Purification and biochemical characterization of a thermostable, alkaliphilic, extracellular α‐amylase fromBacillus subtilisDM‐03, a strain isolated from the traditional fermented food of India

Abstract

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 °C and secretes a significant amount of α‐amylase at pH 8.0 into the culture media. This α‐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 °C and 9.0 respectively, which makes it ideal for use in the detergent industries. Maximum α‐amylase production is obtained by using soluble starch and NH₄Cl as carbon and nitrogen sources respectively. Thermostability of the enzyme is evident from heating the enzyme at 95 °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.