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 |
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| Institution: | 1. Wuxi School of Medicine, Jiangnan University, Wuxi, 214122, China;2. School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China;3. School of Biotechnology, Jiangnan University, Wuxi, 214122, China;1. School of Biotechnology and Health Sciences, Wuyi University, Jiangmen 529020, Guangdong, China;2. Lab of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, Guangdong, China;1. State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, PR China;2. Enzyme and Fermentation Technology Laboratory, College of Light Industry Science and Engineering, Nanjing Forestry University, Nanjing 210037, PR China;1. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China;2. Wuxi School of Medicine, Jiangnan University, Wuxi 214122, PR China;3. School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China;1. Institute of Biochemistry, Department of Biotechnology & Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Str. 4, 17489 Greifswald, Germany;2. Department of Biology, Computer-aided Synthetic Biology, Technische Universität Darmstadt, Schnittspahnstr. 10, 64287 Darmstadt, Germany |
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| 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. |
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| Keywords: | Glucose 1-dehydrogenase Coenzyme regeneration Carbonyl reductase Organic solvent/buffer System Haloketone Enantiopure h alohydrin |
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