Abstract:A codon-optimized gene(named Sygdh) encoding a glucose 1-dehydrogenase of Thermoplasma acidophilum was cloned into the plasmid pET28a and functionally expressed in Escherichia coli. The expressed enzyme was purified to homogeneity using Ni-NTA affinity chromatography with an apparent molecular weight of 41.0 kDa by SDS-PAGE analysis and characterized using the model substrate of D-glucose. The purified recombinant SyGDH was 4.5 U/mg at pH 7.5 and 45 ℃, and highly stable at pH 6.0~8.0 and 55 ℃ or below. It was activated by Zn2+, but strongly inhibited by Cu2+ and not significantly affected by other metal ions tested and EDTA. The Km and Vmax of recombinant SyGDH towards D-glucose were 28.2 mmol/L and 6.5 U/mg, respectively. The results showed that SyGDH exhibits much higher affinity with NADP+ than NAD+ and remarkably resists to several organic solvents. Furthermore, two recombinant strains E. coli BL21/pET28a-S1 and E. coli BL21/pET28a-Sygdh, which include recombinant NADPH-dependent carbonyl reductase and recombinant SyGDH, respectively, were mixed and used for the asymmetric reduction of Ethyl 4-chloroacetoacetate to Ethyl-4-chloro-3-hydroxybutyrate in the coenzyme cycle system. The product yield reached 99.0%, which is 3.11 fold as high as that of reaction without adding recombinant SyGDH strain, confirming that the recombinant SyGDH could be used as NADPH regenerator in biocatalysis oxidation and reduction system.
