ISOLATION AND CHARACTERIZATION OF A L-GLUCITOL DEHYDROGENASE FROM THENEWLY 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 Pseudomona s sp. For growth on L-glucitol an L-glucitol dehydrogenase (L-glucitol : NAD(+) 5-oxidoreductase) is produced, a so far unknown enzyme that c onverts L-glucitol to D-sorbose. The L-glucitol dehydrogenase was puri fied to apparent homogeneity by (NH4)(2)SO4 precipitation, chromatogra phy on phenyl-Sepharose, anion exchange chromatography on Q-Sepharose and gel filtration on Superdex 200 with FPLC. The relative molecular w eight (M(r)) of the native enzyme was determined to be 50000 and its i soelectric point was pH 4.5. SDS-PAGE resulted in one band representin g a polypeptide with an M(r) of 25 000, indicating that the native enz yme is a dimer. L-Glucitol dehydrogenase was specific for NAD(+), but catalyzed the oxidation of several polyols to the corresponding ketose s. The K-m values were determined to be 6.5 mM for L-glucitol, 62 mM f or ribitol, 80 mM for xylitol, 89 mM for galactitol, 200 mM for D-gluc itol, 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 re agents.