新型卤代醇脱卤酶基因的克隆、序列分析及表达

Gene Cloning, Sequence Analyais and Gene Expression of A New Halohydrin Dehalogenase

DOI：10.3969/j.issn.1673-1689.2015.05.008

中文关键词: 卤代醇脱卤酶 宏基因组技术 序列分析 密码子优化 表达

英文关键词: halohydrin dehalogenase,metagenomic technology,sequence analysis,codon optimi- zation,expression

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中文摘要:

卤代醇脱卤酶可以催化合成具有光学纯的环氧化物及β-取代醇等高价值手性药物中间体。作者所在实验室利用宏基因组技术从无锡新区工业集中地污染土壤中筛选了一段编码基因Y,经测序知其片段包含有765 bp的核苷酸,编码254个氨基酸。经Blast软件分析,其氨基酸序列与来自根癌土壤杆菌 (Agrobacterium tumefaciens)的卤代醇脱卤酶HheC同源性为81%,且含有卤代醇脱卤酶类的保守催化三联体Ser132-Thr145-Arg149。推测其属于卤代醇脱卤酶HheC类,命名为SyHheC。根据大肠杆菌Escherichia coli BL21的密码子偏爱性,对基因Y进行密码子优化和人工合成,命名为SyhheC,构建重组表达质粒pET28a-SyhheC,转化感受态E. coli BL21,表达产物经镍柱亲和层析后获得电泳纯的重组卤代醇脱卤酶reSyHheC。SDS-PAGE分析,reSyHheC的相对分子质量为34 000。以2-溴-1-(4-硝基苯基)-乙醇为底物,reSyHheC催化底物产生2-(4-硝基苯基)环氧乙烷的比活性为5.2 U/mg。

英文摘要:

Halohydrin dehalogenase catalyzes the synthesis of optically pure epoxides,β-substituted alcohol and other similar high-value chiral drug intermediates. Our laboratory screened an encoding gene Y from contaminated soil in Wuxi new district industrial park using metagenomic technology. The gene Y contains 765 bp nucleotides and encodes 254 amino acids. According to the Blast analysis,the amino acids sequence shares 81% homology with HheC from Agrobacterium tumefaciens and also has Ser132-Thr145-Arg149 catalytic triad which is the same with the known Halohydrin dehalogenases. Thus,we speculated it belongs to HheC. According to Escherichia coli BL21 codon preference,the optimized gene was synthesized and named as SyhheC. The constructed expression plasmid pET28a-SyhheC was transformed into E. coli BL21. The expressed enzyme with an apparent molecular weight of 34 000 by SDS-PAGE analysis was purified to homogeneity using Ni-NTA affinity chromatography and characterized using the substrate of 2-bromo-1-(4-nitrophenyl)ethanol. The activity of purified reSyHheC was 5.2 U/mg. This study provides a new strategy to excavate new enzymes with excellent characters from the soil.

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