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Purification and biochemical characterization of a thermostable, alkaliphilic, extracellular alpha-amylase from Bacillus subtilis DM-03, a strain isolated from the traditional fermented food of India.

Biotechnology and Applied Biochemistry

chemistry, analysis, alpha-Amylases, Temperature, Substrate Specificity, Species Specificity, Molecular Weight, Kinetics, India, Hydrogen-Ion Concentration, Food Microbiology, Extracellular Fluid, Enzyme Stability, Enzyme Activation, enzymology, classification, Bacillus subtilis, metabolism, Alkalies

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      Bacillus subtilis strain DM-03, which is isolated from starter culture used for the production of alcohol by local Assam tribes, grows optimally at 52-55 degrees C and secretes a significant amount of alpha-amylase at pH 8.0 into the culture media. This alpha-amylase, purified by ion-exchange, gel-filtration and reverse-phase HPLC, constitutes 2.9% of the total extracellular protein. This purified enzyme, named Bsamy-I, has a subunit with molecular mass of 42.8 kDa as determined by SDS/PAGE, and optimum temperature and pH values at 52-55 degrees C and 9.0 respectively, which makes it ideal for use in the detergent industries. Maximum alpha-amylase production is obtained by using soluble starch and NH(4)Cl as carbon and nitrogen sources respectively. Thermostability of the enzyme is evident from heating the enzyme at 95 degrees C for 10 min, which results in a loss of 60% of the original enzyme activity. 4-Bromophenacyl bromide and PMSF at 4 and 1.5 mM concentration respectively completely abolish the enzymic activity, documenting the essential role of histidine and carboxylic residues in the catalytic process.

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