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.... more
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.
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.
