Purification and Properties of NADPH-Linked L-Sorbose Reductase from Gluconobacter melanogenus N44-1.

Abstract

NADPH-Linked L-sorbose reductase from the cytosol fraction of Gluconobacter melanogenus N44-1, which is a 2-keto-L-gulonic acid producer from L-sorbose or D-sorbitol, was purified about 500-fold by DEAE-Cellulose, Blue Sepharose, and DEAE-Sepharose column chromatographies. The purified enzyme was entirely homogeneous on polyacrylamide gel and SDS gel electrophoresis. NADPH and NADP were specifically required for the reduction and oxidation of the substrate, respectively. The apparent molecular weight was 60, 000 by Sephadex G-200 column chromatography, and that of its subunit was 60, 000 by SDS-gel electrophoresis. The pH optimum for the reduction of L-sorbose and D-fructose was 7.0. The pH optimum for the oxidation of D-sorbitol to L-sorbose and D-mannitol to D-fructose was 10.0-10.5.