Enzymes: Some Basic Concepts and Applications in Food Industry

Anil S. Nandane R.V. Prasad

Enzymes are bio-catalyst, which enhances the rate of chemical reactions. Enzymes are used in wide areas but their applications in food processing are also noteworthy. The major advantage of enzyme as a catalyst over their chemical counterpart is their specificity. Even though the enzymes offers numerous advantages there are certain disadvantages like their unstability and high cost of isolation and purification. The present paper is aimed at providing a brief information on some of the technical aspects of enzymes and their applications in food processing industry. Enzymes are known to catalyze about 4,000 biochemical reactions. Not all biochemical catalysts are proteins, since some RNA moleculescalled ribozymes can also catalyze reactions. Enzyme activity can be affected by other molecules. Inhibitors are molecules that decrease enzyme activity, and activators are molecules that increase activity. Drugs and poisons are often enzyme inhibitors. Enzyme activity is also affected by temperature, pH, and the concentration of substrate. Some enzymes are used commercially, for example, in the synthesis of antibiotics. In addition, some household products use enzymes to speed up biochemical reactions (e.g., enzymes in biological washing powders break down protein or fat stains on clothes; enzymes in steak tenderizers break down long meat proteins, making them easier to chew).Enzymes have a number of distinct advantages over conventional chemical catalysts. Foremost amongst these are their specificity and selectivity not only for particular reactions but also in their discrimination between similar parts of molecules or optical isomers. They catalyse only the reactions of very narrow ranges of reactants (substrates), which may consist of a small number of closely related classes of compounds (e.g. trypsin catalyses the hydrolysis of some peptides and esters in addition to most proteins), a single class of compounds (e.g. hexokinase catalyses the transfer of a phosphate group from ATP to several hexoses), or a single compound (e.g. glucose oxidase oxidises only glucose amongst the naturally occurring sugars). This means that the chosen reaction can be catalysed to the exclusion of side-reactions, eliminating undesirable by-products. Thus, higher productivities may be achieved, reducing material costs. As a bonus, the product is generated in an uncontaminated state so reducing purification costs and the downstream environmental burden. Often a smaller number of steps may be required to produce the desired end-product. In addition, certain stereospecific reactions (e.g. the conversion of glucose into fructose) cannot be achieved by classical chemical methods without a

1. INTRODUCTION
Enzymes are proteins that catalyze (i.e. accelerate) chemical reactions. In these reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts these into different molecules, the products. Almost all processes in the 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. Like all catalysts, enzymes work by lowering the activation energy for a reaction and thus dramatically accelerating the rate of the reaction. By binding the transition-state conformation of the substrate/product molecules, the enzyme distorts the bound substrate(s) into their transition state form, thereby reducing the amount of energy required to complete the transition. Most natural enzymes accelerate their reaction many millions of times faster compared to the uncatalyzed reaction. As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. However, enzymes do differ from most other catalysts by being much more specific.

Anil S. Nandane is with Dept of Food Processing Technology, A.D. Patel Institute of Technology, New Vallabh Vidyanagar, Anand, Gujarat. R.V. Prasad is with Dept of Food Processing Technology, A.D. Patel Institute of Technology, New Vallabh Vidyanagar, Anand, Gujarat.

large expenditure of time and effort. Enzymes work under generally mild processing conditions of temperature, pressure and pH. This decreases the energy requirements, reduces the capital costs due to corrosion-resistant process equipment and further reduces unwanted side-reactions. The high reaction velocities and straightforward catalytic regulation achieved in enzyme-catalysed reactions allow an increase in productivity with reduced manufacturing costs due to wages and overheads.

2. SHORT HISTORY AND BACKGROUND OF ENZYMES

The existence of enzymes has been known for well over a century. Some of the earliest studies were performed in 1835 by the Swedish chemist Jon Jakob Berzelius who termed their chemical action catalytic. It was not until 1926, however, that the first enzyme was obtained in pure form, a feat accomplished by James B. Sumner of Cornell University. Sumner was able to isolate and crystallize the enzyme urease from the jack bean. His work was to earn him the 1947 Nobel Prize.John H. Northrop and Wendell M. Stanley of the Rockefeller Institute for Medical Research shared the 1947 Nobel Prize with Sumner. They discovered a complex procedure for isolating pepsin. This precipitation technique devised by Northrop and Stanley has been used to crystallize several enzymes. 2000BC -The Egyptians and Sumerians developed fermentation for use in brewing, bread-baking and cheese-making. 800 BC-Calves' stomachs and the enzyme, chymosin, were used for cheese-making. 1878 -The components of yeast cells which cause fermentation were identified and the term "enzyme" was first used, derived from the Greek term meaning "in yeast". 1926- Enzymes were first shown to be proteins. 1980s-Enzyme preparations were developed to improve the digestibility and nutrient- availability of certain animal feeds. 1982 -The first food application of a product of gene technology, alpha-amylase, took place. 1988 -Recombinant chymosin was approved and introduced in Switzerland, marking an early approval of a product of gene technology for a food use. 1990-Two food processing aids obtained using gene technology: an enzyme for use in cheesemaking in the US, and a yeast used in baking in the UK. For many thousands of years, man has used naturally occurring micro-organisms bacteria, yeasts and moulds - and the enzymes they produce to make foods such as bread, cheese,

beer and wine. For example in bread-making the enzyme, amylase, is used to break down flour into soluble sugars, which are transformed by yeast into alcohol and carbon dioxide. This makes the bread rise. Today, enzymes are used for an increasing range of applications: bakery, cheese making, starch processing and production of fruit juices and other drinks. Here, they can improve texture, appearance and nutritional value, and may generate desirable flavours and aromas. Currently-used food enzymes sometimes originate in animals and plants (for example, a starch-digesting enzyme, amylase, can be obtained from germinating barley seeds) but most come from a range of beneficial micro-organisms. Where enzymes are produced from micro-organisms (the main types include species of Bacillus, Aspergillus, Streptomyces and Kluyveromyces), these are grown by fermentation in large vats or fermenters with capacities of up to 150,000 litres; here, temperature, nutrients and air supplies are adjusted to suit their optimal development. As in other parts of the food chain, strict rules of hygiene are followed. When the process is complete, the fermenter contains a broth which includes enzymes, nutrients and microbes. This is purified by passing it through a series of filters to remove impurities and extract the enzyme.

3. ENZYME NOMENCLATURE AND CLASSIFICATION

Except for some of the originally studied enzymes such as pepsin, rennin, and trypsin, most enzyme names end in "ase". The International Union of Biochemistry (I.U.B.) initiated standards of enzyme nomenclature which recommend that enzyme names indicate both the substrate acted upon and the type of reaction catalyzed. Enzymes are classified according the report of a Nomenclature Committee appointed by the International Union of Biochemistry (1984). This enzyme commission assigned each enzyme a recommended name and a 4-part distinguishing number. It should be appreciated that some alternative names remain in such common usage that they will be used, where appropriate, in this text. The enzyme commission (EC) numbers divide enzymes into six main groups according to the type of reaction catalysed: 1. Oxidoreductases which involve redox reactions in which hydrogen or oxygen atoms or electrons are transferred between molecules.

This extensive class includes the dehydrogenases (hydride transfer), oxidases (electron transfer to molecular oxygen), oxygenases (oxygen transfer from molecular oxygen) and peroxidases (electron transfer to peroxide). For example: glucose oxidase (EC 1.1.3.4, systematic name, -Dglucose:oxygen 1-oxidoreductase). 2. Transferases which catalyse the transfer of an atom or group of atoms (e.g. acyl-, alkyl- and glycosyl-), between two molecules, but excluding such transfers as are classified in the other groups (e.g. oxidoreductases and hydrolases). For example: aspartate aminotransferase (EC 2.6.1.1, systematic name, L-aspartate:2-oxoglutarate aminotransferase; also called glutamicoxaloacetic transaminase or simply GOT). 3. Hydrolases which involve hydrolytic reactions and their reversal. This is presently the most commonly encountered class of enzymes within the field of enzyme technology and includes the esterases, glycosidases, lipases and proteases. For example: chymosin (EC 3.4.23.4, no systematic name declared; also called rennin). 4. Lyases which involve elimination reactions in which a group of atoms is removed from the substrate. This includes the aldolases, decarboxylases, dehydratases and some pectinases but does not include hydrolases. For example: histidine ammonia-lyase (EC 4.3.1.3, systematic name, L-histidine ammonia-lyase; also called histidase). 5.Isomerases which catalyse molecular isomerisations and includes the epimerases, racemases and intramolecular transferases. For example: xylose isomerase (EC 5.3.1.5, systematic name, D-xylose ketol-isomerase; commonly called glucose isomerase). 6. Ligases, also known as synthetases, form a relatively small group of enzymes which involve the formation of a covalent bond joining two molecules together, coupled with the hydrolysis of a nucleoside triphosphate. For example: glutathione synthase (EC 6.3.2.3, systematic name, L-glutamyl-Lcysteine:glycine ligase (ADP-forming); also called glutathione synthetase).

other reagents. They have molecular weights ranging from 10,000 to 2,000,000. Many enzymes require the presence of other compounds - cofactors - before their catalytic activity can be exerted. This entire active complex is referred to as the holoenzyme; i.e., apoenzyme (protein portion) plus the cofactor (coenzyme, prosthetic group or metal-ion-activator) is called the holoenzyme. Apoenzyme + Cofactor = Holoenzyme According to Holum, the cofactor may be: 1. A coenzyme - a non-protein organic substance which is dialyzable, thermostable and loosely attached to the protein part. 2. A prosthetic group - an organic substance which is dialyzable and thermostable which is firmly attached to the protein or apoenzyme portion. 3. A metal-ion-activator - these include K+, Fe++, Fe+++, Cu++, Co++, Zn++, Mn++, Mg++, Ca++, and Mo+++.

COFACTORS
Some enzymes do not need any additional components to show full activity. However, others require non-protein molecules to be bound for activity. Cofactors can be either inorganic (e.g., metal ions and iron-sulfur clusters) or organic compounds, (e.g., flavin and heme). Organic cofactors (coenzymes) are usually prosthetic groups, which are tightly bound to the enzymes that they assist. These tightly-bound cofactors are distinguished from other coenzymes, such as NADH, since they are not released from the active site during the reaction. An example of an enzyme that contains a cofactor is carbonic anhydrase, with a zinc cofactor bound in its active site. These tightlybound molecules are usually found in the active site and are involved in catalysis. For example, flavin and heme cofactors are often involved in redox reactions. Enzymes that require a cofactor but do not have one bound are called apoenzymes. An apoenzyme together with its cofactor(s) is called a holoenzyme (i.e., the active form). Most cofactors are not covalently attached to an enzyme, but are very tightly bound. However, organic prosthetic groups can be covalently bound (e.g., thiamine pyrophosphate in the enzyme pyruvate dehydrogenase). COENZYMES Coenzymes are small molecules that transport chemical groups from one enzyme to another. Some of these chemicals such as riboflavin, thiamine and folic acid are vitamins,

4. CHEMICAL NATURE OF ENZYMES

All known enzymes are proteins. They are high molecular weight compounds made up principally of chains of amino acids linked together by peptide bonds. Enzymes can be denatured and precipitated with salts, solvents and

this is when these compounds cannot be made in the body and must be acquired from the diet. The chemical groups carried include the hydride ion (H+ + 2e-) carried by NAD or NADP+, the acetyl group carried by coenzyme A, formyl, methenyl or methyl groups carried by folic acid and the methyl group carried by S-adenosylmethionine. Since coenzymes are chemically changed as a consequence of enzyme action, it is useful to consider coenzymes to be a special class of substrates, or second substrates, which are common to many different enzymes. For example, about 700 enzymes are known to use the cofactor NADH. Coenzymes are usually regenerated and their concentrations maintained at a steady level inside the cell: for example, NADPH is regenerated through the pentose phosphate pathway and S-adenosylmethionine by methionine adenosyltransferase.

to refer to optimal conditions, especially with regard to pH, ionic strength, temperature, substrate concentration and the presence and concentration of cofactors and coenzymes. However, these so-termed optimal conditions vary both between laboratories and between suppliers. They also depend on the particular application in which the enzyme is to be used. Additionally, preparations of the same notional specific activity may differ with respect to stability and be capable of very different total catalytic productivity (this is the total substrate converted to product during the lifetime of the catalyst, under specified conditions). Conditions for maximum initial activity are not necessarily those for maximum stability. Great care has to be taken over the consideration of these factors when the most efficient catalyst for a particular purpose is to be chosen

5. ENZYME UNITS
The amount of enzyme present or used in a process is difficult to determine in absolute terms (e.g. grams), as its purity is often low and a proportion may be in an inactive, or partially active, state. More relevant parameters are the activity of the enzyme preparation and the activities of any contaminating enzymes. These activities are usually measured in terms of the activity unit (U) which is defined as the amount which will catalyse the transformation of 1 micromole of the substrate per minute under standard conditions. Typically, this represents 106 - 10-11 Kg for pure enzymes and 10-4 - 10-7 Kg for industrial enzyme preparations. Another unit of enzyme activity has been recommended. This is the katal (kat) which is defined as the amount which will catalyse the transformation of one mole of substance per second (1 kat = 60 000 000 U). It is an impracticable unit and has not yet received widespread acceptance. Sometimes non-standard activity units are used, such as Soxhet, Anson and Kilo Novo units, which are based on physical changes such as lowering viscosity and supposedly better understood by industry. Rightfully, such units are gradually falling into disuse. The activity is a measure of enzyme content that is clearly of major interest when the enzyme is to be used in a process. For this reason, enzymes are usually marketed in terms of activity rather than weight. The specific activity (e.g. U Kg-1) is a parameter of interest, some utility as an index of purity but lesser importance. There is a major problem with these definitions of activity; the rather vague notion of "standard conditions". These are meant

6. ENZYME KINETICS
BASIC ENZYME REACTIONS
Enzymes are catalysts and increase the speed of a chemical reaction without themselves undergoing any permanent chemical change. They are neither used up in the reaction nor do they appear as reaction products. The basic enzymatic reaction can be represented as follows

where E represents the enzyme catalyzing the reaction, S the substrate, the substance being changed, and P the product of the reaction. ENERGY LEVELS Chemists have known for almost a century that for most chemical reactions to proceed, some form of energy is needed. They have termed this quantity of energy, "the energy of activation." It is the magnitude of the activation energy which determines just how fast the reaction will proceed. It is believed that enzymes lower the activation energy for the reaction they are catalyzing. The enzyme is thought to reduce the "path" of the reaction. This shortened path would require less energy for each molecule of substrate converted to product. Given a total amount of available energy, more molecules of substrate would be converted when the enzyme is present (the shortened "path") than when it is absent. Hence, the reaction is said to go faster in a given period of time. THE ENZYME SUBSTRATE COMPLEX A theory to explain the catalytic action of enzymes was proposed by the Swedish chemist Savante Arrhenius in 1888. He proposed that the substrate and enzyme formed some intermediate

substance which is known as the enzyme substrate complex. The reaction can be represented as:

If this reaction is combined with the original reaction equation [1], the following results:

The existence of an intermediate enzymesubstrate complex has been demonstrated in the laboratory, for example, using catalase and a hydrogen peroxide derivative. At Yale University, Kurt G. Stern observed spectral shifts in catalase as the reaction it catalyzed proceeded. This experimental evidence indicates that the enzyme first unites in some way with the substrate and then returns to its original form after the reaction is concluded. CHEMICAL EQUILIBRIUM The study of a large number of chemical reactions reveals that most do not go to true completion. This is likewise true of enzymatically-catalyzed reactions. This is due to the reversibility of most reactions. In general:

reactions they catalyze. A few enzymes exhibit absolute specificity; that is, they will catalyze only one particular reaction. Other enzymes will be specific for a particular type of chemical bond or functional group. In general, there are four distinct types of specificity: Absolute specificity - the enzyme will catalyze only one reaction. Group specificity - the enzyme will act only on molecules that have specific functional groups, such as amino, phosphate and methyl groups. Linkage specificity - the enzyme will act on a particular type of chemical bond regardless of the rest of the molecular structure. Stereochemical specificity - the enzyme will act on a particular steric or optical isomer. One of the theories put forward to account for the specificity of the enzyme is the lock-andkey hypothesis of Emil Fisher. According to this concept, there is a structural complementarities between the active site of the enzyme and the substrate. This concept has proved to be correct in accounting for the absolute specificity exhibited by many enzymes. However a major limitation of the lock-key hypothesis is the implication that the entire conformation of the enzyme is rigid. However, proteins are not rigid structures. They are capable of undergoing slight and subtle changes in their configuration. Therefore, Koshland has proposed an induced-fit theory of enzyme action. According to this theory, the complementarity of the active site conformation to the substrate structure is obtained only after the substrate is bound to the enzyme.

where K+1 is the forward reaction rate constant and K-1 is the rate constant for the reverse reaction. Combining the two reactions gives:

8. CONCLUSION
In food production, enzymes have a number of advantages like they are welcomed as alternatives to traditional chemical-based technology, and can replace synthetic chemicals in many processes. This can allow real advances in the environmental performance of production processes, through lower energy consumption and biodegradability. They are more specific in their action than synthetic chemicals. Processes which use enzymes therefore have fewer side reactions and waste by-products, giving higher quality products and reducing the likelihood of pollution. They allow some processes to be carried out which would otherwise be impossible. An example is the production of clear apple juice concentrate, which relies on the use of the enzyme, pectinase. There are some disadvantages in the use of enzymes which cannot be ignored but which are currently being addressed and overcome. In

Applying this general relationship to enzymatic reactions allows the equation:

Equilbrium, a steady state condition, is reached when the forward reaction rates equal the backward rates. This is the basic equation upon which most enzyme activity studies are based.

7. SPECIFICITY OF ENZYMES
One of the properties of enzymes that makes them so important as diagnostic and research tools is the specificity they exhibit relative to the

particular, the high cost of enzyme isolation and purification still discourages their use, especially in areas which currently have an established alternative procedure. The generally unstable nature of enzymes, when removed from their natural environment, is also a major drawback to

their more extensive use. The use of advanced techniques such as micro-encapsulation may further widen the spectrum of industrial applications of enzymes

9. REFERENCES
[01] Harrow, B., and Mazur, A.: Textbook of Biochemistry, 109, Saunders, Philadelphia (1958). N. Shakuntala Manay & M. Shadaksharswami, Food: Facts and Principles, New Age International Publishers, 116-130, 2001

[03] [04] [05]

[02]

[06]

N. Michael Eskin,Uri Cogan Biochemistry of Foods. www.emc.maricopa.edu/faculty farabee/biobk/BioBookEnzym.html www.worthingtonbiochem.com/introBiochem/introEnzym es.html www.en.wikipedia.org/wiki/Enzyme

Table:1 SOURCES OF INDUSTRIALLY IMPORTANT ENZYMES.

Enzyme Amylases Beta-Glucanases Bromelain Cellulases Chymosin Ficin Glucose isojmerase Lipases Papain Pectinases Proteases Pepsin Source Bacillus and Aspergillus spp. Bacillus spp. Pineapple Trichoderma spp. Calf stomach Figs Bacillus and Streptomyces spp. Pseudomonas spp. Papaya latex Aspergillus spp. Bacillus and Aspergillus spp. Stomach of slaughtered animals Stomach of slaughtered animals Applications Starch liquefaction, baking, brewing, textiles, detergents, etc. Brewing and animal feedstuff Meat tenderization, chill-proofing of beer Textile biopolishing, pulp and paper, detergents Cheese manufacture Meat Tenderization Glucose isomerization to fructose Detergents, oils and fats, baking, leather, paper, etc. Meat tenderization, brewing Pectin hydrolysis in fruit juice clarification Detergents, brewing, meat tenderization, baking, bone cleaning, hydrolyzed animal proteins, functional meat proteins, etc. Digestive aid Protein cross-linking and gelation and meat binding. Digestive aid.

Transglutaminases Streptomyces spp. Trypsin

Table : 2 SOME APPLICATIONS OF ENZYMES IN FOOD PRODUCTION

Market Dairy Enzyme Rennet (protease) Lactase Protease Catalases Brewing Alcohol production Baking Cellulases, beta-glucanases, alpha amylases, proteases, maltogenic amylases Amyloglucosidase Alpha-amylases Amyloglycosidases Maltogen amylase (Novamyl) Protease Pentosanase Glucose oxidase Wine and fruit Pectinase juice Glucose oxidase Beta-glucanases Meat Protease Papain Protein Starch Proteases, trypsin, aminopeptidases Alpha amylase, glucoamylases, hemicellulases, maltogenic amylases, glucose isomerases dextranases, beta-glucanases Inulin Inulinases Production of fructose syrups Breakdown of various components Modification and conversion (eg to dextrose or high fructose syrups) Meat tenderising Purpose / function Coagulant in cheese production Hydrolysis of lactose to give lactosefree milk products Hydrolysis of whey proteins Removal of hydrogen peroxide For liquefaction, clarification and to supplement malt enzymes Conversion of starch to sugar Breakdown of starch, maltose production Saccharification Delays process by which bread becomes stale Breakdown of proteins Breakdown of pentosan, leading to reduced gluten production Stability of dough Increase of yield and juice clarification Oxygen removal