宇佐美曲霉环氧化物水解酶基因的克隆与生物信息学分析

Cloning and Bioinformatics Analysis of Epoxide Hydrolase Gene from Aspergillus usamii

DOI：10.3969/j.issn.1673-1689.2013.12.002

中文关键词: 宇佐美曲霉 环氧化物水解酶 侧翼未知DNA序列 基因克隆 生物信息学分析

英文关键词: Aspergillus usami epoxide hydrolase unknown flanking DNA sequence gene cloning bioinformatics analysis

基金项目:国家自然科学基金项目(31101229);国家自然科学基金项目(31271811)

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中文摘要:

环氧化物水解酶(Epoxide hydrolase,EH)是酶法拆分消旋体环氧化物,制备光学活性环氧化物和邻二醇的重要酶之一。通过RT-PCR和新构建的THSO-PCR侧翼未知DNA序列扩增技术,克隆了一种来源于宇佐美曲霉(Aspergillus usamii)E001的EH基因,命名为Aueh2(GenBank No.KF061095),并对该基因进行了相关生物信息学分析。Aueh2的DNA序列长度为2 481 bp,其中包含了5′端和3′端侧翼调控序列,6个内含子序列和编码cDNA序列。开放阅读框序列长度为1 188 bp,编码395个氨基酸,对应的蛋白质命名为AuEH2;该蛋白质为无信号肽的亲水蛋白质,其理论相对分子质量44.6 kD;其三维结构包含EH典型的"α/β"核心催化结构域和"帽子"结构域,活性中心由催化三联体Asp191、His369和Glu343组成。本课题研究成果为深入研究AuEH2及其应用奠定了基础。

英文摘要:

Epoxide hydrolase can be effectively used in the resolution of epoxides for producting optically active epoxides and vicinal diols. A gene encoding a novel epoxide hydrolase from Aspergillus usamii E001 was cloned by using reverse transcription PCR and newly constructed Thairpin structure-mediated PCR amplification(THSO-PCR) techniques. The cloned gene(named Aueh2) is 2,481 bp in length,harboring 5′ and 3′ flanking regulatory regions and the encoded cDNA sequence interrupted by six introns. The open reading frame of Aueh2 encodes a protein of 395-aa(designated AuEH2) with the calculated molecular weight of 44.6 kD. The primary structure analysis of AuEH2 demonstrated that it is a hydrophilic protein,and belongs to the α/β hydrolase fold family. The structure of AuEH2 displays that the catalytic center is situated between the α/β core domain and the lid domain with a catalytic triad consisting of Asp191,His369,and Glu343. The results will lay a foundation for further research of the AuEH2 and its application in depth.

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