ISOLATION AND CHARACTERIZATION OF A L-GLUCITOL DEHYDROGENASE FROM THE NEWLY ISOLATED BACTERIUM PSEUDOMONAS SP AC

A bacterial strain capable of growing on the unnatural sugar alcohol L-glucitol was isolated from a soil sample and identified as Pseudomonas sp. For growth on L-glucitol an L-glucitol dehydrogenase (L-glucitol: NAD(+) 5-oxidoreductase) is produced, a so far unknown enzyme that converts L-glucitol to D-sorbose. The L-glucitol dehydrogenase was purified to apparent homogeneity by (NH4)(2)SO4 precipitation, chromatography on phenyl-Sepharose, anion exchange chromatography on Q-Sepharose and gel filtration on Superdex 200 with FPLC. The relative molecular weight (M(r)) of the native enzyme was determined to be 50000 and its isoelectric point was pH 4.5. SDS-PAGE resulted in one band representing a polypeptide with an M(r) of 25 000, indicating that the native enzyme is a dimer. L-Glucitol dehydrogenase was specific for NAD(+), but catalyzed the oxidation of several polyols to the corresponding ketoses. The K-m values were determined to be 6.5 mM for L-glucitol, 62 mM for ribitol, 80 mM for xylitol, 89 mM for galactitol, 200 mM for D-glucitol, 40 pM for NAD, 75 mM for D-sorbose, and 10 mu M for NADH. The pH optimum for substrate oxidation was 11.5. The activity of L-glucitol dehydrogenase was reduced by chelating reagents as well as by thiol reagents.