Cloning and characterization of cold-active and salt-tolerant α-amylase Amy3 from Pseudoalteromonas sp. K8

Abstract:

[Objective] The present study was to clone a novel alpha-amylase from marine bacterium Pseudoalteromonas sp. K8 isolated from the sediment of Kongsfjorden and characterize the enzyme.[Methods] We cloned a cold-active and salt-tolerant α-amylase Amy3, from Pseudoalteromonas sp. K8. The protein was expressed in Escherichia coli, purified by Ni-NTA and characterized.[Results] Using soluble starch as substrate, the optimum pH of Amy3 was about pH 8.5, and more than 40% of the maximal activity maintained in the pH range of 6.5 to 10. Amy3 showed the maximum activity at 25℃, and retained more than 50% activity at 0℃. It exhibited improved catalytic activity and thermostability in NaCl solution, with maximal activity occurring at 800 mmol/L NaCl/KCl, and more than 50% of maximal activity retained after incubation in 2 mol/L NaCl for 80 h at 25℃. NaCl did not cause significant change of the global tertiary structure of Amy3, whereas influenced the catalysis efficiency. Amy3 hydrolyzed amylopectin preferentially, and could also hydrolyze wheat starch, corn starch and tapioca starch.[Conclusion] The results indicated that the alpha-amylase Amy3 is a cold-active and salt-tolerant α-amylase with potential use in basic research and industry.