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.
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.
Das
Kishore
K
Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur 784 028, India.
Doley
Robin
R
Mukherjee
Ashis Kumar
AK
D000468:Alkalies / Q000378:metabolism; D001412:Bacillus subtilis / Q000145:classification* / Q000201:enzymology*; D004789:Enzyme Activation; D004795:Enzyme Stability; D045604:Extracellular Fluid; D005516:Food Microbiology; D006863:Hydrogen-Ion Concentration; D007194:India; D007700:Kinetics; D008970:Molecular Weight; D013045:Species Specificity; D013379:Substrate Specificity; D013696:Temperature; D000516:alpha-Amylases / Q000032:analysis* / Q000737:chemistry*
D000468:Alkalies; D000516:alpha-Amylases