A recombinant E.coli BL21(DE3)/pET-28a-yacrⅡ was constructed,successfully realizing the expression of(S)-specific carbonyl reductase gene(yacrⅡ)from Yarrowia lipolytica.The recombinant enzyme YaCRⅡ was purified using HisTrap HP affinity chromatography and the properties of pure enzyme were exzamined.The results show that the full length of the gene yacrⅡ was 942 bp.The encoding protein(YaCRⅡ)belonged to a medium chain dehydrogenase/reductase family(MDRs)with a molecular weight of 37 ku.YaCRⅡ was used to catalyze reduction of 2-hydroxy acetophenone to give out(S)-1-phenyl-1,2-ethanediol.The optical purity was 99.9%(e.e.).7.85 U/mg of enzymatic activity was tested in the catalytic conversion.The optimal pH was 5 while the optimal temperature was 50 ℃ in the process.The analysis of the substrate structures showed that YaCRⅡ exhibited higher catalytic activity for 2-hydroxy acetophenones with electron withdrawing group at the para position of benzene ring.This work provided an efficient method to prepare highly optical pure alcohols by reduction of the corresponding ketones using enzymatic catalytic system.- DOI:10.3969/j.issn.1000-1565.2018.02.009解脂酵母羰基还原酶基因的克隆表达及酶学性质张红蕾,裴朝红,张超,韩孟楠,徐志栋,李玮(河北大学 化学与环境科学学院,河北 保定 071002)摘 要:通过构建重组菌E.coli BL21(DE3)/pET28a-yacrⅡ,成功实现了解脂酵母(Yarrowia lipolytica)的羰基还原酶(YaCRⅡ)基因(yacrⅡ)的表达.应用HisTrap HP亲和层析分离纯化YaCRⅡ,并对该酶的酶学性质进行了研究.结果表明,yacrⅡ基因序列长942 bp,编码蛋白的分子质量为 37 ku,属于中链醇脱氢酶家族.该酶可以催化不对称还原2-羟基苯乙酮生成(S)-1-苯基-1,2-乙二醇,酶的比活力为7.85 U/mg,光学纯度为99.9%(e.e.).该酶反应的最适pH值为5.0,最适温度为50 ℃.底物结构分析表明,该酶对苯环对位有吸电子基团的2-羟基苯乙酮有较高的催化活性.该酶的发现为制备高光学纯度的手性醇提供了一个新的有效方法.关键词:解脂酵母;羰基还原酶;重组表达;酶学性质中图分类号:Q814.1 文献标志码:A 文章编号:1000-1565(2018)02-0168-09Over-expression and characterization of a carbonyl reductase gene from Yarrowia lipolyticaZHANG Honglei,PEI Chaohong,ZHANG Chao,HAN Mengnan,XU Zhidong,LI Wei(College of Chemistry and Environmental Science,Hebei University,Baoding 071002,China)Abstract:A recombinant E.coli BL21(DE3)/pET-28a-yacrⅡ was constructed,successfully realizing the expression of(S)-specific carbonyl reductase gene(yacrⅡ)from Yarrowia lipolytica.The recombinant enzyme YaCRⅡ was purified using HisTrap HP affinity chromatography and the properties of pure enzyme were exzamined.The results show that the full length of the gene yacrⅡ was 942 bp.The encoding protein(YaCRⅡ)belonged to a medium chain dehydrogenase/reductase family(MDRs)with a molecular weight of 37 ku.YaCRⅡ was used to catalyze reduction of 2-hydroxy acetophenone to give out(S)-1-phenyl-1,2-ethanediol.The optical purity was 99.9%(e.e.).7.85 U/mg of enzymatic activity was tested in the catalytic conversion.The optimal pH was 5 while the optimal temperature was 50 ℃ in the process.The analysis of the substrate structures showed that YaCRⅡ exhibited higher catalytic activity for 2-hydroxy acetophenones with electron withdrawing group at the para position of benzene ring.This work provided an efficient method to prepare highly optical pure alcohols by reduction of the corresponding ketones using enzymatic catalytic system.- 收稿日期:2017-08-23 基金项目:河北省自然科学基金专项-重点项目(B2016201031);河北省高等学校高层次人才科学研究项目(GCC 2014014);河北大学自然科学研究计划项目(3333112) 第一作者:张红蕾(1985—),女,河北保定人,河北大学博士后,主要从事生物酶催化研究.E-mail:zhl0724@126.com 通信作者:李玮(1963—),男,河北晋州人,河北大学教授,博士生导师,主要从事化学合成及生物酶催化研究. E-mail: liweihebeilab@163.com第2期张红蕾等:解脂酵母羰基还原酶基因的克隆表达及酶学性质
ZHANG Honglei
,
PEI Chaohong
,
ZHANG Chao
,
HAN Mengnan
,
XU Zhidong
,
LI Wei
. Over-expression and characterization of a carbonyl reductase gene from Yarrowia lipolytica[J]. Journal of Hebei University(Natural Science Edition), 2018
, 38(2)
: 168
-176
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DOI: 10.3969/j.issn.1000-1565.2018.02.009
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