Characterization of a robust glucose 1-dehydrogenase, SyGDH, and its application in NADPH regeneration for the asymmetric reduction of haloketone by a carbonyl reductase in organic solvent/buffer system

Abstract To realize coenzyme regeneration in the reduction of haloketones, a codon-optimized gene Sygdh encoding glucose 1-dehydrogenase (SyGDH) was synthesized based on the putative GDH gene sequence (Ta0897) in Thermoplasma acidophilum genomic DNA, and expressed in E. coli BL21(DE3). Recombinant SyGDH was purified to homogeneity by affinity chromatography with the specific activity of 86.3 U/mg protein towards D-glucose at the optimum pH and temperature of 7.5 and 40 °C. It was highly stable in a pH range of 4.5–8.0 and at 60 °C or below, and resistant to various organic solvents. The Km and catalytic efficiency (kcat/Km) of SyGDH towards NADP+ were 0.67 mM and 104.0 mM−1 s−1, respectively, while those towards NAD+ were 157.9 mM and 0.64 mM−1 s−1, suggesting that it preferred NADP+ as coenzyme to NAD+. Additionally, using whole cells of E. coli/Sygdh-Sys1, coexpressing SyGDH and carbonyl reductase (SyS1), as the biocatalyst, the asymmetric reduction of 60 mM m-chlorophenacyl chloride coupled with the regeneration of NADPH in situ was conducted in DMSO/phosphate buffer (2:8, v/v) system, producing (R)-2-chloro-1-(3-chlorophenyl)ethanol with over 99.9% eep and 99.2% yield. Similarly, the reduction of 40 mM α-bromoacetophenone in n-hexane/buffer (6:4, v/v) biphasic system produced (S)-2-bromo-1-phenylethanol with over 99.9% eep and 98.3% yield.