Membrane-bound D-sorbitol dehydrogenase of Gluconobacter suboxydans IFO 3255--enzymatic and genetic characterization.

Gluconobacter strains effectively produce L-sorbose from D-sorbitol because of strong activity of the D-sorbitol dehydrogenase (SLDH). L-sorbose is one of the important intermediates in the industrial vitamin C production process. Two kinds of membrane-bound SLDHs, which consist of three subunits, were reportedly found in Gluconobacter strains [Agric.

5-keto-D-gluconate production is catalyzed by a quinoprotein glycerol dehydrogenase, major polyol dehydrogenase, in gluconobacter species.

Acetic acid bacteria, especially Gluconobacter species, have been known to catalyze the extensive oxidation of sugar alcohols (polyols) such as D-mannitol, glycerol, D-sorbitol, and so on. Gluconobacter species also oxidize sugars and sugar acids and uniquely accumulate two different keto-D-gluconates, 2-keto-D-gluconate and 5-keto-D-gluconate, in the culture medium by the oxidation of D-gluconate. However, there are still many controversies regarding their enzyme systems, especially on D-sorbitol and also D-gluconate oxidations.

Main polyol dehydrogenase of Gluconobacter suboxydans IFO 3255, membrane-bound D-sorbitol dehydrogenase, that needs product of upstream gene, sldB, for activity.

The D-sorbitol dehydrogenase gene, sldA, and an upstream gene, sldB, encoding a hydrophobic polypeptide, SldB, of Gluconobacter suboxydans IFO 3255 were disrupted in a check of their biological functions. The bacterial cells with the sldA gene disrupted did not produce L-sorbose by oxidation of D-sorbitol in resting-cell reactions at pHs 4.5 and 7.

Molecular cloning and functional expression of D-sorbitol dehydrogenase from Gluconobacter suboxydans IF03255, which requires pyrroloquinoline quinone and hydrophobic protein SldB for activity development in E. coli.

The sldA gene that encodes the D-sorbitol dehydrogenase (SLDH) from Gluconobacter suboxydans IFO 3255 was cloned and sequenced. It encodes a polypeptide of 740 residues, which contains a signal sequence of 24 residues. SLDH had 35-37% identity to the membrane-bound quinoprotein glucose dehydrogenases (GDHs) from E.

NADPH-dependent L-sorbose reductase is responsible for L-sorbose assimilation in Gluconobacter suboxydans IFO 3291.

The NADPH-dependent L-sorbose reductase (SR) of L-sorbose-producing Gluconobacter suboxydans IFO 3291 contributes to intracellular L-sorbose assimilation. The gene disruptant showed no SR activity and did not assimilate the once-produced L-sorbose, indicating that the SR functions mainly as an L-sorbose-reducing enzyme in vivo and not as a D-sorbitol-oxidizing enzyme.