| 一种来源于运动替斯崔纳菌KA081020-065的新型卤醇脱卤酶的表达纯化及性质分析 |
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| 引用本文： | 王雷,袁京,姚培圆,程丽华,解美仙,贾荣荣,冯进辉,王敏,吴洽庆,朱敦明.一种来源于运动替斯崔纳菌KA081020-065的新型卤醇脱卤酶的表达纯化及性质分析[J].生物工程学报,2015,31(5):659-669. |
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| 作者姓名： | 王雷 袁京 姚培圆 程丽华 解美仙 贾荣荣 冯进辉 王敏 吴洽庆 朱敦明 |
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| 作者单位： | 1 天津科技大学生物工程学院 工业发酵微生物教育部重点实验室，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 天津市生物催化技术工程中心，天津 300308,2 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 天津市生物催化技术工程中心，天津 300308,2 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 天津市生物催化技术工程中心，天津 300308,3 河北诚信有限责任公司，河北 石家庄 051130,3 河北诚信有限责任公司，河北 石家庄 051130,3 河北诚信有限责任公司，河北 石家庄 051130,2 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 天津市生物催化技术工程中心，天津 300308,1 天津科技大学生物工程学院 工业发酵微生物教育部重点实验室，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 天津市生物催化技术工程中心，天津 300308,2 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 天津市生物催化技术工程中心，天津 300308 |
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| 基金项目： | 中国科学院重点部署项目 (No. KSED-EW-Z-015) 资助。 |
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| 摘 要： | 卤醇脱卤酶(Halohydrin dehalogenase)不仅对于含氯污染物的生物降解、净化环境具有重要意义,而且在手性医药中间体合成中也是一种重要的生物催化剂,但其数量较少。采用基因挖掘在基因组数据库中获得一种来自运动替斯崔纳菌Tistrella mobilis KA081020-065的新型卤醇脱卤酶基因Hhe TM,将该基因克隆、表达在大肠杆菌BL21(DE3)中,利用镍柱亲和层析将该酶进行纯化并研究了其酶学性质。Hhe TM是一种同源四聚体;最适温度为50℃;不同的p H缓冲液对其活性有较大影响;在碱性、30℃以下的条件下稳定性高。在氰根离子存在的条件下,Hhe TM能够催化(S)-4-氯-3-羟基丁酸乙酯合成(R)-4-氰基-3-羟基丁酸乙酯,为酶法合成阿托伐他汀关键中间体提供了一种新的卤醇脱卤酶。
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| 关 键 词： | 卤醇脱卤酶，运动替斯崔纳菌，(R)-4-氰基-3-羟基丁酸乙酯 |
| 收稿时间： | 2014/8/26 0:00:00 |
Expression and characterization of a novel halohydrin dehalogenase from Tistrella mobilis KA081020-065 |
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| Lei Wang,Jing Yuan,Peiyuan Yao,Lihua Cheng,Meixian Xie,Rongrong Ji,Huijin Feng,Min Wang,Qiaqing Wu and Dunming Zhu.Expression and characterization of a novel halohydrin dehalogenase from Tistrella mobilis KA081020-065[J].Chinese Journal of Biotechnology,2015,31(5):659-669. |
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| Authors: | Lei Wang Jing Yuan Peiyuan Yao Lihua Cheng Meixian Xie Rongrong Ji Huijin Feng Min Wang Qiaqing Wu and Dunming Zhu |
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| Institution: | 1 Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China; 2 Tianjin Engineering Center for Biocatalytic Technology, National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China,2 Tianjin Engineering Center for Biocatalytic Technology, National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China,2 Tianjin Engineering Center for Biocatalytic Technology, National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China,3 Hebei Chengxin CO., LTD, Shijiazhuang 051130, Heibei, China,3 Hebei Chengxin CO., LTD, Shijiazhuang 051130, Heibei, China,3 Hebei Chengxin CO., LTD, Shijiazhuang 051130, Heibei, China,2 Tianjin Engineering Center for Biocatalytic Technology, National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China,1 Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China,2 Tianjin Engineering Center for Biocatalytic Technology, National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China and 2 Tianjin Engineering Center for Biocatalytic Technology, National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China |
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| Abstract: | Halohydrin dehalogenase is of great significance for biodegradation of the chlorinated pollutants, and also serves as an important biocatalyst in the synthesis of chiral pharmaceutical intermediates. A putative halohydrin dehalogenase (HheTM) gene from Tistrella mobilis KA081020-065 was cloned and over-expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was purified by Ni-NTA column and characterized. Gel filtration and SDS-PAGE analysis showed that the native form of HheTM was a tetramer. It exhibited the highest activity at 50 °C. The nature and pH of the buffer had a great effect on its activity. The enzyme maintained high stability under the alkaline conditions and below 30 °C. HheTM catalyzed the transformation of ethyl(S)-4-chloro-3-hydroxybutyrate in the presence of cyanide, to give ethyl (R)-4-cyano-3- hydroxybutyrate, a key intermediate for the synthesis of atorvastatin. |
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| Keywords: | halohydrin dehalogenase Tistrella mobilis ethyl(R)-4-cyano-3-hydroxybutyrate |
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