生物技术通报2017，Vol.33Issue(6)：214-222,9.DOI:10.13560/j.cnki.biotech.bull.1985.2016-1195
酮还原酶中立体选择性还原位点的突变及其产物分析
Mutation of Amino Acid Motif Involved in Stereoselectivity by Ketoreductases and Analysis of Its Product
摘要
Abstract
In order to identify whether LDD motif in ketoreductase domain of polyketide synthase from Saccharopolyspora erythraea (EryKR)can account for the stereocontrol of 2-methylcyclohexanone reduction,we constructed the 4 recombinants,Escherichia coli BL21 (pET28a-eryKR1)with heterologous expressing ketoreductase in the first module(EryKR1)of polyketide synthase,E.coli BL21(pET28a-eryKR2)with heterologous expressing ketoreductase in the second module(EryKR2)of polyketide synthase,E.coli BL21(pET28a-TeryKR1) of the site-mutated EryKR1 in which the nucleotide sequence coding for amino acid residues LDD replaced by PQQ,and E.coli BL21 (pET28a-TeryKR2)of the site-mutated EryKR2 while PQQ replaced by LDD. SDS-PAGE demonstrated that ketoreductases were expressed in these 4 recombinants after induction by IPTG. Specific activity of crude enzyme was 1.49 U/mg,0.37 U/mg,0.94 U/mg and 0.31 U/mg, respectively. Gas chromatography analyses of 2-methylcyclohexanone reduction catalyzed by 4 recombinants showed that mutated recombinant E.coli BL21(pET28a-TeryKR1)mainly reduced 2-methylcyclohexanone to trans-2-methylcyclohexanol,unlike the main product of cis-2-methylcyclohexanol catalyzed by wild-type recombinant Escherichia coli BL21(pET28a-eryKR1). Furthermore,main reduction product of mutant E.coli BL21(pET28a-TeryKR2)was cis-2-methylcyclohexanol,rather than the trans-2-methylcyclohexanol by wild-type recombinant E.coli BL21(pET28a-eryKR2),confirming the key role of LDD motif in controlling the stereoselectivity of 2-methylcyclohexanone reduction.关键词
聚酮合成酶/酮还原酶/2-甲基环己酮/突变/立体选择性Key words
polyketide synthase/ketoreductase/2-methylcyclohexanone/mutation/stereoselectivity引用本文复制引用
李凌凌,吕早生,左振宇,杨忠华,刘曜宁,宋采薇..酮还原酶中立体选择性还原位点的突变及其产物分析[J].生物技术通报,2017,33(6):214-222,9.基金项目
国家自然科学基金资助项目(21376184),湖北省科技厅基金项目(2009CDA006),武汉科技大学青年科技骨干培育计划项目(2016XZ014),武汉科技大学大学生科技创新基金研究项目(16ZRA044) （21376184）
