构巢曲霉奎尼酸脱氢酶基因qutB在大肠杆菌中的克隆与表达

Cloning and expression in E.coli of the quinate dehydrogenase qutB gene of Aspergillus nidulans

从莽草酸途径合成奎尼酸(QA),奎尼酸脱氢酶(QDHase)是必须的,大肠杆菌基因组不含有该酶的编码基因,在构巢曲霉(Aspergillusnidulans)中存在qutB基因编码此酶。以构巢曲霉染色体DNA为模板,采用PCR扩增得到qutB基因,在大肠杆菌中进行了克隆表达。结果表明,该基因在λ噬菌体的PRPL串联启动子驱动下实现了QDHase的表达。SDS-PAGE显示,重组菌热诱导后表达出大小约36000的特异蛋白,表达量占菌体总蛋白的19.81%。经酶活性测定,表达产物具有生物学活性,与对照菌株相比,其酶活性提高了27.8倍,为进一步奎尼酸生物合成研究奠定了基础。

For quinate synthesis from the shikimate pathway,a quinate dehydrogenase(QDHase)would be necessary.There is not a gene encoding QDHase in E.coli genome,but the qutB gene of A.nidulans may encode it.In this work,A.nidulans chormosome DNA was used as template to amplify qutB gene by PCR which was cloned and ex-pressed in E.coli.The quinate dehydrogenase was expressed under control of Lambda phase PRPL promoter in DH5α/pBVqutB strain,SDS-PAGE showed that the recombinant bacteria produced a special protein band about 36kD in molecular weight after heat induction,the expression level reached up to19.81%of the total soluble bacte-rial proteins.The expression product had bioactivity and its enzyme activity was raised to27.8fold.This provided a basis for further quinate biosynthesis study.