重组葡萄糖脱氢酶的表达及辅酶再生应用研究

摘要

To solve the regeneration of hydrogen donor NADPH for cyclohexanone reduction catalyzed by the recombinant strain Escherichia coli BL21 (pET28a-eryKR 1)heterologously expressing ketore-ductase domain (EryKR1),the recombinant strain E .coli BL21 (pET28a-gdh)was constructed which cloned the glucose dehydrogenase(GDH)gene gdh from Bacillus subtilis .BLAST analysis showed that nucleotide sequence of the gene gdh in the recombinant strain had the consistency of 100% with that of B .subtilis strain 9902 (accession number EF626962.1 ).SDS-PAGE analysis revealed that GDH could be highly expressed in the recombinant strain induced by IPTG,which accounted for 64%of the total soluble protein.Specific activity of GDH crude enzyme was 137.90 U/mg.Recombinat strain E .coli BL21(pET28a-gdh)was added to the cyclohexanone reduction reaction catalyzed by E . coli BL21(pET28a-eryKR 1).Gas chromatography analysis showed that the yield of cyclohexanol in the two-recombinant strain reaction system was 82.21% and 3.23 times that of the system without E . coli BL21(pET28a-gdh),which confirmed that recombinant GDH could solve the coenzyme regenera-tion problem for cyclohexanone reduction catalyzed by ketoreductase domain.%为解决异源表达酮还原酶域EryKR1的重组大肠杆菌Escherichia coli BL21(pET28a-eryKR1)催化环己酮还原时消耗的氢供体NADPH再生的问题,构建了克隆枯草芽孢杆菌葡萄糖脱氢酶基因gdh的重组菌E.coliBL21(pET28a-gdh),其中的gdh基因经Nucleotide BLAST功能分析显示与枯草芽孢杆菌9902的gdh基因序列(登录号为EF626962.1)的一致性达到100%.SDS-PAGE检测显示该重组菌经IPTG诱导后可以高效表达出葡萄糖脱氢酶(GDH),其表达量占全菌可溶性蛋白质的64%.GDH粗酶液的比酶活为137.90 U/mg.通过气相色谱检测添加了E.coli BL21(pET28a-gdh)的E.coli BL21(pET28a-eryKR1)环己酮还原反应体系中的环己醇含量,结果显示加入重组GDH的双重组菌耦合反应体系中环己醇的产率为82.21%,是未添加GDH的反应体系对应值的3.23倍,表明重组GDH可以为EryKR1还原环己酮系统解决辅酶再生问题.