耐酸耐温α-淀粉酶基因在大肠杆菌中的功能表达

摘要

兼顾大肠杆菌与枯草芽孢杆菌密码子偏好性,优化获得了1个耐酸耐温淀粉酶的基因amyCN1,并将其在大肠杆菌中实现了功能表达.纯化后的重组α-淀粉酶(AM Y1)表征结果表明:在最适pH5.5和最适温度75℃条件下表观米氏常数(Km)值和催化效率(kcat/Km)值分别为20.93 g/L和98.20 L/(g·s)；低浓度的Co2+(1 mmol/L)可以提高30％的淀粉酶活力,Mn2+抑制了大部分活力,包括ca2+在内的其他金属离子影响不显著；高浓度的EDTA(≥50 mmol/L)抑制其活力；生物信息学分析表明,AMY1具有α-淀粉酶家族典型的3-结构域分布,具有1个Ca2+结合位点和5个Zn2+结合位点,活性中心催化氨基酸残基D198和E222位于(β/α)8桶中β折叠片C端一侧.AMY1优良的低pH耐受性和耐温性,有助于木薯淀粉生产燃料乙醇工业液化与糖化同步发酵工艺的实现.%Considering the codon bias in Escherichia coli and Bacillus suhlilis, one gene amyCN1 encoding an aciduric and thermostable α-amylase was optimized and functionally expressed in E. coli. Using the one-step purification procedure of Ni-NTA, the C-terminal 6xHis tag fused AMY1 was purified to purily of 95% assessed by SDS-PACE. The purified recombinant AMY1 showed appealing enzymatic properties under optimum pH of 5. 5, optimum temperature of 75 ℃, Km of 20. 93 g/L and catalytic efficiency (kcat/Km) of 98. 20 L/(g·s) ; low concentration of Co2+ increased the amylase activity by approx. 30% , but Mn + inhibited more than a half of the activity, and other metal ions including Ca2+ showed hardly anything significant to the activities of AMY1, EDTA deteriorated the AMY1 activity, indicating that the metal ions were required for the structural stability and function. The AMY1 adopted the typical arrangement of three domains in a-amylase family; domain A consisting of one (α/β)s barrel locating the catalytic amino acid residual D198 and E222, domain B protruding between the third β strand and third a helix, and domain C composing of all beta sheets. Further theoretical structural model analysis disclosed that there were one Ca2+ binding site and five Zn2+ binding sites located on the AMY1. The aciduric and thermostable features of AMY1 provided the potentiality for the simultaneous liquefaction and saccharifica-tion process for the ethanol production from the cassava starch.