丙烯酰CoA合成酶在大肠杆菌中的表达及其酶活性表征  

Expression and activity characterization of acryloyl-coenzyme A synthase in Escherichia coli

作　　者：黄志兵[1] 宋嘉宁[1] 刘珞[1] 谭天伟[1] 

机构地区：[1]北京化工大学生命科学与技术学院北京市生物加工重点实验室,北京100029

出　　处：《北京化工大学学报（自然科学版）》2013年第3期75-78,共4页Journal of Beijing University of Chemical Technology(Natural Science Edition)

基　　金：国家自然科学基金(21076017);国家"973"计划(2012CB725200)

摘　　要：以橙色绿屈挠菌(Chloroflexus aurantiacus)dsm636基因组DNA为模板,应用PCR技术扩增并克隆了丙烯酰CoA合成酶(ACS)基因,并连接到表达载体pET-22b(+)质粒上,构建了pET-22b(+)-Acs重组质粒,测序结果100%正确。将重组质粒pET-22b(+)-Acs转化到大肠杆菌表达菌株BL21(DE3)中,通过氨苄霉素抗性平板筛选,构建了大肠杆菌BL21(DE3)-pET-22b(+)-Acs基因工程菌。重组菌株经异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,SDS-PAGE电泳分析,目标条带出现在分子量约160000处,表明丙烯酰CoA合成酶基因在大肠杆菌BL21(DE3)中成功表达;体外酶活实验证明,所表达的丙烯酰CoA合成酶是具有活性的。The expression and activity in E.coli of acryloyl-coenzyme A synthase（ACS）,a key enzyme which is able to convert 3-hydroxypropionic acid（3-HP） into acrylic acid,have been studied.The gene Acs encoding acryloyl-coenzyme A synthase was cloned and amplified from the genomic DNA of Chloroflexus aurantiacus dsm636.The PCR product was inserted into the expression vector pET-22b（＋） under the control of T7 promoter.The verified recombinant vector was transformed into the expression strain E.coli BL21（DE3） which is deficient in acryloyl-coenzyme A synthase.The expression of acryloyl-coenzyme A synthase in the recombinant strain was induced by isopropyl-β-D-thiogalactopyranoside（IPTG）,and sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE） showed that the cloned Acs was expressed in the E.coli.The target strip of the expression product had a molecular weight of about 160000.In vitro enzyme activity determination showed that the expressed acryloyl-coenzyme A synthase has active characteristics.

关 键 词：丙烯酸 丙烯酰CoA合成酶基因 3-羟基丙酸 大肠杆菌 

分 类 号：Q78[生物学—分子生物学]