Purification, characterisation and mutagenic enhancement of a thermoactive α-amylase from Bacillus subtilis

Abstract

Bacillus subtilis was isolated from flour mill wastes. It produced a thermostable α-amylase in complex media containing starch. Amylase activity was optimal at the exponential phase and was more strongly expressed with sorghum, yam peel and corn starch than soluble potato starch. The enzyme was purified 24-fold to a specific activity of 2200 U mg-1, with a yield of 10%. It yielded a single band when subjected to SDS-PAGE and an apparent molecular mass of 54780 was determined by mass spectrometry. The enzyme, which was optimally active at 80°C and pH 5.6, released saccharides with a polymerisation degree of 1-6 following hydrolysis of yam peel, sorghum and corn starch. Cells of B. subtilis were exposed to ultraviolet irradiation and N-methyl-N'-nitro-N-nitrosoguanidine. Hyperproductive mutants were obtained by these treatments.