Abstract: An intracellular membrane-bound gluconate dehydrogenase (GADH) with specific activity of 91.43 U/mg and purification fold of 160.4 was purified from Serratia marcescens JUIM03, a 2-ketogluconic acid (2KGA)-producing strain, using a five-step procedure, including ultrasonication, Triton X-114 phase separation, ammonium sulfate fractionation, DEAE-sepharose fast flow and hydroxyapatite column chromatography. The results showed that the membrane-bound GADH consisted of three subunits with molecular masses of 65.0, 45.0 and 23.0 kDa respectively. It was a flavin protein-containing cytochrome C catalyzing the oxidation of gluconic acid to 2KGA. The optimum reaction temperature and pH were 40 ℃ and 5.0 kDa, respectively. It was stable below 30 ℃ and in the pH range of 5.0–6.0, and exhibited strict substrate specificity for D-gluconate with a Km of 1.33 mmol/L. Several metal ions including Fe3+, Cu2+ and Zn2+, organic solvents including ethanol, isopropanol, methanol and acetone, denaturants including SDS and mercaptoethanol, and organic acids including oxamic acid and oxalic acid had significant inhibitory effects on the enzyme activity. This study provides a theoretical basis for the optimization and control of 2KGA biosynthesis in Serratia marcescens.

Key words: Serratia marcescens, 2-ketogluconic acid, membrane-bound gluconate dehydrogenase, purification, enzymatic properties