Abstract: To characterize the novel raw starch digesting α-amylase Amy486,amy486was cloned from the marine bacterium Exiguobacterium 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 Na2SO4, the specific activity toward raw rice starch reached 2209 U/mg. In the presence of 1.0 mol/L Na2SO4, Amy486 maintained more than 60% relative activity at 35 ℃. The enzymatic activity 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.

Key words: α-amylase, raw starch, halophilic enzyme, Ca2+-dependence