生淀粉水解α-淀粉酶Amy486的重组表达和性质分析

Recombinant expression and biochemical characterization of the raw starch digesting α-amylase Amy486

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 Na_(2)SO_(4)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 Na_(2)SO_(4),Amy486在35℃放置500 h,酶活力可保持60%以上。2.5 mmol/L CaCl_(2)可提升酶活力至110%,添加超过5 mmol/L CaCl_(2),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K_(3)0_(2),K_(3)0_(2)E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K_(3)0_(2)E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。

To characterize the novel raw starch digestingα-amylase Amy486,amy486 was cloned from the marine bacterium Exiguobacterium sp.J84 and expressed heterologously.After purified by Ni 2+-NTA affinity chromatography column,the catalytic property,halophilic property and Ca^(2+)-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 Na_(2)SO_(4),the specific activity toward raw rice starch reached 2209 U/mg.In the presence of 1.0 mol/L Na_(2)SO_(4),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 CaCl_(2).K_(3)0_(2)was determined as the binding site of calcium ion.The mutant K_(3)0_(2)E exhibited enhanced binding ability to calcium ion.Amy486 and K_(3)0_(2)E are halophilic raw starch digestingα-amylases and low dependence on calcium ions,which possess potential application in hydrolysis of starch in high salt environment.