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 Na2 SO4 处理可将Amy486 的比酶活由 1.53 U/mg提升至 2209 U/mg.添加 1.0 mol/L Na2 SO4,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.
Abstract
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.
维普
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