宇佐美曲霉阿魏酸酯酶A基因的克隆表达及酶学性质研究

Cloning, Expression and Enzymatic Characterization of Feruloyl Esterase A from Aspergillus usamii

DOI：10.3969/j.issn.1673-1689.2013.07.006

中文关键词: 宇佐美曲霉 阿魏酸酯酶A 基因克隆 表达 毕赤酵母 酶学性质

英文关键词: Aspergillus usamii feruloyl esterase A gene cloning expressing Pichia pastoris enzymatic characterization

基金项目:国家自然科学基金项目(31271811)

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中文摘要:

本研究通过RT-PCR技术从宇佐美曲霉(Aspergillus usamii)E001中克隆了编码阿魏酸酯酶A的成熟肽cDNA(AusfaeA),构建重组表达质粒pPIC9K-AusfaeA,将其经SacⅠ线性化后转化毕赤酵母(Pichia pastoris)GS115,通过抗G418筛选获得高拷贝重组子GS115/AusfaeA,用甲醇诱导表达重组阿魏酸酯酶(reAusFaeA)。SDS-PAGE显示,纯化后的reAusFaeA为单一条带,其表观相对分子质量为36 000;以阿魏酸甲酯为底物,经高效液相色谱法测定,该酶的酶活为29.4 U/mg;reAusFaeA的最适反应温度为45℃,并在45℃以下稳定;最适反应pH为5.0,在pH 4.0~6.0范围内稳定;多数测定的金属离子及EDTA对该酶的活性影响不大。结果表明在P.pastoris中成功实现了AusfaeA的异源表达,该重组酶优良的酶学性质表明其具有较大的工业应用潜力。

英文摘要:

A cDNA gene(AusfaeA),which encodes a feruloyl esterase(FAE) A from Aspergillus usamii E001(abbreviated to AusFaeA),was amplified by RT-PCR.The expressing plasmid pPIC9K-AusfaeA was constructed and linearized with SacⅠ,followed by transforming it into Pichia pastoris GS115 by eletroporation.The recombinant P.pastoris GS115/AusfaeA was screened by G418 and then was induced with methanol to express reAusFaeA.The purified reAusFaeA showed one single band on SDS-PAGE with an apparent molecular weight of 36.0 kDa.The feruloyl esterase activity of the reAusFaeA against methyl ferulate(MFA) reached 29.4 U/mg determined by HPLC.Its optimal temperature and pH were 45 ℃ and 5.0,respectively.It was stable at a temperature of 45 ℃ or below,and at a pH range of 4.0 ~6.0.Its enzymatic acvitity was not signficantly affected by an array of tested metal ions.This indicated that the AusfaeA was successfully expressed in P.pastoris and the excellent properties of the reAusFaeA make it a potential candidate for applications in industrial processes.

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