(73c) Alkaline Protease: a Tool to Clean Environment

The use of extracellular enzymes has been standard in many industries for many years; only recently have they been studied as a means for enhancing bioremediation. Extracellular enzymes are produced during a fermentation process and possess the ability to break down bonds within organic compounds and/or catalyze their transformation into less toxic and more biodegradable forms. Unlike many microbes, enzymes remain effective in a wide range of pH and temperature ranges, particularly if they are immobilized on some carrier, and they can degrade a wide variety of compounds. Alkaline proteases have been demonstrated of being able to reduce pathogen counts, reduce the solids content, and increase deflocculation in sludge. Currently, high production costs inhibit the widespread use of extracellular enzymes for remediation, but bench studies and field studies have shown enzymatic treatment to be feasible options for bioremediation. Microbial proteases, also known as peptidyl-peptide hydrolases, 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). 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 center (serine protease) or are of metallo-type (metalloprotease) and the alkaline serine proteases are the most important group of enzymes exploited commercially. Alkaline proteases are one of the most important groups of enzymes, used in various industrial products/processes as detergents, pharmaceuticals, leather, meat tenderizers, protein hydrolyzates, food products and even in the waste processing. Bacillus sp. - the most widely exploited alkaline proteases producer, are often commercially used in bioremediation mixes, or as probiotic agent in aquaculture. 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 biotreament of leather using an enzymatic approach is preferable as it offers several advantages, e.g. easy control, speed and waste reduction, thus being ecofriendly. Alkaline proteases with elastolytic and keratinolytic activity can 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. 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. 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. Protein-based residues usually represent the most significant potential foulants within food bioprocess sectors, such as milk- and meat-processing operations. The replacement of acid/alkali washes from bioprocess cleaning operations with enzymatic alternative to reduce cleaning costs and potential hazard to bioprocess personnel and the environment and also to increase lifetime of equipment would therefore have significant potential advantages. Alkaline protease from B. subtilis was used 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 and as depilatory agent to remove hair from the drains 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. 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. Use of alkaline protease of Bacillus sp. B21-2 has been reported for the enzymatic hydrolysis of gelatin layers of X-ray films to release silver particles. The alkaline proteases of Bacillus sp. B18 and B. coagulans PB-77 were also efficient in decomposing the gelatinous coating on used X-ray films from which the silver could be recovered. A very pragmatic benefit of enzymatic treatment is that the enzymes themselves are biodegradable proteins, meaning that the enzymes that are not recovered will degrade in the environment after they are no longer needed. Unlike other remediation methods, there is no buildup of biomass or chemicals that must be removed. Although enzymatic technology is very promising, it has limitations. Microbes can reproduce and increase their population in order to consume a large amount of substrate, but extracellular enzymes like alkaline protease cannot. Enzymes cannot reproduce themselves, meaning that any increase in enzyme population must come from outside of the system namely, humans adding more enzymes to the system. It has also been shown that these alkaline proteases lose some reactivity after they interact with pollutants and could eventually become completely inactive. This means that the enzyme concentrations must be monitored and controlled in order to optimize enzyme kinetics for site-specific conditions. Since the enzymes are unable to reproduce, they also do not possess the adaptability that microbes posses through mutations. Mutations allow microbes to be able to metabolize new substrates and to survive in what were formerly considered harsh environments. Even though enzymes can survive in a wide range of environments, they are not able to adapt themselves to survive in environments that are outside this range. The main disadvantage of using alkaline proteases for bioremediation is the high cost of the enzymes themselves. Much of the cost of producing enzymes comes from trying to make as pure an enzyme solution as possible, which performs only its proposed function and has no side activities. Crude enzyme solutions are cheaper to produce, but also tend to have side effects and side activities. The costs are expected to decrease as technology and techniques advance and as cheaper growth substrates are explored for the breeding of the parent bacteria and fungi.