【摘要】 为明晰生淀粉水解α-淀粉酶Amy486独特的催化特征，将克隆自Exiguobacterium sp. J84菌株的amy486进行重组表达，并研究重组酶的酶学性质、嗜盐特性和钙离子依赖性。Amy486最适催化pH为7.5,在pH 6.5～8.5范围内酶活力保持40%以上，最适温度为35℃,30℃半衰期为100 h。1.5 mol/L Na₂SO₄处理可将Amy486的比酶活由1.53 U/mg提升至2 209 U/mg。添加1.0 mol/L Na₂SO₄,Amy486在35℃放置500 h,酶活力可保持60%以上。2.5 mmol/L CaCl₂可提升酶活力至110%,添加超过5 mmol/L CaCl₂,Amy486的相对酶活力降至100%以下，EDTA孵育对Amy486蛋白的酶活力和稳定性影响较小。Amy486与钙离子结合的关键位点为K302,K302E与钙离子的结合能力增强，从而降低该酶对外源钙离子的依赖性。α-淀粉酶Amy486及其突变体酶K302E是具有较高比酶活的生淀粉水解酶，对外源钙离子的依赖性较低，其活力的发挥依赖于适合的盐浓度，可应用于某些高盐环境下的淀粉水解。更多还原

【Abstract】 To characterize the novel raw starch digesting α-amylase Amy486, amy486 was 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 2 209 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 CaCl_2, 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.更多还原