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. Recently, pyrroloquinoline quinone-dependent quinoprotein D-arabitol dehydrogenase and D-sorbitol dehydrogenase have been purified from G. suboxydans, both of which have similar and broad substrate specificity towards several different polyols. In this study, both quinoproteins were shown to be identical based on their immunocross-reactivity and also on gene disruption and were suggested to be the same as the previously isolated glycerol dehydrogenase (EC 1.1.99.22). Thus, glycerol dehydrogenase is the major polyol dehydrogenase involved in the oxidation of almost all sugar alcohols in Gluconobacter sp. In addition, the so-called quinoprotein glycerol dehydrogenase was also uniquely shown to oxidize D-gluconate, which was completely different from flavoprotein D-gluconate dehydrogenase (EC 1.1.99.3), which is involved in the production of 2-keto-Dgluconate. The gene disruption experiment and the reconstitution system of the purified enzyme in this study clearly showed that the production of 5-keto-D-gluconate in G. suboxydans is solely dependent on the quinoprotein glycerol dehydrogenase.Acetic acid bacteria are obligate aerobes well known as vinegar producers and also known to be able to oxidize various sugars and sugar alcohols such as D-glucose, glycerol, D-sorbitol, and so on, in addition to ethanol. Such oxidation reactions are called oxidative fermentation, since they involve incomplete oxidations of such alcohols or sugars accompanied by an accumulation of the corresponding oxidation products in large amounts in the culture medium. Of the two genera of acetic acid bacteria, Gluconobacter species extensively catalyze the oxidation of sugars and sugar alcohols except for ethanol, while Acetobacter species have a high ability to oxidize ethanol to acetic acid. These oxidation reactions of sugars or sugar alcohols seem to be carried out by membrane-bound dehydrogenases linked to the respiratory chain located in the cytoplasmic membrane of the organism (14). Of these oxidative fermentations of acetic acid bacteria, vinegar production from ethanol and 2-keto-D-gluconate (2KGA) production from glucose have each been shown to be carried out by sequential membranebound alcohol and aldehyde dehydrogenases and by glucose and gluconate dehydrogenases, respectively (14).There is still controversy about the mechanism of L-sorbose and 5-keto-D-gluconate (5KGA) production in Gluconobacter species. Three different membrane-bound enzymes have been proposed to be involved in L-sorbose production fr...

A novel enzyme, L-sorbosone dehydrogenase 1 (SNDH1), which directly converts L-sorbosone to L-ascorbic acid (L-AA), was isolated from Ketogulonicigenium vulgare DSM 4025 and characterized. This enzyme was a homooligomer of 75-kDa subunits containing pyrroloquinoline quinone (PQQ) and heme c as the prosthetic groups. Two isozymes of SNDH, SNDH2 consisting of 75-kDa and 55-kDa subunits and SNDH3 consisting of 55-kDa subunits, were also purified from the bacterium. All of the SNDHs produced L-AA, as well as 2-keto-L-gulonic acid (2KGA), from L-sorbosone, suggesting that tautomerization of L-sorbosone causes the dual conversion by SNDHs. The sndH gene coding for SNDH1 was isolated and analyzed. The N-terminal four-fifths of the SNDH amino acid sequence exhibited 40% identity to the sequence of a soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. The C-terminal one-fifth of the sequence exhibited similarity to a c-type cytochrome with a heme-binding motif. A lysate of Escherichia coli cells expressing sndH exhibited SNDH activity in the presence of PQQ and CaCl 2 . Gene disruption analysis of K. vulgare indicated that all of the SNDH proteins are encoded by the sndH gene. The 55-kDa subunit was derived from the 75-kDa subunit, as indicated by cleavage of the C-terminal domain in the bacterial cells. L-Ascorbic acid (L-AA) is an essential nutrient for humans.It is known that L-AA is directly converted from aldonolactones, such as L-gulono-␥-lactone and L-galactono-␥-lactone, by aldonolactone dehydrogenase/oxidases in plants and mammals (except some primates, including humans). The L-gulono-␥-lactone oxidase that catalyzes the conversion of L-gulono-␥-lactone to L-AA was isolated from rat and goat livers and characterized by Nishikimi et al. (19). An L-galactono-␥-lactone oxidase of yeast origin was described by Bleeg (4). In addition, L-sorbosone is also known to be a potential precursor of L-AA in plants. An NADP-dependent L-sorbosone dehydrogenase (SNDH) that converts L-sorbosone to L-AA in spinach leaves has been reported (17), but the enzyme has not been purified or characterized in detail.In current industrial L-AA production processes, as summarized by Hancock and Viola (13), 2-keto-L-gulonic acid (2KGA) is a key intermediate that is chemically converted to L-AA. All of the processes require a large amount of energy and organic solvent, and thus a cheaper and environmentally conscious substitute process, such as enzymatic conversion, is desirable. It has also been reported that L-sorbosone is converted to 2KGA in bacteria. Many microbial L-sorbosone dehydrogenases, including enzymes from Acetobacter liquefaciens (27), Gluconobacter melanogenus UV10 (14), and Gluconobacter oxydans T-100 (23), have been isolated and characterized. Moreover, Ketogulonicigenium vulgare DSM 4025 has been reported to produce aldehyde dehydrogenase (ALDH) (15) and L-sorbose/ L-sorbosone dehydrogenases (SSDHs) (3), which are responsible for sequentially converting L-sorbose to 2KGA via L-sorbosone.The metabolic...

The strain SPO1 producing 13g of 2-keto-L-gulonic acid (2KGA) per liter was isolated as a spontaneous mutant of Gluconobacter melanogenus IFO 3293. For the enhancement of 2KGA productivity, we did further strain improvement studies of the mutant.As a result, the mutant U13, producing about 60g of 2KGAper liter from 100g of L-sorbose per liter, was obtained. In addition, the mutant Z84, producing about 60 g of 2KGAper liter from 100 g of D-sorbitol per liter, was also obtained.During the fermentation from L-sorbose and D-sorbitol, 5 to 10g of L-idonic acid per liter was produced as a by-product, but L-idonic acid was converted to 2KGAbefore the end of fermentation.

D-Sorbitol dehydrogenase was solubilized from the membrane fraction of Gluconobacter suboxydans IFO 3255 with Triton X-100 in the presence of D-sorbitol. Puriˆcation of the enzyme was done by fractionation with column chromatographies of DEAE-Cellulose, DEAE-Sepharose, hydroxylapatite, and Sephacryl HR300 in the presence of Triton X-100.The molecular mass of the enzyme was 800 kDa, consisting of homologous subunits of 80 kDa. The optimum pH of the enzyme activity was 6.0, and the optimum temperature was 309 C.Western blot analysis suggested the occurrence of the enzyme in all the Gluconobacter strains tested.

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