INTRODUCTION:

Although proteases are widespread in nature, microbes serve as a preferred source of these enzymes because of their rapid growth, the limited space required for their cultivation and the ease with which they can be genetically manipulated to generate new enzymes with altered properties that are desirable for various applications. Proteases present in blood serum such as thrombin, plasmin, hageman factor, etc play an important role in blood-clotting, as well as lysis of clots. Other proteases such as elastase and cathepsin G are present in leukocytes playing several different roles in metabolic control. Proteases is one of the fastest "switching on" and "switching off" regulatory mechanisms in the physiology of an organism.

Applications of proteases in food industry

Protease has potential application in dairy industry. Milk contains specific proteins called caseins. Proteases enzymes are added to milk during cheese production, to hydrolyze caseins (specifically kappa casein) which stabilize micelle formation preventing coagulation. Rennet and rennin are used to coagulate milk. The most common enzyme isolated from rennet is chymosin. Bioengineered chymosin may be involved in production of up to 70% of cheese products. Besides casein, milk contains a number of different types of proteins. Denaturing proteins such as lacto albumin and lacto globulin with 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.

Similarly, papain from the leaves and unripe fruit of Carica papaya has been used to tenderise meats. These ancient discoveries have led to the development of various food applications for a wide range of available proteases from many sources, usually microbial. Meat tenderisation by the endogenous proteases in the muscle after slaughter is a complex process which varies with the nutritional, physiological and even psychological state of the animal at the time of slaughter. Meat of older animals remains tough but can be tenderised by injecting inactive papain into the jugular vein of the live animals shortly before slaughter. Injection of the active enzyme would rapidly kill the animal in an unacceptably painful manner. Thus the inactive oxidised disulfide form of the enzyme is used. On slaughter, free thiols that is accumulate in the muscle, activates the papain and thus tenderising the meat.

Proteases are also used in the baking industry. Dough may be prepared more quickly if its gluten is partially hydrolysed. A heat-labile fungal protease is used so that it is inactivated early in the subsequent baking. Weak-gluten flour is required for biscuits in order that the dough can be spread thinly and retains decorative impressions. The gluten in flour derived from these must be extensively degraded if such flour is to be used efficiently for making biscuits or for preventing shrinkage of commercial pie pastry away from their aluminium dishes.

Microbial proteolytic system

Microbial proteases are among the most important hydrolytic enzymes and have been studied extensively since the advent of enzymology. There is renewed interest in the study of proteolytic enzymes, mainly due to there cognition that these enzymes not only play an important role in the cellular metabolic processes but have also gained considerable attention in the industrial community. Intracellular proteases are important for various cellular and metabolic processes, such as sporulation and differentiation, protein turnover, maturation of enzymes and hormones and maintenance of the cellular protein pool. Extracellular proteases are important for the hydrolysis of proteins in cell-free environments and enable the cell to absorb and utilize hydrolytic products (Kalisz 1988).At the same time, these extracellular proteases have also been commercially exploited to assist protein degradation in various industrial processes (Kumar and Takagi1999; Outtrup and Boyce 1990).

Today, proteases account for approximately 40% of the total enzyme sales in various industrial market sectors, such as detergent, food, pharmaceutical, leather, diagnostics, waste management and silver recovery. However, until today, the largest share of the enzyme market has been held by detergent alkaline proteases active and stable in the alkaline pH range. Microbial proteases have been reviewed several times, with emphasis on different aspects of proteases. Proteases are essential constituents of all forms of life on earth, including prokaryotes, fungi, plants and animals. They can be cultured in large quantities in a relatively short time by established methods of fermentation and they also produce an abundant, regular supply of the desired product. Microorganisms account for a two-third share of commercial protease production in the world (Kumar and Takagi 1999). Microbial proteases are classified into various groups, dependent on whether they are active under acidic, neutral, or alkaline conditions and on the characteristics of the active site group of the enzyme, i.e. metallo- (EC.3.4.24), aspartic- (EC.3.4.23), cysteine- or sulphydryl- (EC.3.4.22), or serine-type (EC.3.4.21) (Kalisz 1988; Rao et al. 1998). Alkaline proteases (EC.3.4.21–24, 99) are defined as those proteases which are active in a neutral to alkaline pH range. They either have a serine centre (serine protease) or are of metallo-type (metalloprotease). Alkaline serine proteases are the most important group of enzymes exploited commercially. Alkaline proteases account for a major share of the enzyme market all over the world (Godfrey and West 1996; Kalisz 1988). Alkaline proteases from bacteria find numerous applications in various industrial sectors and different companies worldwide have successfully launched several products based on alkaline proteases.

Food and feed industry

Traditionally, microbial proteases have been exploited in food industries in many ways. Alkaline proteases have been used in the preparation of protein hydrolysates of high nutritional value. The protein hydrolysates play an important role in blood pressure regulation and are used in infant food formulations, specific therapeutic dietary products and the fortification of fruit juices and soft drinks (Neklyudov et al. 2000; Ward 1985). The basic function of proteases is to hydrolyse proteins and this property has been exploited for the preparation of protein hydrolysates of high nutritional value. The alkaline proteases are used in hydrolysate production from various natural protein substrates.

Leather industry

The conventional methods in leather processing involve the use of hydrogen sulfide and other chemicals, creating environmental pollution and safety hazards. Thus, for environmental reasons, the bio treatment of leather using an enzymatic approach is preferable as it offers several advantages, e.g. easy control, speed and waste reduction, thus being ecofriendly (Andersen 1998). Alkaline proteases with elastolytic and keratinolytic activitycan be used in leather-processing industries. Proteases find their use in the soaking, dehairing and bating stages of preparing skins and hides. The enzymatic treatment destroys undesirable pigments, increases the skin area and thereby clean hide is produced. Bating is traditionally an enzymatic process involving pancreatic proteases. However, recently, the use of microbial alkaline proteases has become popular (Varela et al. 1997). Alkaline proteases speed up the process of dehairing, because the alkaline conditions enable the swelling of hair roots; and the subsequent attack of protease on the hair follicle protein allows easy removal of the hair.

Management of industrial and household waste

Proteases solubilize proteinaceous waste and thus help lower the biological oxygen demand of aquatic systems. Recently, the use of alkaline protease in the management of wastes from various food-processing industries and household activities opened up a new era in the use of proteases in waste management. Dalev (1994) used alkaline protease from B. subtilis for the management of waste feathers from poultry slaughterhouses. Waste feathers make up approximately 5% of the body weight of poultry and are considered to be a high protein source for food and feed, provided their rigid keratin structure is completely destroyed. The use of keratinolytic protease for food and feed industry waste, for degrading waste keratinous material from poultry refuse (Ichidaet al. 2001) and as depilatory agent to remove hair from the drains (Takami et al. 1992d) has been reported. A formulation containing proteolytic enzymes from B. subtilis, B. amyloliquefaciens and Streptomyces sp.and a disulfide reducing agent (thioglycolate), that enhances hair degradation and helps in clearing pipes clogged with hair-containing deposits, is currently available in market. It was prepared and patented by Genex (Jacobson et al. 1985).

Photographic industry

Alkaline proteases play a crucial role in the bioprocessing of used X-ray or photographic films for silver recovery. These waste films contain 1.5–2.0% silver by weight in their gelatin layer, which can be used as a good source of silver for a variety of purposes. Conventionally, this silver is recovered by burning the films, which causes undesirable environmental pollution. Furthermore, base film made of polyester cannot be recovered using this method. Since the silver is bound to gelatin, it is possible to extract silver from the protein layer by proteolytic treatments. Enzymatic hydrolysis of gelatin not only helps in extracting silver, but also the polyester film base can be recycled. Alkaline protease from B. subtilis decomposed the gelatin layer within 30 min at 50–60 °C and released the silver (Fujiwara et al. 1989). Ishikawa et al. (1993) have reported the use of alkaline protease of Bacillus sp. B21-2 for the enzymatic hydrolysis of gelatin layers of X-ray films to release silver particles.

Medical usage

Alkaline proteases are also used for developing products of medical importance. Kudrya and Simonenko (1994) exploited the elastolytic activity of B. subtilis316M for the preparation of elastoterase, which was applied for the treatment of burns, purulent wounds, carbuncles, furuncles and deep abscesses. Kim et al. (1996) reported the use of alkaline protease from Bacillus sp. strain CK 11-4as a thrombolytic agent having fibrinolytic activity. Proteases are also useful and important components in biopharmaceutical products such as contact-lens enzyme cleaners and enzymic debriders (Anwar and Saleemuddin, 2000). The proteolytic enzymes also offer a gentle and selective debridement, supporting the natural healing process in the successful local management of skin ulcerations by the efficient removal of the necrotic material (Sjodahl et al. 2002).

Silk degumming

One of the least explored areas for the use of proteases is the silk industry and only a few patents have been filed describing the use of proteases for the degumming of silk (Kanehisa 2000). Sericin, which is about 25% of the total weight of raw silk, covers the periphery of the raw silk fibers, thus providing the rough texture of the silk fibers. This sericin is conventionally removed from the inner core of fibroin by conducting shrink-proofingand twist-setting for the silk yarns, using starch (Kanehisa 2000). The process is generally expensive and therefore an alternative method suggested is the use of enzyme preparations, such as protease, for degumming the silk prior to dyeing. In a recent study, the silk-degumming efficiency of an alkaline protease from Bacillus sp. RGR-14 was studied and results were analyzed gravimetrically (fiber weight reduction) and by scanning electron microscopy (SEM) of treated silk fiber (Puri 2001). After 5 h of incubation of silk fiber with protease from Bacillus sp., the weight loss of silk fiber was 7.5% (Puri 2001).

Proteases in the detergent industry

The history of detergent enzymes dates back to 1914, when two German scientists, Rohm and Haas, used pancreatic proteases and sodium carbonate in washing detergents. The product was named Burnus, after the white arab cloak. The first detergent containing the bacterial enzyme was introduced into the market in 1956 under the trade name Bio-40. However, it was only in 1963 when an alkaline protease, alcalase, was effectively incorporated in detergent powder and was marketed by Novo Industry, Denmark under the trade name Biotex. Unfortunately, detergent proteases faced a setback in the early 1970s, due to unfavorable publicity when some workers developed an allergic reaction during the handling of these enzymes. This problem was solved by the introduction of dust-free encapsulated products. Today, detergent enzymes account for 89% of the total protease sales in the world; and a significant share of the market is captured by subtilisins and/or alkaline proteases from many Bacillus species. The detergent enzyme market has grown nearly 10-fold during the past 20 years.

Beginning in 1995, however, there was considerable need for rationalization in the detergent enzyme industry, owing to the relatively high cost of manufacturing, coupled with increased pressure from detergent manufacturers to drive down raw material costs. One of the important parameters for selection of detergent proteases is the pI value. It is known that detergent proteases perform best when the pH value of the detergent solution in which it works is approximately the same as the pI value for the enzyme. However, there are many more parameters involved in the selection of a good detergent protease, such as compatibility with detergent components, e.g. surfactants, perfumes and bleaches (Bech et al. 1993; Gupta et al. 1999; Kumar et al. 1998), good activity at relevant washing pH and temperature (Aehle et al. 1993; Beg et al. 2002b; Oberoi et al. 2001), compatibility with the ionic strength of the detergent solution, stain degradation and removal potential, stability and shelf life (Showell 1999). Over the past 30 years, the proteases in detergents have changed from being minor additives to being the key ingredients. There is always a need for newer enzymes with novel properties that can further enhance the wash performance of currently used enzyme-based detergents. Conventionally, detergents have been used at elevated washing temperatures, but at present there is considerable interest in the identification of alkaline proteases which are effective over a wide temperature range (Oberoi et al. 2001). In addition, the current consumer demands and increased use of synthetic fibers, which cannot tolerate high temperatures, have changed washing habits towards the use of low washing temperatures (Hasan and Tamiya 1997; Kitayama 1992; Nielsen et al. 1981). This has pushed enzyme manufacturers to look for novel enzyme that can act under low temperatures. Novo Nordisk Bio-industry in Japan has developed a detergent protease called Kannase, which keeps its high efficiency, even at very low temperatures (10-20 °C). Further, a good detergent enzyme should also be stable in the presence of oxidizing agents and bleaches. In general, the majority of the commercially available enzymes are not stable in the presence of bleaching/oxidizing agents. Hence, the latest trend in enzyme-based detergents is the use of recombinant DNA technology to produce bioengineered enzymes with better stability. Bleach and oxidation stability has been introduced through protein engineering by the replacement of certain amino acid residues (Bech et al. 1993; Estell et al. 1985; Wolff et al. 1996; Yang et al. 2000b).

The evaluation of detergent proteases is mainly dependent upon parameters such as the pH and ionic strength of the detergent solution, the washing temperature and pH, mechanical handling, level of soiling and the type of textile. In the case of laundry detergents, protease performance is evaluated by using soiled testfabrics and the efficiency is measured either visually or by measuring the reflectance of light under standard conditions (Durham et al. 1987; Masse and Tilburg 1983; Nielsen et al. 1981; Wolf et al. 1996). In addition, enzyme suppliers and detergent manufacturers are actively pursuing the development of new enzyme activities that address the consumer-expressed need for improved cleaning, fabric care and antimicrobial benefits. However, apart from their use in laundry detergents, they are also popular in the formulation of household dishwashing detergents and both industrial and institutional cleaning detergents (Godfrey and West 1996; Showell1999).

CONCLUSIONS AND FUTURE PROSPECTS:

Several aspects of alkaline proteases have stimulated research on the study of biochemical, regulatory and molecular aspects of proteolytic enzyme systems (Rao et al.1998). Looking into the commercial success of this enzyme class, researchers have now started aiming at the discovery and engineering of novel enzymes that are more robust with respect to their pH and temperature kinetics, using techniques of protein engineering and the identification of active site residues through chemical modifications, X-ray crystallographic data and SDM. In future, protein engineering will offer possibilities of generating proteases with entirely new functions. Hence, although microbial alkaline proteases already play an important role in several industries, their potential is much greater and their applications in future processes are likely to increase in the near future. The pursuit of other, newer approaches, such as novel discovery strategies targeting new dimensions of molecular diversity and technologies to improve performance characteristics by invitro evolutionary changes of protein primary structures will certainly be the major field of development in next few years.

Despite making significant contributions to the economy, the leather industry causes severe environmental pollution owing to the use of various chemicals and the release of a variety of detrimental materials. In leather processing, the first step in the beam house is to remove hairs from hides and skins. Unhairing is defined as the removal of hair from hides. Application of lime sulfide unhairing system, the effluent problems arising from lime are due to its high alkalinity and suspended solids. Besides sulfide, it also liberates toxic hydrogen sulfide, a serious hazard for both tannery workers and sewer men. Tanneries are constantly concerned with the obnoxious odor and the pollution caused by the extremely toxic sodium sulfide used in the dehairing process. The conventional dehairing method involves the use of high proportions of lime and sulfide, which contributes to 80–90% of the total pollution load in the leather industry and generates noxious gases as well as solid wastes, e.g. hydrogen sulfide and lime (Thanikaivelan et al., 2004). Deaths because of this toxic chemical process have also been reported (Balasubramanian&Pugalenthi., 2000 Gupta et al., 2002). Enzymatic unhairing accomplished by proteolytic enzymes is of great commercial importance contributing to more than 40% of the world’s commercially produced enzymes. Approximately 50% of the enzymes produced is used for industrial process (Pepper et al., 1963). Thus tremendous prospect and application of protease enzymes have given importance to this enzyme in terms of research and innovation.

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Received on 10.02.2012 Accepted on 20.04.2012

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