Purification and characterization of extracellular acidophilic -amylase from Bacillus cereus MTCC 10205 isolated from soil

Abstract

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.

The Characteristics of Urease Enzyme of Green Bean Seeds (Vigna radiata L.) and Its Activity as An Antifungal Against Candida albicans

“…However, after reaching the optimal concentration, all the active sites may already be filled with the substrates, and the enzyme has reached a saturation point. Therefore, adding more substrates will not increase the enzyme activity further or even inhibit enzymes [14].…”