突变型聚酮合成酶酮还原酶域的表达及序列分析  

Expression and Sequence Analysis of Mutant Ketoreductase Domain of Polyketide Synthase

作　　者：李凌凌[1] 吕早生[1] 李涛[1] 沈辉[1] 

机构地区：[1]武汉科技大学化学工程与技术学院,湖北武汉430081

出　　处：《化学与生物工程》2011年第8期36-42,共7页Chemistry & Bioengineering

基　　金：湖北省科技厅基金项目(2009CAD006);武汉科技大学校基金资助项目(2006XY14)

摘　　要：为验证糖多孢红霉菌聚酮合成酶模块1的酮还原酶域EryKR1中的控制底物特异性位点,以糖多孢红霉菌基因组DNA为模板,用重叠PCR技术扩增出α6和α7之间的氨基酸残基RHGVIEMP对应的核苷酸序列被替换的EryKR1酶域DNA片段eryKR1M,并克隆到表达载体pET-28a上,构建了质粒pET-ery KR1M,转化到Escherichia coli BL21(DE3)后,获得了重组菌株E.coli BL21(pET-ery KR1M)。经IPTG诱导后,SDS-PAGE电泳分析表明重组大肠杆菌E.coli BL21(pET-eryKR1M)中的重组蛋白质表达量占全菌胞内可溶性蛋白质的5.7%。E.coli BL21(pET-ery KR1M)和异源表达枯草芽孢杆菌葡萄糖脱氢酶基因的重组菌E.coliBL21(pET-gdh1)进行双重组菌耦合,对4-氯乙酰乙酸乙酯、苯乙酮、2-辛酮和环己酮4种底物进行转化还原,利用气相色谱分析转化液,结果显示突变型的重组菌E.coli BL21(pET-ery KR1M)失去野生型重组菌E.coliBL21(pET-ery KR1)2还原环己酮的能力,结合EryKR1酶域与放线菌素聚酮还原酶ActKR的氨基酸序列比对和二维结构比较的结果,推测α6和α7间氨基酸残基RHGVIEMP为EryKR1酶域中的底物结合口袋组成单元,对酶活性保持非常重要。In order to identify the region of ketoreductase domain （EryKR1） in the first module of polyketide synthase from Saccharopolyspora erythraea which accounted for different substrate specificity,the site-mutated gene （eryKRIM）, in which the nucleotide sequence coding amino acid residues RHGVIEMP be- tween a6 and a7 had been replaced, was amplified by overlapping PCR with Saccharopolyspora erythraea ge- nomic DNA as template and cloned into vector pET-28a to construct recombinant plasmid pET-eryKR1M which was then transformed into Escherichia coli BL21（DE3）. Finally recombinant E. coli BL21 （pET-eryKR1M） had been obtained. After induction by IPTG, SDS-PAGE showed that the expressed enzyme EryKR1M accounted for 5.7% of total soluble protein in the recombinant strain. E. coli BE21 （pET-eryKR1M） and E. coli BL21 （pET-gdhl） harboring Bacillus subtilis glucose dehydrogenase gene fermented together with 4-chloro-3-oxobu- tanoate, acetophenone,2-octanone or cyclohexanone as reduction substrate, respectively. The gas chromatography analysis of ferment showed that unlike wild-type recombinant E. coli BL21 （pET-eryKR1）2, mutated reeombinants couldn＇t reduce cyclohexanone. According to amino acid sequence and two-dimensional alignment among Actinorhodin polyketide ketoreductase （ActKR） and EryKR1 domain and the gas chromatography analysis results, the residues RHGVIEMP between a6 and a7 were predicted as part of substrate binding pocket, playing an important role in keeping enzyme activity.

关 键 词：酮还原酶域 聚酮合成酶 糖多孢红霉菌 生物催化 序列分析 

分 类 号：Q789[生物学—分子生物学] Q815