(9/50) 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 7.0, indicating that the dehydrogenase was the main D-sorbitol-oxidizing enzyme in this bacterium. The cells did not produce D-fructose from D-mannitol or dihydroxyacetone from glycerol. The disruption of the sldB gene resulted in undetectable oxidation of D-sorbitol, D-mannitol, or glycerol, although the cells produced the dehydrogenase. The cells with the sldB gene disrupted produced more of what might be signal-unprocessed SldA than the wild-type cells did. SldB may be a chaperone-like component that assists signal processing and folding of the SldA polypeptide to form active D-sorbitol dehydrogenase.  (+info)

(10/50) 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. 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 immuno-cross-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-D-gluconate. 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.  (+info)

(11/50) 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. Biol. Chem. 46 (1982) 135,FEMS Microbiol. Lett. 125 (1995) 45]. We purified a one-subunit-type SLDH (80 kDa) from the membrane fraction of Gluconobacter suboxydans IFO 3255 solubilized with Triton X-100 in the presence of D-sorbitol, but the cofactor could not be identified from the purified enzyme. The SLDH was active on mannitol, glycerol and other sugar alcohols as well as on D-sorbitol to produce respective keto-aldoses. Then, the SLDH gene (sldA) was cloned and sequenced. It encodes the polypeptide of 740 residues, which contains a signal sequence of 24 residues. SLDH had 35-37% identity to those of membrane-bound quinoprotein glucose dehydrogenases (GDHs) from Escherichia coli, Gluconobacter oxydans and Acinetobacter calcoaceticus except the N-terminal hydrophobic region of GDH. Additionally, the sldB gene located just upstream of sldA was found to encode the polypeptide consisting of 126 very hydrophobic residues that is similar to the one-sixth N-terminal region of the GDH. Development of the SLDH activity in E. coli required co-expression of the sldA and sldB genes and the presence of PQQ. The sldA gene disruptant showed undetectable oxidation activities on D-sorbitol in growing culture, and resting-cell reaction (pH 4.5 and 7); in addition, they showed undetectable activities on D-mannitol and glycerol. The disruption of the sldB gene by a gene cassette with a downward promoter to express the sldA gene resulted in formation of a larger size of the SLDH protein and in undetectable oxidation of the polyols. In conclusion, the SLDH of the strain 3255 functions as the main polyol dehydrogenase in vivo. The sldB polypeptide possibly has a chaperone-like function to process the SLDH polypeptide into a mature and active form.  (+info)

(12/50) Purification and characterization of membrane-bound quinoprotein quinate dehydrogenase.

Several bacterial strains carrying quinoprotein quinate dehydrogenase (QDH) were screened through acetic acid bacteria and other bacteria. Strong enzyme activity was found in the membrane fraction of Gluconobacter melanogenus IFO 3294, G. oxydans IFO 3292, G. oxydans IFO 3244, and some strains of Acinetobacter calcoaceticus. Interestingly, in the membrane fraction of A. calcoaceticus AC3, which is unable to produce pyrroloquinoline quinone (PQQ), fairly large amounts of apo-QDH were formed, and were converted to holo-QDH only by the addition of PQQ. It was difficult to detach PQQ from the holo-QDH by EDTA treatment, and EDTA treatment with apo-QDH prior to PQQ addition gave no significant holo-QDH. For QDH purification, Gluconobacter strains were not suitable due to the presence of huge amounts of quinohemoprotein alcohol dehydrogenase (ADH) in the same membrane, which was co-solubilized with QDH and disturbed purification of QDH. Purification of holo-QDH was done with Acinetobacter sp. SA1 instead, which contained no ADH. Apo-QDH was purified from A. aclcoaceticus AC3. This is the first report dealing with QDH purification, and two different criteria of QDH purification were given. A combination of two steps using butyl-Toyopearl and hydroxyapatite columns gave a highly purified holo-QDH which was monodispersed and showed enough purity, though the specific activity did not increase as much as expected. When QDH purification was done with A. calcoaceticus AC3 in the absence of PQQ, purified apo-QDH appeared to be a dimer, which was converted to the monomer on addition of PQQ. Since QDH was highly hydrophobic, one-step chromatography on a DEAE-Sepharose column was tried. Purified holo-QDH of higher specific activity was obtained with a higher yield. The molecular mass of QDH was estimated to be 88 kDa. There was no characteristic absorption spectrum with the purified QDH except for a small bump around 420 nm. QDH oxidized only quinate and shikimate so far examined. The optimal QDH activity was found at pH 6-7 when assayed with artificial electron acceptors. QDH was formed in the presence or absence of quinate in the culture medium, although stronger induction was usually observed in the presence of quinate.  (+info)

(13/50) 3-dehydroquinate production by oxidative fermentation and further conversion of 3-dehydroquinate to the intermediates in the shikimate pathway.

3-Dehydroquinate production from quinate by oxidative fermentation with Gluconobacter strains of acetic acid bacteria was analyzed for the first time. In the bacterial membrane, quinate dehydrogenase, a typical quinoprotein containing pyrroloquinoline quinone (PQQ) as the coenzyme, functions as the primary enzyme in quinate oxidation. Quinate was oxidized to 3-dehydroquinate with the final yield of almost 100% in earlier growth phase. Resting cells, dried cells, and immobilized cells or an immobilized membrane fraction of Gluconobacter strains were found to be useful biocatalysts for quinate oxidation. 3-Dehydroquinate was further converted to 3-dehydroshikimate with a reasonable yield by growing cells and also immobilized cells. Strong enzyme activities of 3-dehydroquinate dehydratase and NADP-dependent shikimate dehydrogenase were detected in the soluble fraction of the same organism and partially fractionated from each other. Since the shikimate pathway is remote from glucose in the metabolic pathway, the entrance into the shikimate pathway from quinate to 3-dehydroquinate looks advantageous to produce metabolic intermediates in the shikimate pathway.  (+info)

(14/50) Identification of strains assigned to the genus Gluconobacter Asai 1935 based on the sequence and the restriction analyses of the 16S-23S rDNA internal transcribed spacer regions.

Thirteen reference strains, including the type strains of the type species of the genus Gluconobacter, Gluconobacter oxydans (NBRC 14819T), Gluconobacter cerinus (NBRC 3267T), and Gluconobacter frateurii (IFO 3264T) were examined for their species identification based on the sequence and the restriction analyses of the 16S-23S rDNA internal transcribed spacer (ITS) regions. A phylogenetic tree constructed by the neighbor-joining method represented three clusters corresponding respectively to the three species, G. oxydans, G. cerinus, and G. frateurii. The type strain of Gluconobacter asaii (NBRC 3276T), which is a junior subjective synonym of G. cerinus, was included completely in the G. cerinus cluster. Several restriction endonucleases discriminating the three species from one another were selected by computer analyses: Bsp1286I, MboII, SapI, Bpu10I, EarI, BsiHKAI, and FatI. On digestion of the PCR products with restriction endonucleases Bsp1286I and MboII, all the restriction patterns coincided with those of the type strains of the three species except for strain NBRC 3251. This strain gave a different pattern from the type strain of G. frateurii, when digested with MboII. However, strain 3251 was included phylogenetically in the G. frateurii cluster. All the reference strains were thus identified at the species level by the sequence and the restriction analyses of the 16S-23S rDNA ITS regions.  (+info)

(15/50) Gluconobacter thailandicus sp. nov., an acetic acid bacterium in the alpha-Proteobacteria.

Four strains of acetic acid bacteria were isolated from flowers collected in Thailand. In phylogenetic trees based on 16S rRNA gene sequences and 16S-23S rDNA internal transcribed spacer (ITS) region sequences, the four isolates were located in the lineage of the genus Gluconobacter and constituted a separate cluster from the known Gluconobacter species, Gluconobacter oxydans, Gluconobacter cerinus, and Gluconobacter frateurii. In addition, the isolates were distinguished from the known species by restriction analysis of 16S-23S rDNA ITS region PCR products using three restriction endonucleases Bsp1286I, MboII, and AvaII. The DNA base composition of the isolates ranged from 55.3-56.3 mol% G+C. The four isolates constituted a taxon separate from G. oxydans, G. cerinus, and G. frateurii on the basis of DNA-DNA similarities. Morphologically, physiologically, and biochemically, the four isolates were very similar to the type strains of G. oxydans, G. cerinus, and G. frateurii; however, the isolates were discriminated in their growth at 37 degrees C from the type strains of G. cerinus and G. frateurii, and in their growth on L-arabitol and meso-ribitol from the type strain of G. oxydans. The isolates showed no acid production from myo-inositol or melibiose, which differed from the type strains of the three known species. The major ubiquinone homologue was Q-10. On the basis of the results obtained, Gluconobacter thailandicus sp. nov. was proposed for the four isolates. The type strain is isolate F149-1(T) (=BCC 14116(T)=NBRC 100600(T)=JCM 12310(T)=TISTR 1533(T)=PCU 225(T)), which had 55.8 mol% G+C, isolated from a flower of the Indian cork tree (Millingtonia hortensis) collected in Bangkok, Thailand.  (+info)

(16/50) Re-identification of Gluconobacter strains based on restriction analysis of 16S-23S rDNA internal transcribed spacer regions.

Thirty Gluconobacter strains maintained at Culture Collection NBRC were re-identified at the species level on the basis of restriction analysis of 16S-23S rDNA internal transcribed spacer (ITS) regions by digestion with two restriction endonucleases MboII and Bsp1286I. The strains examined were divided into seven groups, designated as Group I and Group III-VIII, by the combination of the restriction patterns obtained with the two restriction endonucleases. Group I included seven strains, which gave "G. oxydans patterns" with the two restriction endonucleases and were re-identified as G. oxydans. Group III included 12 strains, which gave "G. frateurii patterns" and were re-identified as G. frateurii. Group IV included six strains, which gave "G. cerinus pattern" with MboII and "G. frateurii pattern" with Bsp1286I and were re-identified as G. frateurii. Group V included one strain (NBRC 3274), which gave respectively "G. frateurii pattern" and "G. cerinus pattern" and was re-identified as G. cerinus. Group VI included one strain (NBRC 3990), which gave respectively "G. oxydans pattern" and an unidentified restriction pattern and was re-identified temporarily as G. oxydans. Group VII included two strains (NBRC 3250 and NBRC 3273), which gave respectively an unidentified restriction pattern and "G. oxydans pattern." Group VIII included one strain (NBRC 3266), which gave unidentified restriction patterns. The three strains of Group VII and Group VIII were suggested to constitute new taxa by sequencing of 16S-23S rDNA ITS regions.  (+info)