Characterization of a sorbose oxidase involved in the biosynthesis of 2-keto-L-gulonic acid from Gluconobacter oxydans WSH-004

Abstract

2-Keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C, can be synthesized from L-sorbose by many microorganisms via “sorbose dehydrogenase” and “sorbosone dehydrogenase (SNDH)”. However, only a few studies were carried out on the catalytic properties of sorbose dehydrogenase in Gluconobacter oxydans. In this study, sorbose dehydrogenase of G. oxydans WSH-004 was identified. Different from previous findings, this enzyme turned out to be a sorbose oxidase (SOX) rather than a sorbose dehydrogenase·H2O2 was determined in the oxidization of L-sorbose to L-sorbosone and it also showed inhibition on the activity of SOX. Phenazine methosulfate (PMS) was the best electron acceptor of SOX and the Km of SOX for L-sorbose was 117 mM when dichlorophenolindophenol (DCIP) was used as the electron acceptor. Finally, the enzymatic catalysis of SOX and SNDH was conducted in vitro. 2-KLG could be produced from L-sorbose by SOX and SNDH with nicotinamide adenine dinucleotide phosphate (NADP+) as a cofactor. These results were useful in the reconstruction of G. oxydans to synthesize 2-KLG in a one-step fermentation process.