| 米曲霉(Aspergillus oryzae)RIB40中烯酮/烯酯还原酶的异源表达及性质分析 |
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| 引用本文： | 张海灵,高秀珍,陈曦,任杰,冯进辉,张同存,吴洽庆,朱敦明.米曲霉(Aspergillus oryzae)RIB40中烯酮/烯酯还原酶的异源表达及性质分析[J].生物加工过程,2013,11(1):41-46. |
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| 作者姓名： | 张海灵 高秀珍 陈曦 任杰 冯进辉 张同存 吴洽庆 朱敦明 |
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| 作者单位： | 天津科技大学生物工程学院;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 |
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| 基金项目： | 国家重点基础发展计划(973计划)资助(2011CB710801) |
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| 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%。
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| 关 键 词： | 不对称还原 烯酮/烯酯还原酶 古老黄色酶 |
Heterologous expression and characterization of enoate reductase from Aspergillus oryzae RIB40 |
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| ZHANG Hailing,GAO Xiuzhen,CHEN Xi,Ren Jie,FENG Jinhui,ZHANG Tongcun,WU Qiaqing,ZHU Dunming.Heterologous expression and characterization of enoate reductase from Aspergillus oryzae RIB40[J].Chinese Journal of Bioprocess Engineering,2013,11(1):41-46. |
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| Authors: | ZHANG Hailing GAO Xiuzhen CHEN Xi Ren Jie FENG Jinhui ZHANG Tongcun WU Qiaqing ZHU Dunming |
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| Institution: | 1.College of Biotechnology,Tianjin University of Science & Technology,Tianjin 300457,China;2.National Engineering Laboratory for Industrial Enzymes,Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China) |
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| Abstract: | 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%. |
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| Keywords: | asymmetric reduction enoate reductase old yellow enzyme |
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