丙烯酰CoA合成酶在大肠杆菌中的表达及其酶活性表征

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。

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

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 (SDSPAGE) 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.