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International Journal of Microbiological Research 4 (2): 203-211, 2013 ISSN 2079-2093 © IDOSI Publications, 2013 DOI: 10.5829/idosi.ijmr.2013.4.2.75170
Fungal Amylase - A Review P. Saranraj and D. Stella Department of Microbiology, Annamalai University, Chidambaram-608 002, Tamil Nadu, India Abstract: The use of enzymes in industrial processes is beginning to deliver its promise. Enzymes have high catalytic rates and work in aqueous solution. Furthermore, their exquisite specificity keeps unwanted side reactions to a minimum, maximizing yield. Because of this, a major problem in the industrial exploitation of enzymes is lack of stability. An example that illustrates both the possibilities and also the limitations of the industrial use of enzymes, is starch processing, which is considered an unqualified success of modern industrial biotechnology. The present review was focused on fungal amylase and this review assesses the following topics: Classification of amylase, fungal amylase, biochemical properties of amylases, fermentation studies on fungal amylase production and uses of amylases. Key words: Enzyme
Amylase
Fungi
Biochemical Properties and Fermentation
INTRODUCTION
brewing, detergent, textile and paper industries with the advent of new frontiers in biotechnology, the spectrum of amylase application has expanded into many other fields such as clinical, medical and analytical chemistry [6]. Hundreds of different species of fungi inhabit the soil, especially near the soil surface where aerobic conditions prevail. Such fungi are active in degrading a wide variety of biological materials present in the soil. Considering that a strain of Aspergillus niger isolated by soil during a screening program for amylase producing microorganisms. Amylases are a group of enzymes that have been found in several microorganisms like bacteria and fungi. Fungal source is confined to terrestrial isolates, mostly to Aspergillus species. Among the microorganisms, many fungi had been found to be good sources of amylolytic (amylase) enzymes. Studies on fungal amylase especially in the developing countries have concerted mainly on Aspergillus niger probably because of the ubiquitous nature and non-fastidious nutritional requirements of the organisms [7]. Bacteria, yeasts and fungi can grow on solid states and find applications in solid state fermentation process. Filamentous fungi are the best adapted for solid state fermentation. The hyphal mode of fungal growth and their good tolerance to low water activity and high osmotic pressure conditions make fungi efficient and competitive in natural microflora for bioconversion of solid substrates. Amylases have been produced by submerged
Microorganisms in particular have been regarded as treasure of useful enzymes. There is a great variation between various genera as to their ability to produce a specific enzyme for the production of particular enzymes varies with the particular medium and pH. In recent years, the potential of using microorganisms as biotechnological sources of industrially relevant enzymes has stimulated interest in the exploration of extracellular enzymatic activity in several microorganisms [1- 3]. The first enzyme produced industrially was an amylase from a fungal source in 1894, which was used for the treatment of digestive disorder [4]. Amylase is an enzyme that breaks down starch into sugar. Amylase is present in human saliva, where it begins the chemical process of digestion. Starch degrading enzyme like amylase have received a great deal of attention because of their perceived technological significance and economic benefits. This enzyme is also used for the commercial production of glucose. In storage tissues such as seeds, starch a polysaccharide of glucose is a hydrolyzing for utilization by the growing seedlings to meet its energy requirement [5]. Microbial amylases have successfully replaced chemical hydrolysis of starch in starch processing industries. Besides their use of starch saccharification, they also find potential application in a number of industrial processes such as in food, baking,
Corresponding Author: P. Saranraj, Project Fellow UGC Major Project Department of Microbiology Annamalai University Annamalainagar-608 002 Tamil Nadu, India. Tel: +9994146964.
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fermentation. In recent years, however, the solid state fermentation (SSF) processes have been increasingly applied for the production of this enzyme. Solid state fermentation compared to submerged fermentation is more simple, require lower capital, has superior productivity, reduce energy requirement, simple fermentation media and absence of rigorous control of fermentation parameters, uses less water and produces lower wastewater, has easier control of bacterial contamination and require a low cost for downstream processing [8].
Both -amylase and -amylase are present in seeds; -amylase is present in an inactive form prior to germination, whereas -amylase and proteases appear once germination has begun. Cereal grain amylase is key to the production of malt. Many microbes also produce amylase to degrade extracellular starches. Animal tissues do not contain -amylase, although it may be present in microorganisms contained within the digestive tract. -Amylase: In addition to cleaving the last (1-4)glycosidic linkages at the nonreducing end of amylose and amylopectin, yielding glucose, -amylase will cleave (1-6) glycosidic linkages. Unlike the other forms of amylase, -amylase is most efficient in acidic environments and has an optimum pH of 3.
Classification of Amylase -Amylase: The -amylases are calcium metalloenzymes, completely unable to function in the absence of calcium. By acting at random locations along the starch chain, -amylase breaks down long-chain carbohydrates, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and "limit dextrin" from amylopectin. Because it can act anywhere on the substrate, -amylase tends to be faster-acting than -amylase. In animals, it is a major digestive enzyme and its optimum pH is 6.7-7.0. In human physiology, both the saliva and pancreatic amylases are -amylases. They are discussed in much more detail at alpha-Amylase. Also found in plants (adequately), fungi (ascomycetes and basidiomycetes) and bacteria (Bacillus). Alpha amylases are one of the important and widely used enzymes whose spectrum of applications has widened in many sectors such as clinical, medicinal and analytical chemistry. Besides their use in starch saccaharification they also find applications in food, baking, brewing, detergent, textile, paper and distilling industry. Alpha amylases (endo-1, 4- -D-glucan glucohydrolase EC 3.2.1.1) constitute the family of endo amylases that randomly cleave the 1,4- -Dglycosidic linkages between adjacent glucose units in the linear amylose chain with retention of -anomeric configuration of the products. Production of fungal alpha amylases has been investigated through submerged (SMF) and solid state fermentation (SSF) [9-11].
Fungal Amylases: Bacteria and fungi secrete amylase to the outside of the cells to carry out extra-cellular digestion. Ellaiah et al. [12] identified amylolytic activity from several fungal species isolated from soil and Aspergillus sp. was found to possess the highest amylase activity [13 ]produced extracellular amylase using bran (wheat bran, rice bran, black gram bran) as carbon source in shake flask cultures of a hemophilic strains of Aspergillus niger. Fungal amylases are used for hydrolyzing carbohydrate, protein and other constitutes of soybeans, wheat into peptides, amino acid, sugars and other low molecular weight compounds. The amylase producing strains of Aspergillus niger have spore bearing heads which are large, tightly packed, globular and may be black or brownish black. They are considered to be mesophilic with optimal temperature for growth between 25°C and 35°C. They are aerobic in nature and can grow over a wide range of hydrogen ion concentration. These organisms can utilize different kinds of agricultural wastes from simple to complex ones, which make them easy to cultivate and maintain in the laboratory [14, 15]. Filamentous fungi are microorganisms secrete large amounts of protein in culture medium. Aspergillus niger has been described as secreting an -amylase and glucoamylase of a number of different molecular weights in submerged culture [16, 17]. Filamentous fungi have been used for the industrial production of a wide variety of native products, such as antibiotic (e.g., penicillin and cephalosporin), organic acid (Citric and acetic acid) and commercial enzymes (e.g., protease, catalase, amylase). Aspergillus niger is an acidifient mould thanks to its -amylase important hydrolytic capacities in the production and its tolerance of acidity (pH < 3), it allows the avoidance of bacterial contamination [18].
-Amylase: Another form of amylase, -amylase is also synthesized by bacteria, fungi and plants. Working from the non-reducing end, -amylase catalyzes the hydrolysis of the second -1,4 glycosidic bond, cleaving off two glucose units (maltose) at a time. During the ripening of fruit, -amylase breaks starch into maltose, resulting in the sweet flavor of ripe fruit. 204
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Biochemical Properties of Amylases Substrate Specificity: As holds true for the other enzymes, the substrate specificity of -amylase varies from microorganism to a microorganism. In general, -amylases display highest specificity towards starch followed by amylose, amylopectin, cyclodextrin, glycogen and maltotriose [30].
The majority of industrial enzymes being used currently belong to the hydrolase group, which utilizes several different natural substrates. Detergent industries are the primary consumers of enzymes, in terms of both volume and value. For years microorganisms have been the principal source of many different enzymes, which were identified after research and currently find their main uses in industrial application several industries employ microbial amylolytic enzymes and a growing industrial application for them is the enzymatic conversion of starch into variants of sugar solution [19-21]. It has been reported that solid state fermentation is the most appropriate process in developing countries due to the advantages it offers. The hyphal mode of fungal growth and their good tolerance to low water activity and high osmotic pressure conditions make fungi efficient and competitive in natural microflora for bioconvension of solid substrates [22, 23]. -Amylase from Aspergillus oryzae was the first microbial enzyme to be manufactured for sale and was named by solid state cultivation for many years. The manufacture had switched to submerged fermentation and such methods have been reviewed. Some difficulties were encountered in making this change since the most effective preparation of some applications contains other enzymes, especially amyloglycosidases and the submerged methods gives a narrower spectrum of additional additives. So, it is worthwhile to isolate a suitable strain of Aspergillus niger for efficient mechanism selection of suitable production media is very essential for growth of microorganisms as well as production of enzyme. The production of alpha-amylase by mouth has been greatly affected by the cultural and nutritional requirement [24- 26]. In most developing countries of the tropics carbohydrate based agricultural products like starchy tubers and cereal occur abundantly. Starchy tubers such as cassava, yam, sweet potato and calcium are important staple foods in the diet of people in most developing countries of the tropics. In these countries, they are widely distributed and more cultivated than cereal. However, despite their importance, a large proportion of their tubers are cost yearly due to inadequate and ineffective storage facilities [27, 28]. Application of these agro-industrial residues in bioprocesses also solve pollution problems, which their disposal may otherwise cause with the advent of biotechnological innovations, mainly in the area of enzyme and fermentation technology, many new areas have opened for their utilization as raw materials for the production of value added fine products 29.
pH Optima and Stability: The pH optima of -amylases vary from 2 to 12. - Amylases from most bacteria and fungi have pH optima in the acidic to neutral range. -Amylase from Alicyclobacillus acidocaldarius showed an acidic pH optimum of 3, in contrast to the alkaline amylase with optima of pH 9 to 10.5 reported from an alkalophilic Bacillus sp. [31]. Extremely alkalophilic -amylase with pH optima of 11 to 12 has been reported from Bacillus sp. In some cases, the pH optimum was observed to be dependent upon temperature as in the case of Bacillus stearothermophilus and on calcium as in the case of Bacillus stearothermophilus. -Amylases are generally stable over a wide range of pH from 4 to 11, however, -amylases with stability in a narrow range have also been reported [30, 32]. Temperature Optima and Stability: The temperature optimum for the activity of amylase is related to the growth of the microorganism. The lowest temperature optimum is reported to be 25 to 30°C for Fusarium oxysporum amylase and the highest of 100 and 130°C from archaebacteria, Pyrococcus furiosus and Pyrococcus woesei, respectively. Temperature optima of enzymes from Micrococcus varians are calcium dependent and that from H. meridiana is sodium chloride dependent [30, 33]. Thermostabilities have not been estimated defector in many studies. Thermostabilities as high as 4 hours at 100°C have been reported for Bacillus licheniformis. Many factors affect thermostability. These include the presence of calcium, substrate and other stabilizers. The stabilizing effect of starch was observed in amylases from Bacillus licheniformis, Lipomyces kononenkoae and Bacillus sp. Thermal stabilization of the enzyme in the presence of calcium has also been reported from time to time [30, 34]. Molecular Weight: Molecular weights of amylases vary from about 10 to 210 kDa. The lowest value, 10 kDa for Bacillus caldolyticus and the highest of 210 kDa for Chloroflexus aurantiacus has been reported. Molecular weights of microbial amylases are usually 50 to 60 kDa as shown directly by analysis of cloned amylase genes and deduced amino acid sequences [30, 35]. 205
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physiology of -amylase production by Aspergillus oryzae during batch cultivation has been done. Accordingly, morphology of Aspergillus oryzae was critically affected by the growth pH [44]. In a series of batch experiments, the authors observed that at pH 3.0 to 3.5, freely dispersed hyphal elements were formed. In the pH range 4 to 5, both pellets and freely dispersed hyphal fragments were observed whereas at pH higher than 6 pellets were the only growth forms recorded. Other groups [45] have recorded similar observations for other strains of Aspergillus oryzae. The optimum growth temperature was found to be 35°C. It is demonstrated that when glucose was exhausted the biomass production stopped whereas the secretion of -amylase increased rapidly [46- 48]. A decline in enzyme production was also accompanied by morphological and metabolic variations during continuous cultivation. The industrial exploitation of SSF for enzyme production has been confined to processes involving fungi and it is generally believed that these techniques are not suitable for bacterial cultivation. The use of the SSF technique in -amylase production and its specific advantages over other methods has been discussed extensively [49- 51]. In the solid state fermentation process, the solid substrate not only supplies the nutrients to the culture, but also serves as an anchorage for the microbial cells. The moisture content of the medium changes during fermentation as a result of evaporation and metabolic activities and thus the optimum moisture level of the substrate is therefore most important [52- 54]. Sumitra Ramachandra et al. [55] carried out the Solid-state fermentation for the production of -amylase using Aspergillus oryzae. Different oil cakes such as coconut oil cake, sesame oil cake, groundnut oil cake and olive oil cake were screened to be used as substrate for the enzyme production and also compared with wheat bran. Groundnut oil cake was found to be the best producer of the enzyme among these. The maximum amount of enzyme was obtained when solid state fermentation was carried out using wheat bran. Groundnut oil cake having initial moisture of 64% and supplemented with lactose and ammonium nitrate at 30°C for 72 hrs uses 2 ml spore suspension. Partial purification of the enzyme using ammonium sulphate fractionation resulted in 2.4-fold increase in the activity. Bertrand Tatsinkou Fossi et al. [56] isolated an amylolytic yeast strain was isolated from starchy soils and its enzyme productivity and activity evaluated. The enzymatic synthesis was optimum at 30°C when the initial pH of fermentation medium was 4.5. After extraction
Carbohydrate moieties raise the molecular weight of some amylases. Glycoproteins have been detected in Aspergillus oryzae, L. kononenkoae, Bacillus stearothermophilus and Bacillus subtilis strains. Glycosylation of bacterial proteins is rare. A carbohydrate content as high as 56% has been reported in S. castelii whereas this is about 10% for other amylases [30, 36]. Inhibitors: Many metal cations, especially heavy metal ions, sulphydryl group reagents, N-bromosuccinimide, hydroxyl mercuribenzoic acid, iodoacetate, BSA, EDTA and EGTA inhibit amylases [30]. Calcium and Stability: Amylase is a metalloenzyme, which contains at least one Ca2+ ion. The affinity of Ca2+ to amylase is much stronger than that of other ions. The amount of bound calcium varies from one to ten. Crystalline Taka- amylase A (TAA) contains ten Ca2+ ions but only one is tightly bound [30, 37]. Calcium free enzymes can be reactivated by adding Ca2+ ions. Some studies have been carried out on the ability of other ions to replace Ca2+ as Sr2+ in Bacillus caldolyticus amylase. Ca2+ in TAA has been substituted by Sr2+ and Mg2+ in successive crystallization in the absence of Ca2+ and in excess of Sr2+ and Mg2+. EDTA inactivated TAA can be reactivated by Sr2+, Mg2+ and Ba2+. In the presence of Ca2+, amylases are much more thermostable than without it. Amylase from Aspergillus oryzae is inactivated in the presence of Ca2+, but retains activity after EDTA treatment. There are also reports where Ca2+ did not have any effect on the enzyme [30,38]. Fermentation Studies on Amylase Production: Solid state fermentation compared to submerged fermentation is more simple, require lower capital has superior productivity, reduced energy requirement, simpler fermentation media and absence of rigorous control of fermentation parameters, uses less water and produces lower waste water, has easier control of bacterial contamination and requires low cost of downstream processing [39, 40]. Industrially important enzymes have traditionally been obtained from submerged fermentation because of the ease of handling and greater control of environmental factors such as temperature and pH. However, solid-state fermentation constitutes an interesting alternative since the metabolites so produced are less costly [41-43]. The effect of environmental conditions on the regulation of extracellular enzymes in batch cultures is well documented. A lot of work on the morphology and 206
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isolated Aspergillus species from various seeds were screened for their ability to produce amylase. A selected strain, Aspergillus sp. showed the highest amylase activity in solid state fermentation. Different substrates were screened for enzyme production, coconut oil cake, groundnut oil cake and rice bran were found to be very good substrates for enzyme production. Different combinations; wheat bran; groundnut oil cake: rice bran (1:2:2) was used which resulted in higher enzyme titre. This combination of substrates was used for further studies on amylase production and characterization. The enzyme amylase was found to be thermostable and active at a wide range of pH. Qunhui Wang et al. [62] investigated the potential of food waste (FW) used as substrate for the glucoamylase production by Aspergillus niger under submerged fermentation. At the optimum concentration of 2.50% (dry basis), smashed food waste (smashed-FW) produced glucoamylase of 126 U/ml after 96 h fermentation, where as 137U/ml of glucoamylase could be attained within the same time from raw food waste (raw-FW) of 3.75%. The particle size showed little effect during enzyme production and could be degraded easily, thus the smash process need not be adopted in this study. The hydrolysis experiment with the produced enzyme demonstrated that about 60 g/l of reducing sugar could be attained from 10% smashed-FW after 2-h hydrolysis. The utilization of food waste for glucoamylase production could save the cost as well as reduce the pollution threatens. Omemu et al.[63] conducted the study with the aim of producing amyloglycosidase enzyme from Aspergillus niger using solid state fermentation and to carry out preliminary characterization of the enzyme produced. Amylolytic Aspergillus niger was isolated from the soil on Remazol Brilliant blue-starch agar and used for enzyme production using rice bran supplemented with soya bean flour in solid state fermentation process. The crude enzyme extract had optimal temperature and pH activities at 60°C and pH 4, respectively. With the exception of cocoyam starch, the enzyme preparation was able to hydrolyze both the cereal (maize) and root starches (yam, cassava, sweet potatoes) tested. Hydrolysis was significantly (P1 mm, 20% > 1mm, 20%; 1mm, 20% and >1 mm, 30% for wheat straw, wheat bran and rye straw respectively. Wheat bran showed highest enzyme production with 160 V/ml under optimum conditions. Hema Anto et al. [58] investigated the Glucoamylase production by solid state fermentation of agro-industrial wastes generated during the processing of paddy to rice flakes along with wheat bran and rice powder by a local soil isolate (Aspergillus sp.). Highest enzyme production was obtained with wheat bran (264 ± 1.44 U/gds) and medium waste (192.1 ± 1.15 U/gds) using 106 spores / ml as inoculum at 28 ± 2°C, pH 5. A combination wheat bran and coarse waste (1:1) gave an enzyme yield as compared to wheat bran alone. Media supplementation with carbon source (0.04g/gds) as sucrose in wheat brand and glucose in coarse and medium waste increased enzyme production to 271.12 ± 0.92; 220.2 ± 0.75 and 208.2 ± 1.99 U/gds respectively. Organic nitrogen supplementation (yeast extract and peptone, 0.02 g/gds) showed a higher enzyme production compared to inorganic source. Kathiresan and Manivannan [59] tested the effects of pH, temperature, incubation time, salinity, sources of carbon and nitrogen in submerged fermentation process in production of amylase by Penicillium fellutanum isolated from coastal mangrove soil. The production medium without the addition of seawater and with provision of maltose as carbon source, peptone as a nitrogen source, incubated for 96 h, maintained with a pH of 6.5 at 30°C, was found optimal for the production of amylase by Penicillium fellutanum. The production of amylolytic enzymes, particularly glucoamylase on solid substrate is more advantageous for the fermentation industry, cereal bran and flours, potato residue and other starchy waste materials have been utilized as fermentation substrate for glucoamylase production by filamentous fungi [60]. Alva et al. [61] 207
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Uses of Amylase: Amylases are among the most important hydrolytic enzymes for all starch based industries and the commercialization of amylases is oldest with first use in 1984, as a pharmaceutical aid for the treatment of digestive disorders. In the present day scenario, amylases find application in all the industrial processes such as in food, detergents, textiles and in paper industry, for the hydrolysis of starch. In this light, microbial amylases have completely replaced chemical hydrolysis in the starch processing industry. They can also be of potential use in the pharmaceutical and fine chemical industries. Today, amylases have the major world market share of enzymes. Several different amylase preparations are available with various enzyme manufacturers for specific use in varied industries [30]. Amylase enzymes find use in bread making and to break down complex sugars such as starch (found in flour) into simple sugars. Yeast then feeds on these simple sugars and converts it into the waste products of alcohol and CO2. This imparts flavour and causes the bread to rise. While Amylase enzymes are found naturally in yeast cells, it takes time for the yeast to produce enough of these enzymes to break down significant quantities of starch in the bread. This is the reason for long fermented doughs such as sourdough. Modern bread making techniques has included amylase enzymes (often in the form of malted barley) into bread improver thereby making the bread making process faster and more practical for commercial use. When used as a food additive Amylase has E number E1100 and may be derived from swine pancreas or mould mushroom. Amylase is also used in clothing and dishwasher detergents to dissolve the starches from fabrics and dishes. Workers in factories that work with amylase for any of the above uses are at increased risk of occupational asthma. 5-9% of bakers have a positive skin test and a fourth to a third of bakers with breathing problems are hypersensitive to amylase. An inhibitor of alpha-amylase called phaseolamin has been tested as a potential diet aid. Blood serum amylase may be measured for purposes of medical diagnosis. A normal concentration is in the range 21-101 U/L. A higher than normal concentration may reflect one of several medical conditions, including acute inflammation of the pancreas (concurrently with the more specific lipase), but also perforated peptic ulcer, torsion of an ovarian cyst, strangulation isles, macroamylasemia and mumps. Amylase may be measured in other body fluids, including urine and peritoneal fluid.
employed in the production of -amylase using submerged and solid state cultivation. The amount of -amylase in enzyme unit (E.U) liberate by each mold was quantified by estimating the amount of reducing sugars produced when the specified quantity of starch was hydrolyzed after incubation at 40°C with known concentration of enzyme solution. Results showed the amylolytic isolates to be Helminthosporium oxysporum, Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus and Penicillium frequestans. The mould Helminthosporium oxysporum liberate 10.77 and 10.42 E.U of -amylase on cassava and yam peels media respectively using submerged cultivation method while Aspergillus flavus produced 11.94 E.U of the enzyme the submerged cultivation methodged cultivation method. Teodoro Suarez-Dieguez et al. [65] studied the effect of pH on the rate of hydrolysis of both sorghum hybrid starches by industrial bacterial amylase and Aspergillus oryzae -amylase. This result suggests that both industrial bacterial amylase and Aspergillus oryzae -amylase had a slightly higher affinity for soluble starch of sorghum montecillo 3 and 1 hybrids, respectively [66]. Arti Gupta et al. [67] characterized the amylase producing Aspergillus niger isolate, optimization of medium composition, cultural conditions for amylase enzyme production, extraction and partial purification of extra cellular enzyme from a potential isolate was investigated using both free and immobilized cells. The alpha amylase of Aspergillus niger had the pH optima ranged at 4-6 and temperature optima ranged at 30-40°C, however the optimum pH, temperature and incubation period for enzyme production was 5.0, 35°C and 5th day for Immobilized cells and 5.0, 30°C and 5th day for free cells respectively. Of the carbon sources, starch was recorded to be the best carbon source for enzyme production. Peptone at 0.03% was the ideal nitrogen source. Sivasakthi et al. [68] evaluated the amylase activity of the amylolytic fungi Aspergillus niger using cassava waste as a feed substrate. Amylase production by Aspergillus niger was detected by the disappearance of blue colour in the Starch agar medium around the microbial colonies after incubation. Cassava was used as the substrates for the amylase production. Solid state fermentation was carried out for the production of amylase using Aspergillus niger. The effect of different carbon sources, nitrogen source, Temperature and pH were determined on enzyme production by Aspergillus niger. Amylase activity was determined by four methods such as DNSA method, Dextrinizing activity method, decrease in Starch-iodine color intensity and Plate assay. 208
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In molecular biology, the presence of amylase can serve as an additional method of selecting for successful integration of a reporter construct in addition to antibiotic resistance. As reporter genes are flanked by homologous regions of the structural gene for amylase, successful integration will disrupt the amylase gene and prevent starch degradation, which is easily detectable through iodine staining.
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CONCLUSION Amylases are among the most important enzymes used in various industries. Research on amylase has progressed very rapidly over the last five decades and potential industrial applications of the enzyme especially in solid waste management have been identified. Major impediments to exploit the commercial potential of amylase are the yield, stability and cost of amylase production. Although, fungal isolates have been extensively studied by many researchers. Further, there arises a need for more efficient amylases in various sectors, which can be achieved either by chemical modification of the existing enzymes or through protein engineering. In the light of modern biotechnology, amylases are now gaining importance in biopharmaceutical applications. Still, their application in food and starch based industries is the major market and thus the demand of amylases would always be high in these sectors.
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