Potential Economic Benefits from Microbial Enzyme - Proteases a highest value commercial enzyme
Enzymes are biological catalysts that increase the rate of chemical reactions taking place within living cells without themselves suffering an overall change. The reactants of enzyme catalyzed reactions are termed substrate and each enzyme is quite specific in character, acting on a particular substrate or substrates to produce a particular product or products. The manufacture of enzymes by organisms especially microorganisms, has been used by humans for thousands of years in the production of foods such as bread, cheese, yoghurt, wine, beer and vinegar. They have a wide range of commercial uses in the food, fuel and pharmaceutical industries, as well as in waste disposal and agriculture.
Commercial Uses of Enzymes
Enzymes are produced by cells either for internal use (intracellular enzymes) or for external use (extracellular enzymes). These enzymes have advantage over inorganic catalysts in commercial processes because they
1. Are highly effective in tiny quantities
2. Function at lower temperature, often needing little or any external heat
3. Operate at low atmospheric pressure, reducing energy needs
4. Are highly specific and therefore produce a very pure product - are essential for food drinks industry
5. Are biodegradable and environmentally acceptable.
Microorganisms are excellent source of enzymes due to their broad biochemical diversity and their susceptibility to genetic manipulation. Among the microbial enzymes, proteases constitute the major share of the enzyme industry because of their number of practical applications in dairy, food, pharmaceutical, leather and detergent industries and collectively account for about 60% of the total worldwide enzyme sales. They are preferred because they possess almost all the characteristics desired for their biotechnological applications. Microbial proteases are produced from high yielding strains by fermentation under controlled conditions.
The proteases constitute a very large and complex group of enzymes which differ in properties such as substrate specificity, active site and catalytic mechanism, pH and temperature activity and stability profiles. Their exquisite specificities provide a basis for their numerous physiological and commercial applications. There is evidence that proteases are involved in the modulation of gene expression, and in enzyme modulation and secretion.
Plant, animal and microbial sources are employed in protease production. The organisms inhabiting protein rich soil tend to utilize more amount of proteinaceous material by producing higher amounts of proteolytic enzymes which are required for the conversion of these substances into peptide bonds and thus to amino acids for their metabolic activities, thereby leading to the disassembly of proteins. Both fungal and bacterial proteases are used in detergent industry. Among the various proteases, bacterial proteases are the most significant, Bacillus sp are specific producers of extra-cellular proteases e.g. Bacillus subtilis, B.amyloliquefaciens, Pseudomonas sp. Most of the bacterial proteases are stable at high temperature and high pH but their production requires cost intensive producers for separation of enzyme from cells
Proteases are classified into 3 - alkaline, neutral and acid proteases based on their pH optima.
Alkaline proteases have an optimum pH greater than or equal to 9.0. They are most commonly used as detergent additives. They are more stable at high temperature and in the alkaline range 9 - 11. They are stable in association with chelating agents and perbonates. B.licheniformis and B.coagulans can be used profitably for large scale production of alkaline protease. On the other hand, alkaline proteases of fungal origin offer an advantage of separation as the mycelia can easily be removed from the final product by simple filtration and the fungus can be grown in inexpensive substrates.
In addition to the use as detergent additives, alkaline proteases are also used in industries like leather, food, photography, pharmaceuticals, waste management etc.
They are secreted by both fungi and bacteria. They are relatively unstable and ions such as Ca++, Na+ and Cl- must be added for more stability. The pH range of activity is fairly narrow and the enzymes are not very stable to increased temperatures. The neutral proteases are also quickly inactivated by alkaline proteases. Because of these limitations, they have restricted industrial application, but do find some uses in leather and food industry for the manufacture of crackers, bread and idli.
They include rennin-like proteases from fungi which are chiefly used in cheese production. The enzymes have a pH optima of 2-4. Acid proteases are used in medicine, in the digestion of soy protein for soy source production and to break down wheat gluten in the baking industry.
Most commercial proteases, mainly neutral and alkaline, are produced by organisms belonging to the genus Bacillus. Bacterial neutral proteases are active in a narrow pH range (pH 5 to 8) and have relatively low thermotolerance. Neutral proteases generate less bitterness in hydrolyzed food proteins than do the animal proteinases and hence are valuable for use in the food industry. Bacterial alkaline proteases are characterized by their high activity at alkaline pH, e.g., pH 10, and their broad substrate specificity. Their optimal temperature is around 60°C. These properties of bacterial alkaline proteases make them suitable for use in the detergent industry. Fungi prooduc a wider variety of enzymes than do bacteria. For example, Aspergillus oryzae produces acid, neutral, and alkaline proteases. The fungal proteases are active over a wide pH range (pH 4 to 11) and exhibit broad substrate specificity.
Uses and Applications of Proteases
The ability of proteases to hydrolyze peptide bonds of proteins is used up widely in the industrial field.
Alkaline proteases are of great importance in detergent industry due to their high thermostability and pH stability. They remove stains such as blood, egg and human sweat. They are more stable during the normal shelf life of the detergent.
Alkaline proteases are of immense use in the leather industry. Proteases are used in the process at the soaking, dehairing and bathing stages. In treatment of fine wooled skins, bacterial protease formulations are used. They play an important role in the production of soft leather.
Alkaline proteases are used for the manufacture of shrinkproof wool. Wool fibres are covered in overlapping scales pointing towards fibre tips. Alkaline proteases prevent the shrinkage of wool by the partial hydrolysis of the fibre tips.
In skin rejuvenation, wrinkle smoothing and dandruff removal, for the removal of atrophied epithelium from the skin surface.
The single major application of alkaline proteases in dairy industry is in cheese making. It accounts for 10% of the total industrial enzyme market.
• Extraction of collagen from skin for collagen replacement therapy.
• Production of amino acids and peptides.
• Due to its gelatinous activity, they are used to decompose the gelatinous coating.
• Used in animal feed industry.
• Used for cleaning DNA during its isolation.
• Used in food industry for meat tenderization and in baking industry for partial hydrolysis of glutan.
• Used in waste management and other bio remediation processes.
• In pharmaceutical industry for preparation of medicines and ointments for debridement of wounds.
• There is a renewed interest in proteases as targets for developing therapeutic agents against relentlessly spreading fatal diseases such as cancer, malaria, and AIDS.
Owing to its potential applications and desirable properties, plenty of research is being done on proteases. Isolation and identification of promising strains, characterization of enzymes and optimization of products is an ongoing process to improve their application. Advances in microbiology and biotechnology have created a favorable condition for the development of proteases.
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