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This review is based on the literatures focused on the enzyme Î±-amylase and their mechanism of action and industrial applications and their important. There are wide sources of amylase including plant, microbial and animal. Bacterial sources has more thermal stability when compared to plant sources or from fungal sources. Depending on the sources the mode of action and conditions required are also varies. In all the industries the application is hydrolysis of starch molecule in to small oligosaccharides and simple sugars. In most the industries it works along with other amylolytic enzymes. Î±-amylase are the most important industrial enzymes widely used in many industries like starch, brewing and baking, and non food industries like textile, paper and detergent. Starch industry major application is in the liquefaction process to produce substrate for other enzyme actions like glucosamylase, pullulanase to produce product like Maltose, Dextrose, Malto dextrins, High fructose syrup. Brewing industry the plant source is utilized by germination of barley, and the developed enzymes activity used for mashing process. Baking industry by using Î±-amylase the there are many advantages including increase in volume and increase in crust colour and increased, and more over it enhances the anti-staling property by preventing retrogradation of starch. But there are many industrial constrains regarding the thermal stability, calcium requirement for the action, inactivation temperature and so on. There are many researches are going on for the production such thermal stable, and modern recombination techniques are used to produce Î±-amylase from yeast cell itself for bio-ethanol production. The researches are showing promising results for the future requirements.
Î±-Amylase are starch hydrolyzing enzymes widely found in microbes, plants and animals. The historical development of the amylase in shows in 9th century application of malt in arrowroot to make sweetener and the commercially used 1930s for a wide applications like brewing, corn syrup production. By the use of Bacillus subtilis and Aspergillus niger for the production of amylase huge quantity started in 1960s (Muralikrishna and Nirmala, 2005). The endo active Î±-amylase act on the Î±-1,4 linkage of both amylase and amylopectin and produce oligosaccharides and then maltotriose, maltose, glucose, and limit dextrins, which cause the liquefaction of the starch (Cui, 2005). The plant source mainly from cereals development especially during germination process and this is applied in brewing industry also important in milling and baking process. The extracellular production of Î±-amylase from microorganism has gained attention for researches (Gupta et al., 2003). Based on the amino acid sequence the Î±-Amylase belongs to Glycoside Hydrolase (GH) 13 family (Skov et al., 2001). The Î±-Amylase is commercially produced from fungal and bacterial sources (Pandey et al., 2000). In starch industry Î±-Amylase is the base enzyme for the production of glucose, fructose, maltose, dextrins, high fructose corn syrup and followed by the other hydrolyzing enzymes (Haki, and Rakshit, 2003). Brewing is a best example for the first application of applied enzymology. The Î±-amylase is developed by the malting process of the barley and this is the one of best utilization of plant source for starch hydrolysis to supply fermentable product to yeast. And also the use Î±-amylase in bread industry also old practice by adding the plant source the malted barley flour. It is not only helped to improve the volume of the bread but also its keeping quality by its anti staling property. And also it increases the colour of the bread because of production of more reducing sugar from starch (Goesaert et al., 2009). And also use in other non food applications like textile industry for reducing the starch viscosity for the application of thread strengthening (Gupta et al., 2003) and in paper industry for viscosity and other property control of starches for coating application and also commercial detergent application (Rodríguez et al., 2006) Î±-amylase widely used. There are many researches are going to improve its performance like thermal stability by recombination (Haki and Rakshit, 2003), immobilization (Türünç et al., 2009) techniques. And also the recombination techniques is applied on yeast to secret directly amylase enzyme hence it can convert starch to alcohol (Shigechi et al., 2002).
SOURCES & PRODUCTION
Î±-Amylases are ubiquitous in distribution, with plants, bacteria and fungi being the predominant sources. Î±-Amylases are universally distributed throughout the animal, plant and microbial kingdoms. However, enzymes from fungal and bacterial sources have dominated applications in industrial sectors (Pandey et al., 2000).
3.2 Media and Growth Conditions
The production of a-amylase by submerged fermentation (SmF) and solid state fermentation (SSF) has been thoroughly investigated. composition of the growth medium, pH of the medium, phosphate concentration, inoculum age, temperature, aeration, carbon source and nitrogen source. Î±-amylase is a induced enzyme by microorganism in presence of maltose or starch. Few days starved mycelia shows optimal maltose induction (Yabuki et al., 1977).
Other than the starch and maltose the growth media need to be adjusted for the other nutrients like nitrogen sources, phosphates, ions, etcâ€¦The organics sources of nitrogen can be from extracts of yeast (Hamilton et al., 1999), soybean flour soybean meal, meat extracts and inorganic sources like ammonium sulphate or ammonium nitrate can be used to increase the productivity. The addition of phosphate also shows enhanced growth and significant increase in the production of Î±-amylase. Ions like Na+, Mg2+, Co2+ in the medium has positive effect on the production of Î±-amylase, where as the Ca2+ has inhibitory effect for Î±-amylase production (Pazlarova and Votruba, 1996).
The physical conditions like temperature, pH, Agitation are also need to be controlled for the optimal production of the Î±-amylase. The stability of the amylase produced in the medium can be affected by the change pH during the growth. So the pH control is essential for the optimal productivity. For the production of Î±-amylase from bacteria for both Solid State Fermentation and Submerged Fermentation the optimal pH is 6-7. The pH requirement will depends the source organism. For example A.oryzae DAE 1679 & E1212 are 3.2-4.2 and 7-8 respectively. And also pH indicates the end of the enzyme synthesis. The growth of organism is depends on the temperature hence the productivity. Production from fungi the temperature vary from 25-55oC depends on the species where as bacteria it is quite wider like 36 - 80oC depends on species. The agitation rate up to 300rpm normally applied to control the mixing and oxygen transfers (Gupta et al., 2003).
Other studies shows the production of Î±-amylase from Aspergillus oryzae by using the low cost medium gruel as inducer, maltodextrin and many quantity if nitrogen sources leads the higher expression of Î±-amylase (Kammoun et al., 2008) and also from Coconut Oil Cake (Ramachandran et al., 2004) as substrate along with the supplementation of starch and peptone also shows increased production.
3.3 Fermentation process
Studies shows that production of high amount of Î±-amylase when the glucose get exhausted and production stopped. Continuous and fed-batch fermentation shows more output than single batch fermentation with a controlled feeding of maltose and production of Î±-amylase also depends the feed rate of the inducer. The dilution rate also have influence on the production of biomass and Î±-amylase so the it need to be controlled for optimal production of enzyme. More production of biomass may leads to the production of less enzyme (Imai et al., 1993).
The purification method applied is depends on the type of organism and extent of the purity requirements, like commercial grade may require less purity whereas pharmaceutical grade may require high level of purity. The process involved in purification generally are culture separation from broth, concentration by precipitation, chromatographical separation, ion exchange and gel filtration.
MECHANISM OF ACTION
Starch: is the second largest biopolymer next to cellulose and the major storage material in higher plants. It is mainly composed of amylose and amylo pectin. In amylose it is the linier linkage of glucose molecules by Î±-1,4 glycosidic linkage. Where as in amylopectin in addition to this Î±-1,4 glycosidic linkage there will be branching by Î±-1,6 glycosidic linkage. In amylose the degree of polymerization (DP) is between 100 and 10,000.
The amylases are classified based on the mode of action as endoamylase and exoamylases. The endo amylases act internally and in random manner. Action these enzymes results in the cleavage of Î±-1,4 glycosidic bonds in amylose, amylopectin and related molecules, which results the formation oligosaccharides and Î±-limit dextrins. This results in rapid decrease in the starch slurry viscosity, hence it is called starch liquification, which will reduce the iodine staining power also.
Exoamylase act on non reducing end and cleave the maltose / glucose molecule from non reducing ends. The end product of the action is maltose and glucose and Î²-limit dextrins.
Source: Goesaert et al., (2009)
The major application of the Î±-amylase is in starch industry for the production of starch hydrolyzed product such as glucose, fructose, maltose, dextrins, high fructose corn syrup which consists about 30% of the world's enzyme market (Van der Maarel et al., 2002). The starch is first gelatinized for dissolution of the starch granules and by action of Î±-amylase the liquefaction takes place by partial hydrolysis and further hydrolysis leads to the production of glucose and maltose by sccharification by action of Î²-amylase. The hydrolysis process takes place after gelatinization, which normally takes place at high temperature. So the cooling is required before addition of the enzyme depends on gelatinization temperature and enzyme thermal stability. If the thermal stability of the enzyme is less the gelatinized mass need to be cooled to a low temperature. There are several thermostable Î±-amylase are isolated from different sources are shown below (Haki, and Rakshit, 2003).
Source: G.D. Haki, S.K. Rakshit (2003)
Glucose and high fructose syrup (HFS): Glucose and high fructose corn syrup can be produced from one of the starch source from corn, potato, rice, sorghum, wheat and cassava. The HFS is used as sweetener in big quantity in beverage industry(Gupta et al., 2003). The study shows that the glucose and HFS are manufactured from cleaned cassava and sweet potato by pasting with water and adjusted for pH (6.5) and temperature (90oC) and then added the Î±-amylase for liquification. After liquefaction again the temperature (60oC) and pH(4.0) adjusted suitable condition for the action of amyloglucosidase.
Making beer is the one of the oldest application of applied enzymology. The major enzymes involved in brewing is amylase which convert the malt starch into fermentable sugars and then by action of yeast enzyme which convert the fermentable sugars to alcohol. Here the major role is from plant derived amylase developed during malting process and also few enzymes produced by micro flora from malt. For high gravity brewing thermostable enzymes are desirable.
As a part of energy security the recyclable fuel demand thrusting. Currently corn starch is widely used for the production of bio ethanol. But future focus is on cellulosic material which is abundant in earth. But the yeast cannot hydrolyze the starch in to sugars, it can only convert the simple sugars to ethanol. So breaking down of higher polymer into small fermentable sugars is necessary. Ethanol can be produced directly by yeast by recombination that produces amylase enzyme. Studies shows that the yeast transformed with Î±-amylase and glucoamylase genes can be replace the external addition of the starch hydrolyzing enzymes. But a complete efficiency the addition of Î±-amylase is necessary (Jamai et al., 2007).
The malt flour along with wheat flour used to improve the quality of bread because of its Î±-amylase content. Legally it approved by U.S and U.K in 1955 and1963 respectively. Addition of Î±-amylase will retard the staling by preventing starch retrogradation. But the excess amount is required to add to survival of enzyme during gelatinization to have remarkable effect on retrogradation. But excess amount will make the dough sticky. But thermostable Î±-amylase can survive during starch gelatinization may present until the end of baking process. So it will produce 4-9 degree polymerized dextrin and shows anti-staling activity (Gupta et al., 2003). The falling number analysis is done for simple and rapid analysis of the flour amylase content and to control the quality of the wheat flour. The post harvest sprouting will cause the reduction in falling number (Mares and Mrva, 2008).
Dough Volume and Crust Colour: In good quality wheat flour the amount of Î±-amylase will be less, but it can be adjusted by adding malt flour or Î±-amylase. By addition of this it will increase the yeast favorable fermentable sugars and reducing sugars. Reducing sugar will involve in the Maillard reaction and hence improve the golden brown crust colour and flavor of the bread. And also the it reduces the dough viscosity while baking during gelatinization and hence crumb settling will retard so the volume of the bread will increases. So the thermostable Î±-amylase has more effect on this process.
Anti-staling Effect: The solid form of structure is due to gelatinization and pasting of starch and setting of gluten protein, which is caused by the combination of moisture, heat and time of the baking. This will result the starch granules will loose the crystalline structure and become amorphous. But certain granular structure can be retained, some amylose will leach out of this granule. There is a chance to form amylose rich region in starch granule. The final structure is formed by the formation of permanent gluten network with gelatinized starch granule. The soft crumb structure and slicability of the bread is due to partially leached amylose together with gluten network are formed During cooling. During storage of the bread the water migration from within the crumb, and crumb to crust occurs. The water content in the amorphous region of the crumb is decreased by water is immobilization within the amylopectin crystalline moieties. This immobilized water can no longer plasticise so crumb firmness will occurs, which decrease the gluten flexibility also. By adding Î±-amylase which will retard the re-crystallination and hence staling time will be extended. But the anti-staling effect of the Î±-amylase is depends on the kind and source of enzyme. The conventional endo acting Î±-amylase from Bacillus subtilis has limited action on retrogradation of amylopectin, hence the anti-staling property will be less. Whereas the maltogenic amylase from Bacillus stearothermophilus form a permanent amylose network, hence the initial firmness will be higher, but during storage the re-crystallization property of the amylopectin will be completely reduced. So anti-staling property during storage will be higher for maltogenic amylase from Bacillus stearothermophilus ( Goesaert et al., 2009)
Other (Paper, Textile & detergent)
For cotton based yarns many modified starches are used. The enzyme hydrolysis done on native and modified starches like starch acetate, starch phosphate and cationic starch (Fan et al., 2008). To make the yarn more strength and prevent from breaking while weaving a protective layer is applied to the threads. And further it can be removed easily. So the desizing of these starch is done by applying Î±-amylase, hence it would not attack the fibers. So the dextrin produced by the action of the Î±-amylase are water soluble and can be washed off easily (Gupta et al., 2003).
In paper industry is used for coated paper for modification of starches. The sizing makes stiffen the paper and prevent mechanical damage. Good coated paper has good erasability also. The coating is done by passing the paper through rollers which transfer the starch slurry on the paper. The viscosity of the starch slurry is depends on the kind and quality of the paper quality. To reduce the viscosity often Î±-amylase is added, and it is depends on the source of starch and Î±-amylase used. The commercial enzymes available brands like TermamylÂ®, Fungamyl, BANÂ® (Novozymes, Denmark).
In detergents by using Î±-amylase the temperature of cleaning water can be reduced and also it reduces the use of environmental threat other chemicals along with other enzymes from renewable sources. As they are biodegradable the risk on aquatic life is nil and treatment of the water will be easy. Normally Î±-amylase from Bacillus licheniformis are used in commercial detergent production (Rodríguez et al., 2006). The use of chemicals also harm the cloths and hands also. 90% of liquid detergent contains Î±-amylase. The stability is depends on the low amount of Ca2+ presence (Gupta et al., 2003). Commercially the Î±-amylase is available for detergent industry under brands such as TermamylÂ®, Stainzyme, NovoprimeÂ® D 659, TermamylÂ® Ultra 300 (Novozymes, Denmark). The amylase will remove the stain from starchy material.
There are many studies are conducted on Î±-amylase enzyme production by modern biotechnology. In brewing the study shows that the recombination done by disrupting Î±-acetolactate synthase gene and introducing the Î±-amylase gene on an amylolytic brewing yeast Saccharomyces pastorianus gained ability to utilize starch as carbon source and able to reduce diacetyle, which is an off flavouring compound in beer without interrupting the brewing performance (Liu et al., 2004). The attempt for the production of Calcium independent and acid stable Î±-amylase have done by protein engineering to prevent use of Calcium in starch and brewing industry. Calcium oxalate formed during the process may cause the blocking of pipe line and heat exchangers. The researches leads to develop Calcium independent Termamyl LCâ„¢ by site-directed mutagenesis (Haki and Rakshit, 2003). The development of novel yeast which has ability to convert starch to ethanol by surface engineering on Saccharomyces cerevisiae. This recombinant yeast shows that higher starch hydrolysis and ethanol production by secretion and co-display also. Both are have improved action of ethanol production from starch (Shigechi et al., 2002). To save the operational cost in industry it is able to produce ethanol directly from low temperature cooked starch by using surface engineered yeast yeast strain YF207/pGA11/pAA12 with co-expressing Rhizopus oryzae glucoamylase and Bacillus stearothermophilus Î±-amylase are developed (Shigechi et al., 2004). The thermal stability of Î±-amylase can be increased by using of enzyme immobilization technology, enzyme Î±-amylase immobilized by cyclic carbonate material, which bind the enzyme by covalent bonds, without changing the pH (Türünç et al., 2009) .
Amylases are the most important enzyme used in industrial process. Many industries like starch industries they are the key additives for the production of dextrose, glucose syrup, dextrin and high fructose malt syrup. In brewing industry also the amylolytic brewing yeast Saccharomyces pastorianus gained ability to directly utilize starch as carbon source and able to reduce diacetyle production. Highly thermal stable bacterial amylase also gained attention in baking industry for its anti-staling activity, which retard or prevent the starch retrogradation hence prevent staling of breads. It is necessary to have favorable conditions for the maximum performance of the enzyme. So mostly the process conditions and the enzyme optimum conditions will not match. Hence the enzyme sources or genetic modification need to be optimized to get required level of performance.