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

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4mg/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96h。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2.015U的醇腈酶。

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.