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

摘要

A cDNA gene (AusfaeA),which encodes a feruloyl esterase (FAE) A from A spergillus usamii E001 (abbreviated to AusFaeA),was amplified by RT-PCR.The expressing plasmid pPIC9K-AusfaeA was constructed and linearized with Sac Ⅰ,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 re AusFaeA 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 A usfaeA was successfully expressed in P.pastoris and the excellent properties of the reAusFaeA make it a potential candidate for applications in industrial processes.%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-A usfaeA,将其经Sac Ⅰ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/A usfaeA,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36000;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.