise stiff and rigid fabrics like denim. In the traditional stonewashing process, the blue denim was faded by the abrasive action of pumice stones on the garment surface. Nowadays, denim finishers are using a special cellulase. Cellulase works by loosening the indigo dye on the denim in a process know as 'Bio-Stonewashing'. A small dose of enzyme can replace several kilograms of pumice stones. The use of less pumice stones results in less damage to garment, machine and less pumice dust in the laundry environment.


opened up new possibilities in denim finishing by increasing the variety of finishes available. For example, it is now possible to fade denim to a greater degree without running the risk of damaging the garment. Productivity can also be increased because laundry machines contain fewer stones or no stones and more garments.


Enzymes are required for the production of cheeses, yogurt and other dairy products, while others are used in a more specialized fashion to improve texture or flavour. Five of the more common types of enzymes and their role in the dairy industry are described below.

Milk contains proteins, specifically caseins, that maintain its liquid form. Proteases are enzymes that are added to milk during cheese production, to hydrolyze caseins, specifically kappa casein, which stabilizes micelle formation preventing coagulation. Rennet and rennin are general terms for any enzyme used to coagulate milk. Technically rennet is also the term for the lining of a calf's fourth stomach. The most common enzyme isolated from rennet is chymosin. Chymosin can also be obtained from several other animal, microbial or vegetable sources, but indigenous microbial chymosin (from fungi or bacteria) is ineffective for making cheddar and other hard cheeses. Limited supplies

of calf rennet have prompted genetic engineering of microbial chymosin by cloning calf prochymosin genes into bacteria. Bioengineered chymosin may be involved in production of up to 70% of cheese products.

Milk contains a number of different types of proteins, in addition to the caseins. Cow milk also contains whey proteins such as lactalbumin and lactoglobulin. The denaturing of these whey proteins, using proteases, results in a creamier yogurt product. Destruction of whey proteins is also essential for cheese production. During production of soft cheeses, whey is separated from the milk after curdling, and may be sold as a nutrient supplement for body building, weight loss, and lowing blood pressure, among other things. There have even been reports of dietary whey for cancer therapies, and having a role in the induction of insulin production for those with Type 2 diabetes. Proteases are used to produce hydrolyzed whey protein, which is whey protein broken down into shorter polypeptide sequences. Hydrolyzed whey is less likely to cause allergic reactions and is used to prepare supplements for infant formulas and medical uses. 3.


Lactase is a glycoside hydrolase enzyme that cuts lactose into its constituent sugars, galactose and glucose. Without sufficient production of lactase enzyme in the small intestine, humans become lactose intolerant, resulting in discomfort (cramps, gas and diarrhea) in the digestive tract upon ingestion of milk products. Lactase is used commercially to prepare lactose-free products, particularly milk, for such individuals. It is also used in preparation of ice cream, to make a creamier and sweeter-tasting product. Lactase is usually prepared from Kluyveromyces sp. of yeast and Aspergillus sp. of fungi.

The enzyme Catalase has found limited use in one particular area of cheese production. Hydrogen peroxide is a potent oxidizer and toxic to cells. It is used instead of pasteurization, when making certain cheeses

such as Swiss, in order to preserve natural milk enzymes that are beneficial to the end product and flavour development of the cheese. These enzymes would be destroyed by the high heat of pasteurization. However, residues of hydrogen peroxide in the milk will inhibit the bacterial cultures that are required for the actual cheese production, so all traces of it must be removed. Catalase enzymes are typically obtained from bovine livers or microbial sources, and are added to convert the hydrogen peroxide to water and molecular oxygen.


Lipases are used to break down milk fats and give characteristic flavors to cheeses. Stronger flavored cheeses, for example, the Italian cheese, Romano, are prepared using lipases. The flavor comes from the free fatty acids produced when milk fats are hydrolyzed. Animal lipases are obtained from kid, calf and lamb, while microbial lipase is derived by fermentation with the fungal species Mucor meihei. Although microbial lipases are available for cheese-making, they are less specific in what fats they hydrolyze, while the animal enzymes are more partial to short and medium-length fats. Hydrolysis of the shorter fats is preferred because it results in the desirable taste of many cheeses.


Juices extracted from ripe fruit contain a significant amount of pectin. Pectin imparts a cloudy appearance to the juice that many consumers do not find appealing, it also makes fruit juice unstable.

Enzymes increase processing capacity and improve economy in the fruit juice and wine industries. The most commonly used enzymes in these industries are pectinases. Pectinases increase juice yields and accelerate juice clarification. Pectinases are naturally occurring enzymes that act on pectin yielding a crystal clear juice with the appearance, stability, mouth-feel, taste, and texture characteristics preferred by consumers. While pectinases naturally occur in most fruits used to make juice, the manufacturer often adds more to produce clear juice in a reasonable span of time.


One of the oldest applications of industrial enzymes is processing hides and skins for leather. Hides and skins contain proteins and fat in between collagen fibers and before tanning; these substances should be partially and fully removed. The proteins can be removed by proteases and lipases as well as other chemicals can remove the fat. Today, proteases and lipases are mainly used for soaking, bating and enzyme assisted un-hairing. Using lipases to dissolve and remove fat is a recent development and lipases are now extensively used for leather processing in many parts of the world.

To make leather pliable, the hides and skins require an enzymatic treatment before tanning know as bating. During bating, scud is loosened and other unwanted proteins are removed. Bating de-swells swollen pelts and prepares leather for tanning. It makes the grain surface of the finished leather clean, smooth and fine. Bating with proteases is an indispensable operation of leather processing to obtain best quality of leather and cannot be substituted with a chemical process. Traditional methods for bating employed manure of dog, pigeon or hen. These were very unpleasant, unreliable and slow methods. Bio-technical developments in science have now completely replaced these methods with use of industrial enzymes (mostly proteases).

Soaking is first important operation of leather processing. Hides and skins received into a tannery are in the four conditions, as green or fresh, as wet salted, as dry salted or as dried. It is advisable to carry out soaking for all types of skin and hides to obtain best quality leather. Soaking cleans hides and skins by removing dirt, blood, flesh, grease, dung etc. and most importantly, re-hydrates them to bring skins as far as possible back to state of green hides. Soaking agents fall into three categories, like Chemical Agents, Surface-active agents and enzymatic agents. Enzymatic agents are biocatalyst. Specific protease and lipase enzymes enhance water uptake by dissolving intrafibrillary proteins that cement fibres together and disperse fats and oils together with dirt and other contaminants present on skin.

The conventional and most wide spread way to remove hair from bovine hides is to use lime and sodium sulphide in a hair-burning process. They dissolve the hair and open up the fibre structure. Most importantly, enzyme-assisted un-hairing results in a cleaner grain

surface and improved area yield and softness. Proteases are used in unhairing process.

DEGREASINGLipases are a type of enzyme that specifically degrades fat and so cannot damage the leather itself. Lipases hydrolyse not just the fat on the outside of the hides and skins, but also the fat inside the skin structure. Once most of the natural fat has been removed, subsequent chemical treatments such as tanning, re-tanning and dyeing have a better effect. The main advantages of using lipases are a more uniform colour and a cleaner appearance. Lipases also improve the production of hydrophobic (waterproof) leather; Lipases represent a more environmentally sound method of removing fat.


Animal feed is composed of plant material, cereals and vegetable proteins, which cannot be fully digested and utilized by animals. However, feed utilization and digestion can often be increased by the addition of external enzymes to the feed. Many cereals have a proportion of their energy in the form of nonstarch polysaccharides (NSPs), more commonly known as fibre. Enzymes are to break down these NSPs, which lead in increase of metabolisable energy and protein utilization.

In some cereals, a large part of the NSP is soluble and causes high viscosity in the small intestine of a monogastric animal. As a result, digestion becomes impaired. Selected microbial enzymes can partially degrade this NSP, lowering viscosity in the intestine and improving feed utilization. Many vegetable protein sources, such as soybean meal, also contain NSP. The addition of selected microbial enzymes can be used to break down the NSP and make it available to the animal. Just as with cereals, the metabolisable energy and protein utilization for vegetable protein sources can be improved by using the correct combination of amylases and proteases. In almost all plant material used for animal feed, a large part of the mineral phosphorus is bound in the form of phytic acid, which cannot be degraded by monogastric animals. Phytase liberates part of the bound phosphorus and makes it possible to reduce the phosphorus content of the feed by 25-30%.


The main cost in the raising of poultry is the feed, which is mainly cereal-based. Maize (corn) has a low content of soluble NSPs and is considered to be an ideal cereal. Other cereals contain higher amounts of NSPs that normally impair feed utilization. It is possible to partially degrading these NSPs with selected enzymes acting on specific raw materials. For example, when using a standard dose of the xylanase

enzymes for poultry feed, the metabolisable energy value for wheat increases. Some cereals are also avoided in poultry diets due to the adverse effects caused by NSP. Barley is a prime example because inclusion of more than 10% barley in broiler diets gives rise to wet and sticky droppings as well as reduced growth rates. This is due to beta glucan, a soluble, high viscosity NSP located in the cell walls of the barley grain. By adding microbial enzymes to the barley based feed, the NSP could be degraded giving an improvement in droppings, better feed utilization and faster growth rate.

Global sales of enzymes for industrial use totaled $1,498.0 million in 2004 and will finish out 2005 at $1,557.0 million, according to a new study released by Business Communications Co., Inc. (BCC, Norwalk, CT). Global sales are projected to reach $1,820.3 million, for an average annual growth rate of 4.0% (compounded) during this period. Prospects for industrial applications of enzymes look bright, according to Philip Rotheim, author of the study. Providing the expected growth are increased market penetration in existing applications, new applications, and new technologies improving the performance characteristics to make enzymes more attractive to industry. Food and feed applications dominate industrial enzyme sales on a worldwide basis In the detergents sector, enzyme market penetration is nearly complete, especially in laundry detergents. Dishwashing detergents still offer room for growth

In the textiles sector, enzymes for cellulosic material dominates. Owing to a fashion change, the market for enzymes that process denim, which creates the "stonewashed" look, is expected to stall. On the other hand, new types of cellulosic fiber being introduced to the market are expected to pull up the market for enzymes need for their processing.


AAGR %, Market Sector Food and animal feed Detergents/cleaners Textiles, leather, fur Pulp and paper Chemicals Total 2003 705.0 475.2 161.0 97.6 59.2 1,498.0 2004 729.7 498.0 164.2 104.3 60.8 2005 833.1 600.9 182.7 136.0 67.6 97-02 3.5 4.8 2.0 6.9 2.7 19.9

1,557.0 1,820.3

Source: BCC report C-147NA: "Enzymes for Industrial Applications

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