Isolation and characterization of α-amylase from marine Pseudomonas sp. K6-28-040
Liu J., Zhang Z., Zhu H., Dang H., Lu J., Cui Z.
The α-amylase of marine Pseudomonas sp. K6-28-040 was purified through a series of three steps and the purity of enzymes was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results showed that, the enzyme was purified 4.7-fold with a specific activity of 134.6 U/mg protein and a yield of 44%. When it was subjected to SDS-PAGE, a single band near 58 kDa appeared. The optimum temperature and pH were 50°C and 7.0, respectively. The addition of Ca2+, Mn2+ and Co2+ could improve the enzyme activity, while Cu2+, Hg2+, Fe3+ and Al3+ decreased the activity. The enzyme was inhibited by ethylenediaminetetraacetic acid (EDTA), ethylenebis(oxonitrilo)]tetra-acetate (EGTA), SDS and dimethyl sulfoxide (DMSO), but was not affected by phenylmethane-sulfonyl fluoride (PMSF) and 1,4-dithiothreitol (DTT). Km and Vmax values of the purified enzyme for soluble starch were 1.73 ± 0.3 mg/ml and 1.24 ± 0.02 mg/ml/min, respectively. The degradation ability of wild type α-amylase on starch granules was examined by thin layer chromatography. The final purified enzyme had an isoelectric point of 7.5-7.8 and α-helix of 28%, β-sheet of 32% and random coil of 40%. © 2011 Academic Journals.