重组葡萄糖脱氢酶的表达及辅酶再生应用研究

Expression of recombinant glucose dehydrogenase and its application to coenzyme regeneration

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.coli BL21(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137.90U/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。

To solve the regeneration of hydrogen donor NADPH for cyclohexanone reduction catalyzed by the recombinant strain Escherichia coli BL21（pET28a-eryKR1）heterologously expressing ketoreductase 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 of100% 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-eryKR1）.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 regeneration problem for cyclohexanone reduction catalyzed by ketoreductase domain.