Abstract
2-Ketogluconate reductase (2KGR) from the cell free extract of Gluconobacter liquefaciens (IFO 12388) was purified about 1000-fold by a procedure involving ammonium sulfate fractionation and column chromatographies using DEAE-cellulose, hydroxylapatite, and Sephadex gel. The purified enzyme gave a single band on polyacrymamide gel electrophoresis. NADP was specifically required for the oxidation reaction of gluconic acid. Using gel filtration a molecular weight of about 110, 000 was estimated for the enzyme. The pH optimum for the oxidation of gluconic acid (GA) to 2-ketogluconic acid (2KGA) by the enzyme was 10.5 and for the reduction of 2KGA was 6.5. The optimum temperature of the enzyme was 50°C for both reactions of oxidation and reduction. The enzyme was stable at pH between 5.0 and 11.0 and at temperature under 50°C. The enzyme activity was strongly inhibited with p-chloromercuribenzoate and mercury ions, but remarkably stimulated by manganese ions (1×10-3M). Km value of the enzyme for GA was 1.3×10-2M and for 2KGA was 6.6×10-3M. Km values for NADP and NADPH2 were 1.25×10-5 and 1.52×10-5M, respectively.
