| A strain producing amylase was isolated from the sediments in the Southern Okinawa Trough. It was identified as a strain of Pseudomonas sp. based on morphology, physiology, and 16S rDNA gene sequence analysis. The new isolated strain was named as Pseudomonas sp. C40.The fermentation parameters were optimized in the shake-flask culture of marine Pseudomonas sp. C40 to increase the amylase production using Plackett–Burman design and response surface methodology. The experimental results indicated that the optimized composition of medium was 2.00 g L-1 starch, 3.99 g L-1 peptone, 3.86 g L-1 yeast extract and 3.81% salinity, 0.33 g L-1 CaCl2. The optimal fermentation conditions were pH 6.0, 35oC and 180 rpm.The cell-free supernatant was subjected to ammonium sulfate precipitation, dialysis, and ultrafiltration concentration. The active fractions were applied to a Sephadex G-75 column. The purity of amylase obtained was greater than 95% and the recovery of amylase was close to 40.8%.The native molecular mass of amylase estimated by gel chromatography and electrophoresis was about 58 kDa. The optimal pH for enzyme activity was found to be 7.0, and at this pH the enzyme exhibited maximum activity at 50oC. Among various metal ions examined, Co2+ exerted a positive effect on amylase activity, whereas Mg2+, Ca2+, Fe2+, Al3+, Fe3+, Zn2+, Ni2+ and Cu2+ were found to be inhibitors. PMSF was found to have no effect on the amylase activity, but EDTA and EGTA were strong inhibitors. These results indicated that the amylase was a metalenzyme and it had no serine in the active center. The Km and Vmax values of the purified amylase were 1.73mg.mL-1 and 1.24mg.mL-1min-1, respectively.
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