Amylase enzyme

INTRODUCTION


Much of the history of biochemistry of enzyme research, biological catalysis was first recognized and described in the late 1700s, in the studies on the digestion of meat by secretions of stomach and research continued in the 1800s with examinations of the conversion of starch into sugar by saliva and various plant extracts. In the 1850’s Louis Pasture concluded that fermentation of sugar into alcohol by yeast is catalyzed by “ferments”. He postulated that these ferments were inseparable from the structure of living yeast cells a view called vitalism that prevailed for many years. Then in 1897 Eduard Buchner discovered that yeast extracts can ferment sugar to alcohol, proving that fermentation was promoted by molecules that continue to function when removed from the cell. Fredrick W. Kuhne called these molecular enzymes.
The industrial enzyme producers sell enzyme for a wide variety of application. The estimated value of world market is presently about US$ 2.7 billion and estimated to increase by 4% annually through 2012. Detergents (37%), Textile (12%), starch (11%), baking (8%) and animal feed (6%) are the main industries, which use about 75% of the industrially produced enzymes. Amylase enzyme constitutes a class of industrial enzymes, having approx 25% of the enzyme market. An extracellular amylase, specially raw starch digesting amylase has found important application in bio conversion of starches and stay based substrates. The level of α-amylase activity in various human body fluids is of microbial α amylases are used as industrial enzymes. Starches regarding amylolytic enzymes are of great significance in biotechnological application regarding from food, fermentation, textile to paper industries. Although amylases can be derived from several sources, such as plants, animals and microorganisms, the enzymes from microbial sources generally meet industrial demands and had made significant contribution to the production foods and beverages in last three decades. The microbial amylases have almost completely replaced chemical hydrolysis of starch processing industry as earlier described by (Das S, et al . 2011).
An amylase is an enzyme that catalyzes the hydrolysis of starch into sugars. Amylase is present in the saliva of the humans and some other mammals, where it begins the chemical process of digestion. The pancreas and salivary gland make α-amylase to hydrolyze to glucose to supply energy to the body. Plants and some other bacteria also produce amylase. As diastase, amylase was the first enzyme to be discovered and isolated by (Anselme Payen, 1833).  All amylase are glycoside hydrolases and act on α-1,4 -glycosidic bonds.
Members of the genus Bacillus are heterogeneous and they are very versatile in their adaptability to the environment. There are various factors that influence the nature of their metabolic process and enzymes produced. Bacillus species produce a large variety of extra cellular enzymes such as amylase, which have significant industrial importance (Cordeiro, et al., 2003).
Amylase are example of hydrolyses and functions in the hydrolysis of molecules. Amylases are enzymes, which hydrolyze starch molecules to give diverse products including dextrin’s and progressively smaller polymers composed of glucose units (Windish and Mhatre., 1965).
Enzymes are organic catalyst produced by living organism. The reactant in an enzyme catalyzed reaction is called substrate. Most enzymes are larger than their substrate. The active site of an enzyme is that small portion of the molecule which is responsible for the catalytic action of enzyme. Enzymes provide a chemical pathway that has a lower activation energy that the same reaction un-catalyzed.  As the enzymes names is often derived from its substrate or the reaction it catalyzes, with the word ending with- ase for instant amylase. On the basis of international union of biochemistry and molecular biology has classified enzymes as Oxidoreductase, Transferase, Hydrolases, Isomerases, and Ligases.
Starch hydrolyzing enzymes, such as amylase are amongst the most important industrial enzymes and account for over 25% of industrial enzymes (Reddy et al., 2003, Cordeiro, et al., 2003). Amylases are used in numerous biotechnological processes, including biofuel, textile, medicine, chemicals and analytical fields, paper and detergent production (Kokab et al., 2003; Regupati et al., 2007, Saxena et al., 2007).
Amylase belongs to groups, which is called amylolytic enzymes. Amylotic enzymes represent a group of catalytic proteins of great importance to food industry. They were also one of the first enzymes to be produced commercially by microorganism. Amylase refers to a group of enzymes whose catalytic function is to hydrolyze (breakdown) sugar and starch. Amylases digest carbohydrates (polysaccharides) into small disaccharide units, eventually converting them into monosaccharaides such as glucose. People who are fat intolerant often eat sugar and carbohydrates to make up for the lack of fat in their diet. If their diet is excessive in carbohydrates, they may develop an amylase deficiency.
Different types of amylase are present depending upon the bond they break in starch molecule. They are α-amylases, β-amylases and iso- amylases etc.

CLASSIFICTION OF AMYLASE

Alpha amylase - The α-amylase calcium metallo completely unable to function in the absence of calcium by acting at random location along the attach chain α- amylase break down carbohydrate ultimately yielding maltoriose and maltose the α- amylase trend to be faster than β-amylase.

Beta amylase - Beta amylase is another form of amylase. It is also synthesized by bacteria, fungi and plant working from non-reducing end. β amylase catalyzes the hydrolysis of the second α-1-4- glycoside bond, cleaving off two glucose unit at time. During the ripening the fruits, beta amylase breaks the starch into sugar.

Gamma amylase- It is also called   1,4 α- glycosidic glucoamylse. In addition to cleaving the last1,4 α- glycosidic linkage at the non-reducing end of amylase and amylopectin yielding glucose, amylase can cleave α- 1,6 1,4 α- glycosidic linkage.

The common features of α- amylase family-
Hydrolysis activity where the enzyme act on the α- glycosidic bond and hydrolysis this bond to produce α- anomeric, monosaccharides or oligosaccharides or trans glycosylation- activity where α-1,6 1,4 α- glycosidic linkage are formed or a combination of activities.
They possess a (β/α) 8 or TIM barrel containing the catalytic site residues.
They possess four conserved regions in their primary sequence. Some of these conserved amino acids from catalytic site and some are involved in the stability of the conserved amino acids TIM barrel topology (Kurikiand Imanaka, 1999; Van Der Maarel et al., 2002).
Amylase constitutes a class of industrial enzymes having approximately 25% of the enzymes market. The spectrum of amylase application has widened in many other fields, such as clinical, medical and analytical chemistries, as well as their wide spread application in starch scarification and in the textile, fermentation, paper, brewing and distilling industries (Pandey, et al., 2000). Amylase is universally distributed through-out the animal, plant and microbial kingdoms. However, enzymes from the fungal and the bacterial sources have dominated application in industrial sectors. The genus Bacillus produces a large variety of extracellular enzymes of which amylases and proteases are of significant industrial importance.

INDUSTRIAL APPLICATIONS

Starch conversion
The most wide spread application of 2-amylase are in the starch industry, which are used for starch hydrolysis in the staff liquefication process that converts starch into fructose and glucose syrups. The enzymatic conversion of all starch includes- gelatinization, which involves the dissolution of starch granules, thereby forming viscous and scarification, amylase of Bacillus amyloiquefaciens was used but it has been replaced by the α-amylase of Bacillus stearo thermophillus or Bacillus licheniformis.
Fuel alcohol production
Ethanol is the most utilized liquid biofuel. For the ethanol production, starch is the most wide used substrate due to its low price and easily available raw material in most regions of the world. In the production, starch has to be solubilized and then submitted to two enzymatic steps in order to obtain fermentable sugars. The bioconversion of starch into ethanol involves liquefication and saccharification, where starch is converted sugars using an amylolytic microorganism or enzymes such as α- amylase, followed by fermentation, where sugar is converted ethanol using an ethanol fermenting microorganism such as yeast Saccharomycescerevisiae.
Textile industries
Amylases are used in textile industry for resizing process. Sizing agents like starch are applied to yarn before fabric production to ensure fat and secure weaving process. Starch is a very attractive size, because it is cheap, easily available in most of the regions of the world, and it can be remove easily. Starch is later removed from the woven fabric in a wet-process in the textile industry.


Food industry
With the development of modern biotechnology, the food industry has undergone great changes. There are many reports about genetic engineering enzymes that have been used in the food industry. Amylases are extensively used in processed food industries such as backing industry, preparation of digestive acids, and production of cakes, fruit juices and starch syrups. These enzymes can be added to the dough of bread to degrade the starch. The flour converted into smaller, which are subsequently fermented by the yeast.
Detergent industry
They are the primary consumers of enzymes, in terms of both volume and value. The use of enzyme in detergents formulations enhances the detergents tough strains and making the detergent environmentally safe. Amylases are the second type of enzyme used in the formulation of enzymatic detergents, and 90% of all liquid detergent contains these enzymes. These enzymes catalysis the hydrolysis of glycosidic linkages in starch polymers, commonly found in foods such as pasta, chocolate, baby food, barbecue sauce and gravy.
Flour Additive
Amylase are used 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 flavor and causes the bread to rise. While amylases are found naturally in yeast cells, it takes time for yeast to produce enough of these enzymes to break down significant quantities of starch in the bread. This is the reason for long fermented dough such as sour dough. Modern bread making techniques have included amylases into bread improver, thereby making the process faster and more practical for commercial use.
Paper Industries
The use of α-amylases in the pulp and paper industry is for the modification of starch of coated paper, i.e. forth production of low viscosity, high molecular weight starch. The coating treatment serves to make the surface of paper sufficiently smooth and strong, to improve the writing quality of the paper. Amylase have been purified earlier from various Bacillus species such as Bacillus megatarium (Oyeleke, S.B. et al., 2010), from Bacillus subtilis (Riaz, N. et al., 2003) from Bacillus licheniformis SPT 27 (Aiyer, P.V.D., 2004).
                

    














                   


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