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Enzyme Technology Introduction
Enzymes are proteins that catalyze chemical reactions, the enzymes are involves in both biosynthetic and degradative, occurring in living cells. Some RNA molecules have catalytic activity, these are known as ribozymes. The use of purified enzymes for generating a useful product or service constitutes enzyme technology. Enzyme activities are exploited in almost all biotechnological activities; the enzymes may themselves be present in living cells or in an isolated and purified state. Purified enzymes are employed in industrial processes, medicine, research and DNA technology. The bulk nearly one-third, of the enzymes are used for cheese production and in detergents.
The term 'enzyme' was introduced by Kuhne in 1878, although the first observation of enzyme activity in a test tube was reported by Payen and Persoz in 1833. During 1890 Fisher suggested the 'lock and key' model of enzyme action, while mathematical model of enzyme action was proposed by Michaelis and Menten in 1913. In 1926, Sumner crystallized for the first time an enzyme (urease). The transition state theory of enzyme action was put forth by pauling in 1948, and in 1951 Pauling and Corey discovered the a-helix and 3-sheet structure of enzymes. Sanger, in 1953, determined the amino acid sequence of a protein (insulin). In 1986, Cech discovered catalytic RNA (ribozyme), While Lemer and Schutlz developed catalytic antibodies (abzyme).
The enzymes may or may not have anon protein molecule attached to them. (i) Some enzymes contain covalently bound carbohydrate groups, which do not affect the catalytic activity, but may influence enzyme stability or solubility. (ii) Many enzymes have metal ions, while some others possess low weight organic molecules; these are called cofactors, and are essential for enzyme activity. An organic cofactor is commonly known as coenzymes. Cofactors and coenzymes may be covalently or non-covalently attached to protein molecule, called apoenzyme. An apoenzyme lacks catalytic activity in the absence of its specific cofactor. When a cofactor is tightly bound to the apoenzyme that it is difficult to remove it without damaging the enzyme, the cofactor is often called prosthetic group. Both coenzymes and cofactors generally contribute to enzyme activity as well as stability. The complex of an apoenzyme and the cofactor is known as holoenzyme.
Characteristics of Enzymes:
Enzymes are similar to catalyst in the following respects;
- they lower the activation energy of reaction,
- they do not participate in the reaction, and return in their original form at the end of reaction,
- they only increase the rate of reaction;
- they are highly specific.
- all enzymes are protein but all proteins are not enzyme.
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A comparison between enzymes and non-biological catalysts
|Specificity||Highly specific; binding to substrate may be less specific, but the catalyzed reaction is highly specific||Non-specific|
|Rate of reaction||Enhanced by a factor of 103 - 106||Enhancement only a fraction of that by enzymes|
|Regulation||Enzymes subject to a variety of regulations, which increase/decrease the rate of reaction||They are not subject to regulation|
|Saturation||Enzymes have a maximum rate of reaction with respect to substrate concentration||Most catalysts do not show substrate saturation|
|Chemical nature||Enzymes are often proteins (RNA with enzymatic reaction is called ribozyme)||Metals and nonmetals are inorganic molecules|
|Temperature, pressure and pH||Mild biologically compatible||Often high temperature and high pressure|
|Side reaction (other than main reaction)||Do not occur||Occur|
Enzymes Vs Whole Cells:
Purified enzymes offer certain advantages over whole cells, which are follows.
- An enzyme catalyses only a specific reaction, and there is no risk of by-product formation or product breakdown as may be the case with whole cells.
- Whole cells convert a proportion of the substrate into biomass, which is not the case with enzymes.
- The optimum conditions for an enzyme may different from those for the organism producing it; a purified enzyme permits the operations to be performed under the optimal conditions.
- Often purification of the product is much easier than when whole cells are used; this is because there is no interference from the molecules present in the complex medium used for production based on whole cells, the by-products produced by the cells and the constituents of the cells.
However, purified enzymes have limitation of high cost, instability, an enzyme catalizes only a single reaction, while most industrial products are generated after a series of biochemical reactions, which can be perform only by whole cells. In some cases, the by-products produced by whole cells may add to the quality of the product, e.g., in case of wines, various aldehydes, ketones, acids and tannins, etc., increase its qualities. Therefore whole cells are preferred to enzymes in all biotechnological processes where the use of latter does not offer an economic advantage.
Production of enzymes:
Enzymes are obtained from animal tissues, plants, bacteria and fungi, including yeast. The bulk of enzymes, both in the terms of quantity and variety, are derived from microorganism, higher plant being the distant second and animals being the least important. Only animal enzyme to be produced in quantities greater than 2 ton /year is rennet or chymotrypsin obtained from calf stomach. The bulk of plant enzymes are hydrolytic in nature, e.g., papain a-amylase, -amylases, 3-gIucanase, etc. Most of the enzymes are used by food industry. Therefore, initially plant and animal enzymes were preferred over microbial enzymes mainly from considerations of safety and fear of contamination by microorganism, toxins etc.
Increased demands, shortages in supplies of enzymes from plants and animal sources, and difficulties in maintaining a continued supply of these enzymes prompted a much closer and more pragmatic evaluation of the microbial enzymes. These enzymes have found increasing applications even in such areas where enzyme of animal origin were once exclusively used, cheese production. The rennet (proteinase) produced by Mucor miehei is now widely used for cheese production, while rennet from calf stomach was exclusively used in earlier days. Microbes as a source of enzymes have the advantage of large scale production by fermentation, ease in isolation of specially those enzymes, which are excreted into the medium, e.g., most hydrotases, the variety of enzymes produced and the ease in genetic manipulation to enhance enzyme yields and even to modify the enzyme using, if desired, recombinant DNA technology.