Cloning, Expression, Purification, and Characterization of Cold-Adapted α-Amylase from Pseudoalteromonas arctica GS230.

cold-adapted α-amylase ( ParAmy) gene from Pseudoalteromonas arctica GS230 was cloned, sequenced, and expressed as an N-terminus His-tag fusion protein in E. coli. A recombinant protein was produced and purified with DEAE-sepherose ion exchange chromatography and Ni affinity chromatography. The molecular weight of ParAmy was estimated to be 55 KDa with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). With an optimum temperature for activity 30 °C, ParAmy showed 34.5% of maximum activity at 0 °C and its activity decreased sharply at above 40 °C. ParAmy was stable in the range of pH 7-8.5 at 30 °C for 1 h. ParAmy was activated by Mn, K and Na, and inhibited by Hg, Cu, and Fe. N-Bromosuccinimid showed a significant repressive effect on enzyme activity. The K and V values of the α-amylase for soluble starch were 7.28 mg/mL and 13.07 mg/mL min, respectively. This research suggests that Paramy has a good potential to be a cold-stable and alkalitolerant amylase in detergent industry. [ABSTRACT FROM AUTHOR]