Purification and Properties of 5-Ketogluconate Reductase from Gluconobacter liquefaciens

Abstract

5-Ketogluconate reductase (5 KGR) from the cell free extract of Gluconobacter liquefaciens (IFO 12388) was partially purified about 120-fold by a procedure employing ammonium sulfate fractionation, and DEAE-cellulose-, hydroxylapatite- and DEAE-Sephadex A-50-column chromatographies. NADP was specifically required for the oxidative reaction of gluconic acid. The optimum pH for the oxidation of gluconic acid (GA) to 5-ketogluconic acid (5 KGA) by the enzyme was 10.0 and for the reduction of 5 KGA was 7.5. The optimum temperature of the enzyme was 50°C for both reactions of oxidation and reduction. The enzyme was considerably unstable and lost all of its activity within 3 days. The enzyme activity was strongly inhibited with p-chloromercuribenzoate and mercury ion, but remarkably stimulated by EDTA (1×10^-3M). Apparent Km values were 1.8×10^-2M for GA, 0.9×10^-3M for 5 KGA, 1.6×10^-5M for NADP, and 1.1×10^-5M for NADPH².