Recombinant expression and biochemical characterization of the raw starch digesting α-amylase Amy486
To characterize the novel raw starch digesting α-amylase Amy486,amy486 was cloned from the marine bacterium Exig-uobacterium sp.J84 and expressed heterologously.After purified by Ni2+-NTA affinity chromatography column,the catalytic property,halophilic property and Ca2+-dependence of Amy486 were analyzed.The optimum pH of Amy486 was 7.5 and it maintained above 40%residual activity in the pH range of 6.5-8.5.The optimum temperature was 35℃and Amy486 was more stable at lower temperature,with a half-life of about 100 h at 30℃.With the addition of 1.5 mol/L Na2 SO4,the specific activity toward raw rice starch reached 2209 U/mg.In the presence of 1.0 mol/L Na2 SO4,Amy486 maintained more than 60%relative activity at 35℃.The enzymatic ac-tivity can be enhanced up to 110%in the presence of 2.5 mmol/L CaCl2,and the activity was inhibited with the addition of more than 5 mmol/L CaCl2.K302 was determined as the binding site of calcium ion.The mutant K302E exhibited enhanced binding ability to calcium ion.Amy486 and K302E are halophilic raw starch digesting α-amylases and low dependence on calcium ions,which possess potential application in hydrolysis of starch in high salt environment.
万方数据
维普
