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

作　　者：李凌凌[1] 吕早生[1] 左振宇[1] 杨忠华[1] 刘曜宁[1] 宋采薇 

机构地区：[1]武汉科技大学化学与化工学院,武汉430081

出　　处：《生物技术通报》2017年第6期214-222,共9页Biotechnology Bulletin

基　　金：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)

摘　　要：为验证糖多孢红霉菌聚酮合成酶中酮还原酶(Ery KR)的LDD模式序列是否为控制2-甲基环己酮立体选择性还原的位点,构建了分别异源表达聚酮合成酶模块1的酮还原酶(Ery KR1)、模块2的酮还原酶(Ery KR2)、LDD残基替换为PQQ(LDD→PQQ)的EryKR1及PQQ替换为LDD(PQQ→LDD)的Ery KR2的重组大肠杆菌Escherichia coli BL21(p ET28a-eryKR1)、E.coliBL21(pET28a-ery KR2)、E.coli BL21(p ET28a-Tery KR1)和E.coli BL21(p ET28a-Tery KR2)。SDS-PAGE实验证明,经IPTG诱导后4个重组菌中都表达出相应的酮还原酶。粗酶液的比酶活分别为1.49 U/mg、0.37 U/mg、0.94 U/mg和0.31 U/mg。利用气相色谱分别检测4个重组菌还原2-甲基环己酮体系中产物的立体结构,结果显示与野生型Ery KR1的还原产物以顺式-2-甲基环己醇为主不同,LDD→PQQ的突变型Ery KR1催化2-甲基环己酮的还原产物主要为反式-2-甲基环己醇,而PQQ→LDD的突变型Ery KR2的主要还原产物也由野生型Ery KR2的反式-2-甲基环己醇转变成顺式-2-甲基环己醇,证实了LDD模式序列确实为酶中控制2-甲基环己酮立体选择性还原的位点。In order to identify whether LDD motif in ketoreductase domain of polyketide synthase from Saccharopolyspora erythraea（Ery KR）can account for the stereocontrol of 2-methylcyclohexanone reduction,we constructed the 4 recombinants,Escherichia coli BL21（p ET28a-ery KR1）with heterologous expressing ketoreductase in the first module（Ery KR1）of polyketide synthase, E.coli BL21（p ET28 aery KR2）with heterologous expressing ketoreductase in the second module（Ery KR2）of polyketide synthase,E.coli BL21（p ET28a-Tery KR1）of the site-mutated Ery KR1 in which the nucleotide sequence coding for amino acid residues LDD replaced by PQQ,and E.coli BL21（p ET28a-Tery KR2）of the site-mutated Ery KR2 while PQQ replaced by LDD. SDS-PAGE demonstrated that ketoreductases were expressedin 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 recombinantE.coli BL21（p ET28a-Tery KR1）mainly reduced 2-methylcyclohexanone to trans-2-methylcyclohexanol,unlike the main product of cis-2-methylcyclohexanol catalyzed by wild-type recombinant Escherichia coli BL21（p ET28a-ery KR1）. Furthermore,main reduction product ofmutant E.coli BL21（p ET28a-Tery KR2）was cis-2-methylcyclohexanol,rather than the trans-2-methylcyclohexanol by wild-type recombinant E.coli BL21（p ET28a-ery KR2）,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 

分 类 号：O629.8[理学—有机化学]