重组的葡萄糖脱氢酶催化辅酶的再生性质

Cofactor regeneration of recombinant glucose dehydrogenase

为解决生物催化环己酮还原反应中的辅酶再生问题,扩增了枯草芽孢杆菌中的葡萄糖脱氢酶基因(gdh),构建了表达载体pET-gdh,转化到Escherichiacoli BL21(DE3)后,获得了重组菌株E.coliBL21(pET-gdh1).经IPTG诱导后,葡萄糖脱氢酶粗酶液的比酶活达18.4U/mg蛋白,SDS-PAGE电泳分析表明重组蛋白表达量占全菌胞内可溶性蛋白质的54.1%.添加E.coliBL21(pET-gdh1)到以异源表达糖多孢红霉菌聚酮合成酶模块1的酮还原酶域基因的重组菌E.coliBL21(pET-eryKR2)为催化剂的环己酮还原体系中,利用气相色谱分析转化液,显示此条件下产物环己醇的得率为93.24%,是未添加E.coliBL21(pET-gdh1)转化反应产率的3.4倍,说明E.coliBL21(pET-gdh1)能为此环己酮还原系统完成还原态辅酶NADPH的再生.

To solve the cofactor regeneration of cyclohexanone reduction by biocatalysis,glucose dehydrogenase（GDH） gene from Bacillus subtilis was amplified by polymerase chain reaction and cloned into vector pET-28a to construct recombinant plasmid pET-gdh,which was transformed into Escherichia coli BL21（DE3）.Then recombinant E.coli BL21 （pET-gdh1） was obtained.After induction by IPTG,the specific activity of crude GDH from recombinant was 18.4 U/mg.SDS-PAGE showed that GDH accounted for 54.1% of total soluble protein.E.coli BL21 （pET-gdh1） was added to the cyclohexanone reduction reaction mixture which used E.coli BL21 （pET-eryKR2） harboring the gene coding ketoreductase domain in the first module of polyketide synthase from Saccharopolyspora erythraea as catalysts.This ferment analysis by gas chromatography showed that the yield of cyclohexanol was 93.24% and about 3.4 fold as high as that of the reaction without addition of E.coli BL21 （pET-gdh1）.This result predicated that E.coli BL21（pET-gdh1） could be used as NADPH regenerator in this cyclohexanone reduction system.