| 1. |
Hoshino T,
Sugisawa T,
Shinjoh M,
Tomiyama N,
Miyazaki T,
( 2003 ) Membrane-bound D-sorbitol dehydrogenase of Gluconobacter suboxydans IFO 3255--enzymatic and genetic characterization. PMID : 12686146 : DOI : 10.1016/s1570-9639(03)00071-2 Abstract >>
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.
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2. |
Matsushita K,
Fujii Y,
Ano Y,
Toyama H,
Shinjoh M,
Tomiyama N,
Miyazaki T,
Sugisawa T,
Hoshino T,
Adachi O,
( 2003 ) 5-keto-D-gluconate production is catalyzed by a quinoprotein glycerol dehydrogenase, major polyol dehydrogenase, in gluconobacter species. PMID : 12676670 : DOI : 10.1128/aem.69.4.1959-1966.2003 PMC : PMC154820 Abstract >>
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.
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3. |
Matsushita K,
Nagatani Y,
Shinagawa E,
Adachi O,
Ameyama M,
( 1991 ) Reconstitution of the ethanol oxidase respiratory chain in membranes of quinoprotein alcohol dehydrogenase-deficient Gluconobacter suboxydans subsp. alpha strains. PMID : 1646200 : DOI : 10.1128/jb.173.11.3440-3445.1991 PMC : PMC207957 DOI : 10.1128/jb.173.11.3440-3445.1991 PMC : PMC207957 Abstract >>
The ethanol oxidase respiratory chain of Gluconobacter suboxydan was characterized by using G. suboxydans subsp. alpha, a variant species of G. suboxydans incapable of oxidizing ethanol. The membranes of G. suboxydans subsp. alpha exhibited neither alcohol dehydrogenase, ethanol oxidase, nor glucose-ferricyanide oxidoreductase activity. Furthermore, the respiratory chain of the organism exhibited an extremely diminished amount of cytochrome c and an increased sensitivity of the respiratory activity for cyanide or azide when compared with G. suboxydans. The first-subunit quinohemoprotein and the second-subunit cytochrome c of alcohol dehydrogenase complex in the membranes of G. suboxydans subsp. alpha were shown to be reduced and deficient, respectively, by using heme-staining and immunoblotting methods. Ethanol oxidase activity, lacking in G. suboxydans subsp. alpha, was entirely restored by reconstituting alcohol dehydrogenase purified from G. suboxydans to the membranes of G. suboxydans subsp. alpha; this also led to restoration of the cyanide or azide insensitivity and the glucose-ferricyanide oxidoreductase activity in the respiratory chain without affecting other respiratory activities such as glucose and sorbitol oxidases. Ethanol oxidase activity was also reconstituted with only the second-subunit cytochrome c of the enzyme complex. The results indicate that the second-subunit cytochrome c of the alcohol dehydrogenase complex is essential in ethanol oxidase respiratory chain and may be involved in the cyanide- or azide-insensitive respiratory chain bypass of G. suboxydans.
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4. |
Matsushita K,
Nagatani Y,
Shinagawa E,
Adachi O,
Ameyama M,
( 1991 ) Reconstitution of the ethanol oxidase respiratory chain in membranes of quinoprotein alcohol dehydrogenase-deficient Gluconobacter suboxydans subsp. alpha strains. PMID : 1646200 : DOI : 10.1128/jb.173.11.3440-3445.1991 PMC : PMC207957 DOI : 10.1128/jb.173.11.3440-3445.1991 PMC : PMC207957 Abstract >>
The ethanol oxidase respiratory chain of Gluconobacter suboxydan was characterized by using G. suboxydans subsp. alpha, a variant species of G. suboxydans incapable of oxidizing ethanol. The membranes of G. suboxydans subsp. alpha exhibited neither alcohol dehydrogenase, ethanol oxidase, nor glucose-ferricyanide oxidoreductase activity. Furthermore, the respiratory chain of the organism exhibited an extremely diminished amount of cytochrome c and an increased sensitivity of the respiratory activity for cyanide or azide when compared with G. suboxydans. The first-subunit quinohemoprotein and the second-subunit cytochrome c of alcohol dehydrogenase complex in the membranes of G. suboxydans subsp. alpha were shown to be reduced and deficient, respectively, by using heme-staining and immunoblotting methods. Ethanol oxidase activity, lacking in G. suboxydans subsp. alpha, was entirely restored by reconstituting alcohol dehydrogenase purified from G. suboxydans to the membranes of G. suboxydans subsp. alpha; this also led to restoration of the cyanide or azide insensitivity and the glucose-ferricyanide oxidoreductase activity in the respiratory chain without affecting other respiratory activities such as glucose and sorbitol oxidases. Ethanol oxidase activity was also reconstituted with only the second-subunit cytochrome c of the enzyme complex. The results indicate that the second-subunit cytochrome c of the alcohol dehydrogenase complex is essential in ethanol oxidase respiratory chain and may be involved in the cyanide- or azide-insensitive respiratory chain bypass of G. suboxydans.
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5. |
( 2013 ) Draft Genome Sequence of Dihydroxyacetone-Producing Gluconobacter thailandicus Strain NBRC 3255. PMID : 23580707 : DOI : 10.1128/genomeA.00118-13 PMC : PMC3624681 DOI : 10.1128/genomeA.00118-13 PMC : PMC3624681 Abstract >>
Here, we report the draft genome sequence of the acetic acid bacterium Glucnobacter thailandicus strain NBRC 3255. The draft genome sequence is composed of 109 contigs in 3,305,227 bp and contains 3,225 protein-coding genes. Two paralogous sets of sldAB operons, which are responsible for dihydroxyacetone production from glycerol, were identified.
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6. |
( 2013 ) Draft Genome Sequence of Dihydroxyacetone-Producing Gluconobacter thailandicus Strain NBRC 3255. PMID : 23580707 : DOI : 10.1128/genomeA.00118-13 PMC : PMC3624681 DOI : 10.1128/genomeA.00118-13 PMC : PMC3624681 Abstract >>
Here, we report the draft genome sequence of the acetic acid bacterium Glucnobacter thailandicus strain NBRC 3255. The draft genome sequence is composed of 109 contigs in 3,305,227 bp and contains 3,225 protein-coding genes. Two paralogous sets of sldAB operons, which are responsible for dihydroxyacetone production from glycerol, were identified.
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