Amylase from Bacillus cereus MTCC 10205 was purified 20.41 with 11.82% recovery by ammonium sulfate precipitation, gel filtration chromatography through Sephadex G-100 and ion-exchange chromatography on diethylaminoethyl (DEAE)-cellulose. The final enzyme preparation was pure to near homogeneity as judged by native-polyacrylamide gel electrophoresis (PAGE). The enzyme had a molecular weight of 55 kDa as determined by gel filtration and a single band of 55 kDa as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis showing it to be a monomer. The purified enzyme had temperature optima of 55°C and pH optima of 5.5. The enzyme retained 72% of its original activity after 90 min of incubation and exhibited gradual loss in activity when incubated at higher temperature. At 60°C after 90 min of incubation, the enzyme was completely inactive. The enzyme appeared to be quite stable at 4°C as it could be stored upto five days with 10% loss in activity, whereas at 35°C, the enzyme lost 28% of its activity just after three days of storage. Inhibition studies revealed SH groups to be involved at the active site of the enzyme.
Key words: Amylase, Bacillus cereus, gel-filtration, purification, sodium dodecyl sulphate.
SM, Starch medium; DNSA, 3, 5- dinitrosalicylic acid; DEAE, diethyl aminoethyl; PAGE, polyacrylamide gel electrophoresis; PHMB, para hydroxyl mercuric-benzoate; PMSF, phenyl methoxy sulfonyl fluoride; DTNB, 5, 5-dithiobis-2-nitrobenzoic acid; EDTA, ethylenediaminetetraacetic acid; β-ME, β-mercaptoethanol.
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