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Yuzo Yamada - One of the best experts on this subject based on the ideXlab platform.
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Gluconobacter nephelii sp nov an acetic acid bacterium in the class alphaproteobacteria
International Journal of Systematic and Evolutionary Microbiology, 2011Co-Authors: Jintana Kommanee, Somboon Tanasupawat, Pattaraporn Yukphan, Yuzo Yamada, Taweesak Malimas, Yuki Muramatsu, Yasuyoshi NakagawaAbstract:Three strains, RBY-1T, PHD-1 and PHD-2, were isolated from fruits in Thailand. The strains were Gram-negative, aerobic rods with polar flagella, produced acetic acid from ethanol and did not oxidize acetate or lactate. In phylogenetic trees based on 16S rRNA gene sequences and 16S–23S rRNA gene internal transcribed spacer (ITS) sequences, the strains formed a cluster separate from the type strains of recognized species of the genus Gluconobacter. The calculated 16S rRNA gene sequence and 16S–23S rRNA gene ITS sequence similarities were respectively 97.7–99.7 % and 77.3–98.1 %. DNA G+C contents ranged from 57.2 to 57.6 mol%. The strains showed high DNA–DNA relatedness of 100 % to one another, but low DNA–DNA relatedness of 11–34 % to the tested type strains of recognized Gluconobacter species. Q-10 was the major quinone. On the basis of the genotypic and phenotypic data obtained, the three strains clearly represent a novel species, for which the name Gluconobacter nephelii sp. nov. is proposed. The type strain is RBY-1T ( = BCC 36733T = NBRC 106061T = PCU 318T), whose DNA G+C content is 57.2 mol%.
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Gluconobacter japonicus sp nov an acetic acid bacterium in the alphaproteobacteria
International Journal of Systematic and Evolutionary Microbiology, 2009Co-Authors: Taweesak Malimas, Somboon Tanasupawat, Pattaraporn Yukphan, Mai Takahashi, Yuki Muramatsu, Mika Kaneyasu, Wanchern Potacharoen, Yasuyoshi Nakagawa, Morakot Tanticharoen, Yuzo YamadaAbstract:Five strains, NBRC 3271(T), NBRC 3272, NBRC 3263, NBRC 3260 and NBRC 3269 were examined genetically, phylogenetically, phenotypically and chemotaxonomically. The DNA G+C contents of the five strains were 55.1-56.4 mol%. The five strains had low levels of DNA-DNA hybridization of 13-51 % to the type strains of Gluconobacter frateurii, Gluconobacter thailandicus, Gluconobacter oxydans, Gluconobacter cerinus, Gluconobacter albidus and Gluconobacter kondonii and formed a cluster that was separate from the type strains of the six Gluconobacter species given above in phylogenetic trees based on 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences. The five strains weakly produced dihydroxyacetone from glycerol, but not 2,5-diketo-d-gluconate or a water-soluble brown pigment from d-glucose and contained ubiquinone-10. The five strains were assigned as representing a novel species of the genus Gluconobacter, for which the name Gluconobacter japonicus sp. nov. is proposed. The type strain is NBRC 3271(T) (=BCC 14458(T)=strain 7(T), K. Kondo). Cells of the type strain are motile by means of polar flagella and the DNA G+C content is 56.4 mol%.
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identification of acetobacter Gluconobacter and asaia strains isolated in thailand based on 16s 23s rrna gene internal transcribed spacer restriction and 16s rrna gene sequence analyses
Microbes and Environments, 2009Co-Authors: Somboon Tanasupawat, Pattaraporn Yukphan, Yuzo Yamada, Taweesak Malimas, Yasuyoshi Nakagawa, Jintana KommaneeAbstract:Twenty-six strains of acetic acid bacteria were isolated from fruits, flowers and related materials collected in Thailand. They were divided into three genera, Acetobacter, Gluconobacter and Asaia, by phenotypic characterization and 16S rRNA gene sequence analyses. On the basis of 16S-23S rRNA gene internal transcribed spacer (16S-23S rDNA ITS) restriction and 16S rRNA gene sequence analyses, fourteen isolates assigned to the genus Acetobacter were divided into five groups: 1) Group 1A or A. tropicalis (one isolate); 2) Group 2A or A. orientalis (four isolates); 3) Group 3A or A. pasteurianus (five isolates); 4) Group 4A or A. syzygii (one isolate); and 5) Group 5A or A. ghanensis (three isolates). The eleven isolates assigned to the genus Gluconobacter were divided into three groups: 6) Group 1B or G. frateurii (four isolates); 7) Group 2B or G. japonicus (six isolates); and 8) Group 3B or unidentified (one isolate). The remaining isolate was placed into: 9) Group 1C or unidentified, which was assigned to the genus Asaia and considered to constitute a new species on the basis of the 16S rRNA gene sequence analysis and DNA-DNA hybridization.
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Gluconobacter sphaericus ameyama 1975 comb nov a brown pigment producing acetic acid bacterium in the alphaproteobacteria
Journal of General and Applied Microbiology, 2008Co-Authors: Taweesak Malimas, Somboon Tanasupawat, Pattaraporn Yukphan, Mai Takahashi, Yuki Muramatsu, Mika Kaneyasu, Wanchern Potacharoen, Yasuyoshi Nakagawa, Morakot Tanticharoen, Yuzo YamadaAbstract:Strain NBRC 12467 T was examined genetically, phylogenetically, phenotypically, and chemotaxonomically. The DNA G+C content of the strain was 59.5 mol%. The strain represented low levels of DNA-DNA hybridization of 49-9% to the type strains of eight Gluconobacter species. The strain formed a cluster along with the type strains of G. albidus and G. kondonii in phylogenetic trees based on 16S rRNA gene sequences. In a phylogenetic tree based on 165-23S rRNA gene ITS sequences, however, the strain formed an independent cluster from the type strains of the eight Gluconobacter species. Such phylogenetic relationships were supported by the calculated pair-wise 16S rRNA gene and 16S-23S rRNA gene ITS sequence similarities. The strain was distinguished from the type strains of the eight Gluconobacter species by 16S-23S rRNA gene ITS restriction analysis using five restriction endonucleases. The strain produced a water-soluble brown pigment and 2,5-diketo-D-gluconate from D-glucose, differing from the type strains of the eight Gluconobacter species, and acid from meso-erythritol very weakly, differing from the type strains of the remaining seven Gluconobacter species except for the type strain of G. roseus, but not from maltose, differing from the type strain of G. oxydans, and had Q-10. For the strain, which was once classified as G. oxydans subsp. sphaericus, Gluconobacter sphaericus (Ameyama 1975) comb. nov. is proposed. The type strain is NBRC 12467 T , which is also deposited as BCC 14448 T .
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Gluconobacter thailandicus sp. nov., an acetic acid bacterium in the alpha-Proteobacteria.
The Journal of general and applied microbiology, 2004Co-Authors: Somboon Tanasupawat, Duangtip Moonmangmee, Osao Adachi, Chitti Thawai, Pattaraporn Yukphan, Takashi Itoh, Yuzo YamadaAbstract: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°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 F149-1T (=BCC 14116T=NBRC 100600T=JCM 12310T=TISTR 1533T=PCU 225T), which had 55.8 mol% G+C, isolated from a flower of the Indian cork tree (Millingtonia hortensis) collected in Bangkok, Thailand.
Tatsuo Hoshino - One of the best experts on this subject based on the ideXlab platform.
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membrane bound d sorbitol dehydrogenase of Gluconobacter suboxydans ifo 3255 enzymatic and genetic characterization
Biochimica et Biophysica Acta, 2003Co-Authors: Tatsuo Hoshino, Masako Shinjoh, Noribumi Tomiyama, Teruhide Sugisawa, Taro MiyazakiAbstract: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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5 keto d gluconate production is catalyzed by a quinoprotein glycerol dehydrogenase major polyol dehydrogenase in Gluconobacter species
Applied and Environmental Microbiology, 2003Co-Authors: Kazunobu Matsushita, Masako Shinjoh, Hirohide Toyama, Yoshitaka Ano, Yoshikazu Fujii, Noribumi Tomiyama, Taro Miyazaki, Teruhide Sugisawa, Tatsuo HoshinoAbstract: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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molecular cloning and functional expression of d sorbitol dehydrogenase from Gluconobacter suboxydans ifo3255 which requires pyrroloquinoline quinone and hydrophobic
Bioscience Biotechnology and Biochemistry, 2002Co-Authors: Taro Miyazaki, Masako Shinjoh, Noribumi Tomiyama, Tatsuo HoshinoAbstract: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. 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 a polypeptide consisting of 126 very hydrophobic residues that is similar in sequence to the one-sixth N-terminal region of the GDH. For the development of the SLDH activity in E. coli, co-expression of the sldA and sldB genes and the presence of pyrrloquinolone quinone as a co-factor were required.
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nadph dependent l sorbose reductase is responsible for l sorbose assimilation in Gluconobacter suboxydans ifo 3291
Journal of Bacteriology, 2002Co-Authors: Masako Shinjoh, Masaaki Tazoe, Tatsuo HoshinoAbstract: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.
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purification and properties of membrane bound d sorbitol dehydrogenase from Gluconobacter suboxydans ifo 3255
Bioscience Biotechnology and Biochemistry, 2002Co-Authors: Teruhide Sugisawa, Tatsuo HoshinoAbstract:D-Sorbitol dehydrogenase was solubilized from the membrane fraction of Gluconobacter suboxydans IFO 3255 with Triton X-100 in the presence of D-sorbitol. Purification 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 30°C.Western blot analysis suggested the occurrence of the enzyme in all the Gluconobacter strains tested.
Kazunobu Matsushita - One of the best experts on this subject based on the ideXlab platform.
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the 5 ketofructose reductase of Gluconobacter sp strain chm43 is a novel class in the shikimate dehydrogenase family
Journal of Bacteriology, 2021Co-Authors: Thuy Minh Nguyen, Osao Adachi, Kazunobu Matsushita, Masaru Goto, Shohei Noda, Minenosuke Matsutani, Yuki Hodoya, Naoya Kataoka, Toshiharu YakushiAbstract:Gluconobacter sp. strain CHM43 oxidizes mannitol to fructose and then oxidizes fructose to 5-keto-d-fructose (5KF) in the periplasmic space. Since NADPH-dependent 5KF reductase was found in the soluble fraction of Gluconobacter spp., 5KF might be transported into the cytoplasm and metabolized. Here, we identified the GLF_2050 gene as the kfr gene encoding 5KF reductase (KFR). A mutant strain devoid of the kfr gene showed lower KFR activity and no 5KF consumption. The crystal structure revealed that KFR is similar to NADP+-dependent shikimate dehydrogenase (SDH), which catalyzes the reversible NADP+-dependent oxidation of shikimate to 3-dehydroshikimate. We found that several amino acid residues in the putative substrate-binding site of KFR were different from those of SDH. Phylogenetic analyses revealed that only a subclass in the SDH family containing KFR conserved such a unique substrate-binding site. We constructed KFR derivatives with amino acid substitutions, including replacement of Asn21 in the substrate-binding site with Ser that is found in SDH. The KFR-N21S derivative showed a strong increase in the Km value for 5KF but a higher shikimate oxidation activity than wild-type KFR, suggesting that Asn21 is important for 5KF binding. In addition, the conserved catalytic dyad Lys72 and Asp108 were individually substituted for Asn. The K72N and D108N derivatives showed only negligible activities without a dramatic change in the Km value for 5KF, suggesting a catalytic mechanism similar to that of SDH. With these data taken together, we suggest that KFR is a new member of the SDH family. IMPORTANCE A limited number of species of acetic acid bacteria, such as Gluconobacter sp. strain CHM43, produce 5-ketofructose, a potential low-calorie sweetener, at a high yield. Here, we show that an NADPH-dependent 5-ketofructose reductase (KFR) is involved in 5-ketofructose degradation, and we characterize this enzyme with respect to its structure, phylogeny, and function. The crystal structure of KFR was similar to that of shikimate dehydrogenase, which is functionally crucial in the shikimate pathway in bacteria and plants. Phylogenetic analysis suggested that KFR is positioned in a small subgroup of the shikimate dehydrogenase family. Catalytically important amino acid residues were also conserved, and their relevance was experimentally validated. Thus, we propose KFR as a new member of shikimate dehydrogenase family.
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change in product selectivity during the production of glyceric acid from glycerol by Gluconobacter strains in the presence of methanol
AMB Express, 2013Co-Authors: Shun Sato, Kazunobu Matsushita, Dai Kitamoto, Toshiharu Yakushi, Naoki Morita, Hiroshi HabeAbstract:To enhance the value-added use of methanol-containing raw glycerol derived from biodiesel fuel production, the effect of methanol supplementation on glyceric acid (GA) production by Gluconobacter spp. was investigated. We first conducted fed-batch fermentation with Gluconobacter frateurii NBRC103465 using raw glycerol as a feeding solution. GA productivity decreased with increasing dihydroxyacetone (DHA) formation when the raw glycerol contained methanol. The results of this experiment and comparative experiments using a synthetic solution modeled after the raw glycerol indicate that the presence of methanol caused a change in the concentrations of GA and DHA, two glycerol derivatives produced during fermentation. Other Gluconobacter spp. also decreased GA production in the presence of 1% (v/v) methanol. In addition, purified membrane-bound alcohol dehydrogenase (mADH) from Gluconobacter oxydans, which is a key enzyme in GA production, showed a decrease in dehydrogenase activity toward glycerol as the methanol concentration increased. These results strongly suggest that the observed decrease in GA production by Gluconobacter spp. resulted from the methanol-induced inhibition of mADH-mediated glycerol oxidation.
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heterologous overexpression and characterization of a flavoprotein cytochrome c complex fructose dehydrogenase of Gluconobacter japonicus nbrc3260
Applied and Environmental Microbiology, 2013Co-Authors: Shota Kawai, Toshiharu Yakushi, Maiko Godatsutsumi, Kenji Kano, Kazunobu MatsushitaAbstract:A heterotrimeric flavoprotein-cytochrome c complex fructose dehydrogenase (FDH) of Gluconobacter japonicus NBRC3260 catalyzes the oxidation of d-fructose to produce 5-keto-d-fructose and is used for diagnosis and basic research purposes as a direct electron transfer-type bioelectrocatalysis. The fdhSCL genes encoding the FDH complex of G. japonicus NBRC3260 were isolated by a PCR-based gene amplification method with degenerate primers designed from the amino-terminal amino acid sequence of the large subunit and sequenced. Three open reading frames for fdhSCL encoding the small, cytochrome c, and large subunits, respectively, were found and were presumably in a polycistronic transcriptional unit. Heterologous overexpression of fdhSCL was conducted using a broad-host-range plasmid vector, pBBR1MCS-4, carrying a DNA fragment containing the putative promoter region of the membrane-bound alcohol dehydrogenase gene of Gluconobacter oxydans and a G. oxydans strain as the expression host. We also constructed derivatives modified in the translational initiation codon to ATG from TTG, designated TTGFDH and ATGFDH. Membranes of the cells producing recombinant TTGFDH and ATGFDH showed approximately 20 times and 100 times higher specific activity than those of G. japonicus NBRC3260, respectively. The cells producing only FdhS and FdhL had no fructose-oxidizing activity, but showed significantly high d-fructose:ferricyanide oxidoreductase activity in the soluble fraction of cell extracts, whereas the cells producing the FDH complex showed activity in the membrane fraction. It is reasonable to conclude that the cytochrome c subunit is responsible not only for membrane anchoring but also for ubiquinone reduction.
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use of a Gluconobacter frateurii mutant to prevent dihydroxyacetone accumulation during glyceric acid production from glycerol
Bioscience Biotechnology and Biochemistry, 2010Co-Authors: Hiroshi Habe, Tokuma Fukuoka, Kazunobu Matsushita, Dai Kitamoto, Yuko Shimada, Masayuki Itagaki, Hiroshi Yanagishita, Kunihiro Watanabe, Toshiharu Yakushi, Keiji SakakiAbstract:To prevent dihydroxyacetone (DHA) by-production during glyceric acid (GA) production from glycerol using Gluconobacter frateurii, we used a G. frateurii THD32 mutant, ΔsldA, in which the glycerol dehydrogenase subunit-encoding gene (sldA) was disrupted, but ΔsldA grew much more slowly than the wild type, growth starting after a lag of 3 d under the same culture conditions. The addition of 1% w/v D-sorbitol to the medium improved both the growth and the GA productivity of the mutant, and ΔsldA produced 89.1 g/l GA during 4 d of incubation without DHA accumulation.
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screening of thermotolerant Gluconobacter strains for production of 5 keto d gluconic acid and disruption of flavin adenine dinucleotide containing d gluconate dehydrogenase
Applied and Environmental Microbiology, 2009Co-Authors: Ittipon Saichana, Duangtip Moonmangmee, Osao Adachi, Kazunobu Matsushita, Hirohide ToyamaAbstract:We isolated thermotolerant Gluconobacter strains that are able to produce 5-keto-d-gluconic acid (5KGA) at 37°C, a temperature at which regular mesophilic 5KGA-producing strains showed much less growth and 5KGA production. The thermotolerant strains produced 2KGA as the major product at both 30 and 37°C. The amount of ketogluconates produced at 37°C was slightly less than the amount produced at 30°C. To improve the yield of 5KGA in these strains, we disrupted flavin adenine dinucleotide-gluconate dehydrogenase (FAD-GADH), which is responsible for 2KGA production. Genes for FAD-GADH were cloned by using inverse PCR and an in vitro cloning strategy. The sequences obtained for three thermotolerant strains were identical and showed high levels of identity to the FAD-GADH sequence reported for the genome of Gluconobacter oxydans 621 H. A kanamycin resistance gene cassette was used to disrupt the FAD-GADH genes in the thermotolerant strains. The mutant strains produced 5KGA exclusively, and the final yields were over 90% at 30°C and 50% at 37°C. We found that the activity of pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase, which is responsible for 5KGA production, increased in response to addition of PQQ and CaCl2 in vitro when cells were grown at 37°C. Addition of 5 mM CaCl2 to the culture medium of the mutant strains increased 5KGA production to the point where over 90% of the initial substrate was converted. The thermotolerant Gluconobacter strains that we isolated in this study provide a promising new option for industrial 5KGA production.
Hirohide Toyama - One of the best experts on this subject based on the ideXlab platform.
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screening of thermotolerant Gluconobacter strains for production of 5 keto d gluconic acid and disruption of flavin adenine dinucleotide containing d gluconate dehydrogenase
Applied and Environmental Microbiology, 2009Co-Authors: Ittipon Saichana, Duangtip Moonmangmee, Osao Adachi, Kazunobu Matsushita, Hirohide ToyamaAbstract:We isolated thermotolerant Gluconobacter strains that are able to produce 5-keto-d-gluconic acid (5KGA) at 37°C, a temperature at which regular mesophilic 5KGA-producing strains showed much less growth and 5KGA production. The thermotolerant strains produced 2KGA as the major product at both 30 and 37°C. The amount of ketogluconates produced at 37°C was slightly less than the amount produced at 30°C. To improve the yield of 5KGA in these strains, we disrupted flavin adenine dinucleotide-gluconate dehydrogenase (FAD-GADH), which is responsible for 2KGA production. Genes for FAD-GADH were cloned by using inverse PCR and an in vitro cloning strategy. The sequences obtained for three thermotolerant strains were identical and showed high levels of identity to the FAD-GADH sequence reported for the genome of Gluconobacter oxydans 621 H. A kanamycin resistance gene cassette was used to disrupt the FAD-GADH genes in the thermotolerant strains. The mutant strains produced 5KGA exclusively, and the final yields were over 90% at 30°C and 50% at 37°C. We found that the activity of pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase, which is responsible for 5KGA production, increased in response to addition of PQQ and CaCl2 in vitro when cells were grown at 37°C. Addition of 5 mM CaCl2 to the culture medium of the mutant strains increased 5KGA production to the point where over 90% of the initial substrate was converted. The thermotolerant Gluconobacter strains that we isolated in this study provide a promising new option for industrial 5KGA production.
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Biochemical and Spectroscopic Properties of Cyanide-Insensitive Quinol Oxidase from Gluconobacter oxydans
Journal of biochemistry, 2009Co-Authors: Tatsushi Mogi, Hirohide Toyama, Yoshitaka Ano, Tomoko Nakatsuka, Atsushi Muroi, Hideto Miyoshi, Catharina T. Migita, Kazuro Shiomi, Satoshi OmuraAbstract:Cyanide-insensitive quinol oxidase (CioAB), a relative of cytochrome bd, has no spectroscopic features of hemes b(595) and d in the wild-type bacteria and is difficult to purify for detailed characterization. Here we studied enzymatic and spectroscopic properties of CioAB from the acetic acid bacterium Gluconobacter oxydans. Gluconobacter oxydans CioAB showed the K(m) value for ubiquinol-1 comparable to that of Escherichia coli cytochrome bd but it was more resistant to KCN and quinone-analogue inhibitors except piericidin A and LL-Z1272gamma. We obtained the spectroscopic evidence for the presence of hemes b(595) and d. Heme b(595) showed the alpha peak at 587 nm in the reduced state and a rhombic high-spin signal at g = 6.3 and 5.5 in the air-oxidized state. Heme d showed the alpha peak at 626 and 644 nm in the reduced and air-oxidized state, respectively, and an axial high-spin signal at g = 6.0 and low-spin signals at g = 2.63, 2.37 and 2.32. We found also a broad low-spin signal at g = 3.2, attributable to heme b(558). Further, we identified the presence of heme D by mass spectrometry. In conclusion, CioAB binds all three ham species present in cytochrome bd quinol oxidase.
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energy metabolism of a unique acetic acid bacterium asaia bogorensis that lacks ethanol oxidation activity
Bioscience Biotechnology and Biochemistry, 2008Co-Authors: Hirohide Toyama, Osao Adachi, Kazunobu MatsushitaAbstract:Acetic acid bacteria (AAB) are known as a vinegar producer on account of their ability to accumulate a high concentration of acetic acid due to oxidative fermentation linking the ethanol oxidation respiratory chain. Reactions in oxidative fermentation cause poor growth because a large amount of the carbon source is oxidized incompletely and the harmful oxidized products are accumulated almost stoichiometrically in the culture medium during growth, but a newly identified AAB, Asaia, has shown unusual properties, including scanty acetic acid production and rapid growth, as compared with known AAB as Acetobacter, Gluconobacter, and Gluconacetobacter. To understand these unique properties of Asaia in more detail, the respiratory chain and energetics of this strain were investigated. It was found that Asaia lacks quinoprotein alcohol dehydrogenase, but has other sugar and sugar alcohol-oxidizing enzymes specific to the respiratory chain of Gluconobacter, especially quinoprotein glycerol dehydrogenase. It was a...
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l sorbose reductase and its transcriptional regulator involved in l sorbose utilization of Gluconobacter frateurii
Journal of Bacteriology, 2007Co-Authors: Wichai Soemphol, Duangtip Moonmangmee, Hirohide Toyama, Osao Adachi, Kazunobu MatsushitaAbstract:Upstream of the gene for flavin adenine dinucleotide (FAD)-dependent d-sorbitol dehydrogenase (SLDH), sldSLC, a putative transcriptional regulator was found in Gluconobacter frateurii THD32 (NBRC 101656). In this study, the whole sboR gene and the adjacent gene, sboA, were cloned and analyzed. sboR mutation did not affect FAD-SLDH activity in the membrane fractions. The SboA enzyme expressed and purified from an Escherichia coli transformant showed NADPH-dependent l-sorbose reductase (NADPH-SR) activity, and the enzyme was different from the NADPH-SR previously reported for Gluconobacter suboxydans IFO 3291 in molecular size and amino acid sequence. A mutant defective in sboA showed significantly reduced growth on l-sorbose, indicating that the SboA enzyme is required for efficient growth on l-sorbose. The sboR mutant grew on l-sorbose even better than the wild-type strain did, and higher NADPH-SR activity was detected in cytoplasm fractions. Reverse transcription-PCR experiments indicated that sboRA comprises an operon. These data suggest that sboR is involved in the repression of sboA, but not in the induction of sldSLC, on d-sorbitol and that another activator is required for the induction of these genes by d-sorbitol or l-sorbose.
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Purification and Properties of NADP-Dependent Shikimate Dehydrogenase from Gluconobacter oxydans IFO 3244 and Its Application to Enzymatic Shikimate Production
Bioscience Biotechnology and Biochemistry, 2006Co-Authors: Osao Adachi, Hirohide Toyama, Yoshitaka Ano, Kazunobu MatsushitaAbstract:NADP-Dependent shikimate dehydrogenae (SKDH, EC 1.1.1.25) was purified from Gluconobacter oxydans IFO 3244. SKDH showed a single protein band on native-PAGE accompanying enzyme activity. It required NADP exclusively and catalyzed only the shuttle reaction between shikimate and 3-dehydroshikimate. The optimum pH for shikimate oxidation and 3-dehydroshikimate reduction was found at pH 10 and 7 respectively. SKDH proved to be a useful catalyst for shikimate production from 3-dehydroshikimate.
Masako Shinjoh - One of the best experts on this subject based on the ideXlab platform.
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membrane bound d sorbitol dehydrogenase of Gluconobacter suboxydans ifo 3255 enzymatic and genetic characterization
Biochimica et Biophysica Acta, 2003Co-Authors: Tatsuo Hoshino, Masako Shinjoh, Noribumi Tomiyama, Teruhide Sugisawa, Taro MiyazakiAbstract: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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5 keto d gluconate production is catalyzed by a quinoprotein glycerol dehydrogenase major polyol dehydrogenase in Gluconobacter species
Applied and Environmental Microbiology, 2003Co-Authors: Kazunobu Matsushita, Masako Shinjoh, Hirohide Toyama, Yoshitaka Ano, Yoshikazu Fujii, Noribumi Tomiyama, Taro Miyazaki, Teruhide Sugisawa, Tatsuo HoshinoAbstract: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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molecular cloning and functional expression of d sorbitol dehydrogenase from Gluconobacter suboxydans ifo3255 which requires pyrroloquinoline quinone and hydrophobic
Bioscience Biotechnology and Biochemistry, 2002Co-Authors: Taro Miyazaki, Masako Shinjoh, Noribumi Tomiyama, Tatsuo HoshinoAbstract: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. 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 a polypeptide consisting of 126 very hydrophobic residues that is similar in sequence to the one-sixth N-terminal region of the GDH. For the development of the SLDH activity in E. coli, co-expression of the sldA and sldB genes and the presence of pyrrloquinolone quinone as a co-factor were required.
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nadph dependent l sorbose reductase is responsible for l sorbose assimilation in Gluconobacter suboxydans ifo 3291
Journal of Bacteriology, 2002Co-Authors: Masako Shinjoh, Masaaki Tazoe, Tatsuo HoshinoAbstract: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.
