米曲霉(Aspergillus oryzae)RIB40中烯酮/烯酯还原酶的异源表达及性质分析  

Heterologous expression and characterization of enoate reductase from Aspergillus oryzae RIB40

作　　者：张海灵[1,2] 高秀珍[2] 陈曦[2] 任杰[2] 冯进辉[2] 张同存[1] 吴洽庆[2] 朱敦明[2] 

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院天津工业生物技术研究所工业酶国家工程实验室,天津300308

出　　处：《生物加工过程》2013年第1期41-46,共6页Chinese Journal of Bioprocess Engineering

基　　金：国家重点基础发展计划(973计划)资助(2011CB710801)

摘　　要：以米曲霉(Aspergillus oryzae)RIB40基因组DNA为模版,通过PCR扩增其烯酮烯酯还原酶(AspER)基因(asper)后连接到表达载体pET32a(+)上,在大肠杆菌BL21(DE3)中以可溶形式表达。通过Ni-NTA亲和色谱层析纯化后,蛋白纯度提高1.9倍,回收率为60.62%。根据分子筛凝胶层析结果推算,AspER以二聚体形式存在。性质分析表明此酶为依赖于NADPH的氧化还原酶,最适pH为7.0～8.0,最适温度为40℃。对2-环己烯酮Km和kcat值分别为(2.45±0.36)mmol/L和(4.4±0.4)×103s-1。底物谱分析发现AspER对马来酰亚胺及其衍生物有较高的活性,其中对2-甲基马来酰亚胺的转化率和e.e.值均高于99%。The gene encoding enoate reductase of Aspergillus oryzae RIB40, asper, was amplified from the genome DNA and cloned on pET32a（＋）. Encoded protein was expressed as a soluble form in E. coli BL21 （DE3）. Recombinant native protein was purified by Ni-NTA affinity chromatography. The native enzyme was NADPH-dependent and existed as a dimer determined by gel filtration. The enzyme exibited maximam activity at pH 7.0 to pH 8.0 at 40 qc. The purified enzyme was active to malelmide and its derivatives. The conversion and the enantiomeric excess toward 2-methylmaleimide were both higher than 99%.

关 键 词：不对称还原 烯酮 烯酯还原酶 古老黄色酶 

分 类 号：Q78[生物学—分子生物学] Q81