中国木薯醇腈酶在汉逊酵母中的表达

Expression of Hydroxynitrile Lyase from Chinese Subspecies of Manihot Esculenta in Hansenula Polymorpha

摘要:

α-醇腈酶(α-hydroxynitrilelyase, HNL)是手性醇腈化合物生物合成十分有用的工具, 能够催化羰基化合物和HCN立体选择性的加工形成手性醇腈化合物。应用PCR扩增得到HNL基因, 连接到pMD18-T 中进行测序, 然后通过EcoRⅠ和NotⅠ将其连接到汉逊酵母表达载体pHMOXGαA中。通过在含有4 mg/mL的G418的YPD培养基上进行筛选获得阳性重组子, 用0.5%的甲醇诱导96 h。酶活测定和SDS-PAGE分析显示HNL在汉逊酵母NCYC495(leu1.1)中得到正确表达, 每毫升发酵液中获得2.015 U的醇腈酶。

Abstract:

α-Hydroxynitrilelyase (HNL), a vital tool in the bio-synthesis of optically active cyanohydrin, which can catalyze carbonyls and HCN selective forming optically active cyanohydrin. HNL gene was amplified by PCR using pET30a-HNL as template and then ligated into pMD18-T vector. Validated HNL gene by sequenced was linked into pHMOXGαA vector. Positive recombinants grown in the medium containing 4 mg/mL of G418 were screened using PCR. Bio-active α-hydroxynitrilelyase expressed in Hansenula polymorpha after 0.5% methanol induction was obtained with 2.015 U determined by analysis of enzyme activity and SDS-PAGE.