知识与财富的链接
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 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-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.
