Enhanced Production and Partial Purification of Glucoamylase from Mutated Bacillus sp FME

In this study, a potent newly-isolated glucoamylase producing actively growing cells of Bacillus sp. FME was subjected to UV irradiation and ethidium bromide (EtBr) mutagenesis. The promising colonies were further screened for glucoamylase production via plate assays and submerged enzyme production at the flask level. Amongst all of the tested colonies, the best mutant, Bacillus sp. FME 2, selected from UV irradiation (20 min) and EtBr (1 mg/mL), was shown to be the most promising. The yield of glucoamylase generated by the mutant strain was approximately 3.0 fold and 1397 U/mL with an incubation period of 24 h, which was larger than the yield generated by the wild-type strain. The glucoamylase was partially purified using ammonium sulphate precipitation followed by gel exclusion chromatography. The enzyme was partially purified 3.0-fold to homogeneity with a final recovery of 66% and a specific activity of 1145 U/mg protein for the mutant strain. The molecular mass was approximately 67.1 kDa, as determined by SDS-PAGE. The active band was observed as a clear colorless area on zymogram analysis, which indicated an absence of glucoamylase isoforms. The results of thin-layer chromatography identified this enzyme as a glucoamylase.