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Kenji Soda - One of the best experts on this subject based on the ideXlab platform.
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Paradoxical thermostable enzymes from psychrophile: molecular characterization and potentiality for biotechnological application
Journal of Molecular Catalysis B-enzymatic, 2003Co-Authors: Tadao Oikawa, Takayuki Kazuoka, Kenji SodaAbstract:Abstract NAD(P) + -dependent aldehyde dehydrogenase (EC 1.2.1.5) and aspartase (EC 4.3.1.1) in the cells of an atypical psychrophile from Antarctic seawater, Cytophaga sp. KUC-1, were paradoxically thermostable, although they derived from a psychrophile. Both enzymes showed the highest activity at about 55 °C, and also active even under cold conditions. The enzymes contained more Ile residues than the enzymes from mesophiles. The Ile/Ile + Val + Leu ratio of the Cytophaga thermostable enzymes was much higher than that of the enzymes from mesophiles. As compared with the enzymes from other microorganisms, the Cytophaga thermostable enzymes have the structural differences in the C-terminal region of the enzymes. Therefore, the C-terminal region might be important for the paradoxical thermostability of the enzymes. The psychrophilic microorganism produces not only psychrophilic enzyme, but thermostable enzyme with psychrophilicity. Therefore, the psychrophilic microorganism is one of the candidates for isolation of novel biocatalysts, which have potential for various industrial applications.
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Novel Psychrophilic and Thermolabile l-Threonine Dehydrogenase from Psychrophilic Cytophaga sp. Strain KUC-1
Journal of bacteriology, 2003Co-Authors: Takayuki Kazuoka, Tadao Oikawa, Shouhei Takigawa, Noriaki Arakawa, Yoshiyuki Hizukuri, Ikuo Muraoka, Kenji SodaAbstract:A psychrophilic bacterium, Cytophaga sp. strain KUC-1, that abundantly produces a NAD+-dependent l-threonine dehydrogenase was isolated from Antarctic seawater, and the enzyme was purified. The molecular weight of the enzyme was estimated to be 139,000, and that of the subunit was determined to be 35,000. The enzyme is a homotetramer. Atomic absorption analysis showed that the enzyme contains no metals. In these respects, the Cytophaga enzyme is distinct from other l-threonine dehydrogenases that have thus far been studied. l-Threonine and dl-threo-3-hydroxynorvaline were the substrates, and NAD+ and some of its analogs served as coenzymes. The enzyme showed maximum activity at pH 9.5 and at 45°C. The kinetic parameters of the enzyme are highly influenced by temperatures. The Km for l-threonine was lowest at 20°C. Dead-end inhibition studies with pyruvate and adenosine-5′-diphosphoribose showed that the enzyme reaction proceeds via the ordered Bi Bi mechanism in which NAD+ binds to an enzyme prior to l-threonine and 2-amino-3-oxobutyrate is released from the enzyme prior to NADH. The enzyme gene was cloned into Escherichia coli, and its nucleotides were sequenced. The enzyme gene contains an open reading frame of 939 bp encoding a protein of 312 amino acid residues. The amino acid sequence of the enzyme showed a significant similarity to that of UDP-glucose 4-epimerase from Staphylococcus aureus and belongs to the short-chain dehydrogenase-reductase superfamily. In contrast, l-threonine dehydrogenase from E. coli belongs to the medium-chain alcohol dehydrogenase family, and its amino acid sequence is not at all similar to that of the Cytophaga enzyme. l-Threonine dehydrogenase is significantly similar to an epimerase, which was shown for the first time. The amino acid residues playing an important role in the catalysis of the E. coli and human UDP-glucose 4-epimerases are highly conserved in the Cytophaga enzyme, except for the residues participating in the substrate binding.
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Thermostable aldehyde dehydrogenase from psychrophile, Cytophaga sp. KUC-1: enzymological characteristics and functional properties.
Biochemical and Biophysical Research Communications, 2002Co-Authors: Yuko Yamanaka, Takayuki Kazuoka, Kazuya Yamanaka, Tadao Oikawa, Masahiro Yoshida, Kenji SodaAbstract:Abstract We found the occurrence of NAD(P) + -dependent aldehyde dehydrogenase (EC1.2.1.5) in the cells of a psychrophile from Antarctic seawater, Cytophaga sp. KUC-1, and purified to homogeneity. About 50% of the enzyme activity remained even after heating at 50 °C for 65 min and the highest activity was observed in the range of 55–60 °C. The enzyme was thermostable and thermophilic, although it was derived from a psychrophile. The circular dichroism at 222 nm of the enzyme showed a peak at 32 °C. This temperature was closely similar to the transition temperature in the Arrhenius plots. The stereospecificity for the hydride transfer at C4-site of nicotinamide moiety of NADH was pro-R . The gene encoding the enzyme consisted of an open reading frame of 1506-bp encoding a protein of 501 amino acid residues. The significant sequence identity (61%) was found between the Cytophaga and the Pseudomonas aeruginosa enzymes, although their thermostabilities are completely different.
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psychrophilic valine dehydrogenase of the antarctic psychrophile Cytophaga sp kuc 1 purification molecular characterization and expression
FEBS Journal, 2001Co-Authors: Tadao Oikawa, Takayuki Kazuoka, Kazuya Yamanaka, Noriyuki Kanzawa, Kenji SodaAbstract:1We found the occurrence of valine dehydrogenase in the cell extract of a psychrophilic bacterium, Cytophaga sp. KUC-1, isolated from Antarctic seawater and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be ≈ 154 kDa by gel filtration and that of the subunit was 43 kDa by SDS/PAGE: the enzyme was a homotetramer. The enzyme required NAD+ as a coenzyme, and catalyzed the oxidative deamination of l-valine, l-isoleucine, l-leucine and the reductive amination of α-ketoisovalerate, α-ketovalerate, α-ketoisocaproate, and α-ketocaproate. The reaction proceeds through an iso-ordered bi–bi mechanism. The enzyme was highly susceptible to heat treatment and the half-life at 45 °C was estimated to be 2.4 min. The kcat/Km (µm−1·s−1) values for l-valine and NAD+ at 20 °C were 27.48 and 421.6, respectively. The enzyme showed pro-S stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of coenzyme. The gene encoding valine dehydrogenase was cloned into Escherichia coli (Novablue), and the primary structure of the enzyme was deduced on the basis of the nucleotide sequence of the gene encoding the enzyme. The enzyme contains 370 amino-acid residues, and is highly homologous with S. coelicolor ValDH (identity, 46.7%) and S. fradiae ValDH (43.1%). Cytophaga sp. KUC-1 ValDH contains much lower numbers of proline and arginine residues than those of other ValDHs. The changes probably lead to an increase in conformational flexibility of the Cytophaga enzyme molecule to enhance the catalytic activity at low temperatures.
J P Bowman - One of the best experts on this subject based on the ideXlab platform.
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description of cellulophaga algicola sp nov isolated from the surfaces of antarctic algae and reclassification of Cytophaga uliginosa zobell and upham 1944 reichenbach 1989 as cellulophaga uliginosa comb nov
International Journal of Systematic and Evolutionary Microbiology, 2000Co-Authors: J P BowmanAbstract:A group of strains with potent extracellular enzymic activity were isolated from the surfaces of the chain-forming sea-ice diatom Melosira and from an unidentified macrophyte collected from the Eastern Antarctic coastal zone. 16S rDNA sequence analysis indicated that the strains belonged to the genus Cellulophaga and showed greatest similarity to the species Cellulophaga baltica (sequence similarity 97%). Phenotypic characteristics, DNA base composition and DNA-DNA hybridization values clearly separate the Antarctic strains from Cellulophaga baltica and other Cellulophaga species. Thus, the strains form a distinct and novel species and have the proposed name Cellulophaga algicola sp. nov. (type strain IC166T = ACAM 630T). In addition, it was recognized that the species Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989, a species phylogenetically remote from the type species of the genus Cytophaga, possessed 16S rDNA sequences and phenotypic and chemotaxonomic traits similar to those of other Cellulophaga species. Thus, it was proposed that the species Cytophaga uliginosa be renamed as Cellulophaga uliginosa comb. nov.
Tadao Oikawa - One of the best experts on this subject based on the ideXlab platform.
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Paradoxical thermostable enzymes from psychrophile: molecular characterization and potentiality for biotechnological application
Journal of Molecular Catalysis B-enzymatic, 2003Co-Authors: Tadao Oikawa, Takayuki Kazuoka, Kenji SodaAbstract:Abstract NAD(P) + -dependent aldehyde dehydrogenase (EC 1.2.1.5) and aspartase (EC 4.3.1.1) in the cells of an atypical psychrophile from Antarctic seawater, Cytophaga sp. KUC-1, were paradoxically thermostable, although they derived from a psychrophile. Both enzymes showed the highest activity at about 55 °C, and also active even under cold conditions. The enzymes contained more Ile residues than the enzymes from mesophiles. The Ile/Ile + Val + Leu ratio of the Cytophaga thermostable enzymes was much higher than that of the enzymes from mesophiles. As compared with the enzymes from other microorganisms, the Cytophaga thermostable enzymes have the structural differences in the C-terminal region of the enzymes. Therefore, the C-terminal region might be important for the paradoxical thermostability of the enzymes. The psychrophilic microorganism produces not only psychrophilic enzyme, but thermostable enzyme with psychrophilicity. Therefore, the psychrophilic microorganism is one of the candidates for isolation of novel biocatalysts, which have potential for various industrial applications.
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Novel Psychrophilic and Thermolabile l-Threonine Dehydrogenase from Psychrophilic Cytophaga sp. Strain KUC-1
Journal of bacteriology, 2003Co-Authors: Takayuki Kazuoka, Tadao Oikawa, Shouhei Takigawa, Noriaki Arakawa, Yoshiyuki Hizukuri, Ikuo Muraoka, Kenji SodaAbstract:A psychrophilic bacterium, Cytophaga sp. strain KUC-1, that abundantly produces a NAD+-dependent l-threonine dehydrogenase was isolated from Antarctic seawater, and the enzyme was purified. The molecular weight of the enzyme was estimated to be 139,000, and that of the subunit was determined to be 35,000. The enzyme is a homotetramer. Atomic absorption analysis showed that the enzyme contains no metals. In these respects, the Cytophaga enzyme is distinct from other l-threonine dehydrogenases that have thus far been studied. l-Threonine and dl-threo-3-hydroxynorvaline were the substrates, and NAD+ and some of its analogs served as coenzymes. The enzyme showed maximum activity at pH 9.5 and at 45°C. The kinetic parameters of the enzyme are highly influenced by temperatures. The Km for l-threonine was lowest at 20°C. Dead-end inhibition studies with pyruvate and adenosine-5′-diphosphoribose showed that the enzyme reaction proceeds via the ordered Bi Bi mechanism in which NAD+ binds to an enzyme prior to l-threonine and 2-amino-3-oxobutyrate is released from the enzyme prior to NADH. The enzyme gene was cloned into Escherichia coli, and its nucleotides were sequenced. The enzyme gene contains an open reading frame of 939 bp encoding a protein of 312 amino acid residues. The amino acid sequence of the enzyme showed a significant similarity to that of UDP-glucose 4-epimerase from Staphylococcus aureus and belongs to the short-chain dehydrogenase-reductase superfamily. In contrast, l-threonine dehydrogenase from E. coli belongs to the medium-chain alcohol dehydrogenase family, and its amino acid sequence is not at all similar to that of the Cytophaga enzyme. l-Threonine dehydrogenase is significantly similar to an epimerase, which was shown for the first time. The amino acid residues playing an important role in the catalysis of the E. coli and human UDP-glucose 4-epimerases are highly conserved in the Cytophaga enzyme, except for the residues participating in the substrate binding.
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Thermostable aldehyde dehydrogenase from psychrophile, Cytophaga sp. KUC-1: enzymological characteristics and functional properties.
Biochemical and Biophysical Research Communications, 2002Co-Authors: Yuko Yamanaka, Takayuki Kazuoka, Kazuya Yamanaka, Tadao Oikawa, Masahiro Yoshida, Kenji SodaAbstract:Abstract We found the occurrence of NAD(P) + -dependent aldehyde dehydrogenase (EC1.2.1.5) in the cells of a psychrophile from Antarctic seawater, Cytophaga sp. KUC-1, and purified to homogeneity. About 50% of the enzyme activity remained even after heating at 50 °C for 65 min and the highest activity was observed in the range of 55–60 °C. The enzyme was thermostable and thermophilic, although it was derived from a psychrophile. The circular dichroism at 222 nm of the enzyme showed a peak at 32 °C. This temperature was closely similar to the transition temperature in the Arrhenius plots. The stereospecificity for the hydride transfer at C4-site of nicotinamide moiety of NADH was pro-R . The gene encoding the enzyme consisted of an open reading frame of 1506-bp encoding a protein of 501 amino acid residues. The significant sequence identity (61%) was found between the Cytophaga and the Pseudomonas aeruginosa enzymes, although their thermostabilities are completely different.
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psychrophilic valine dehydrogenase of the antarctic psychrophile Cytophaga sp kuc 1 purification molecular characterization and expression
FEBS Journal, 2001Co-Authors: Tadao Oikawa, Takayuki Kazuoka, Kazuya Yamanaka, Noriyuki Kanzawa, Kenji SodaAbstract:1We found the occurrence of valine dehydrogenase in the cell extract of a psychrophilic bacterium, Cytophaga sp. KUC-1, isolated from Antarctic seawater and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be ≈ 154 kDa by gel filtration and that of the subunit was 43 kDa by SDS/PAGE: the enzyme was a homotetramer. The enzyme required NAD+ as a coenzyme, and catalyzed the oxidative deamination of l-valine, l-isoleucine, l-leucine and the reductive amination of α-ketoisovalerate, α-ketovalerate, α-ketoisocaproate, and α-ketocaproate. The reaction proceeds through an iso-ordered bi–bi mechanism. The enzyme was highly susceptible to heat treatment and the half-life at 45 °C was estimated to be 2.4 min. The kcat/Km (µm−1·s−1) values for l-valine and NAD+ at 20 °C were 27.48 and 421.6, respectively. The enzyme showed pro-S stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of coenzyme. The gene encoding valine dehydrogenase was cloned into Escherichia coli (Novablue), and the primary structure of the enzyme was deduced on the basis of the nucleotide sequence of the gene encoding the enzyme. The enzyme contains 370 amino-acid residues, and is highly homologous with S. coelicolor ValDH (identity, 46.7%) and S. fradiae ValDH (43.1%). Cytophaga sp. KUC-1 ValDH contains much lower numbers of proline and arginine residues than those of other ValDHs. The changes probably lead to an increase in conformational flexibility of the Cytophaga enzyme molecule to enhance the catalytic activity at low temperatures.
Takayuki Kazuoka - One of the best experts on this subject based on the ideXlab platform.
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Paradoxical thermostable enzymes from psychrophile: molecular characterization and potentiality for biotechnological application
Journal of Molecular Catalysis B-enzymatic, 2003Co-Authors: Tadao Oikawa, Takayuki Kazuoka, Kenji SodaAbstract:Abstract NAD(P) + -dependent aldehyde dehydrogenase (EC 1.2.1.5) and aspartase (EC 4.3.1.1) in the cells of an atypical psychrophile from Antarctic seawater, Cytophaga sp. KUC-1, were paradoxically thermostable, although they derived from a psychrophile. Both enzymes showed the highest activity at about 55 °C, and also active even under cold conditions. The enzymes contained more Ile residues than the enzymes from mesophiles. The Ile/Ile + Val + Leu ratio of the Cytophaga thermostable enzymes was much higher than that of the enzymes from mesophiles. As compared with the enzymes from other microorganisms, the Cytophaga thermostable enzymes have the structural differences in the C-terminal region of the enzymes. Therefore, the C-terminal region might be important for the paradoxical thermostability of the enzymes. The psychrophilic microorganism produces not only psychrophilic enzyme, but thermostable enzyme with psychrophilicity. Therefore, the psychrophilic microorganism is one of the candidates for isolation of novel biocatalysts, which have potential for various industrial applications.
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Novel Psychrophilic and Thermolabile l-Threonine Dehydrogenase from Psychrophilic Cytophaga sp. Strain KUC-1
Journal of bacteriology, 2003Co-Authors: Takayuki Kazuoka, Tadao Oikawa, Shouhei Takigawa, Noriaki Arakawa, Yoshiyuki Hizukuri, Ikuo Muraoka, Kenji SodaAbstract:A psychrophilic bacterium, Cytophaga sp. strain KUC-1, that abundantly produces a NAD+-dependent l-threonine dehydrogenase was isolated from Antarctic seawater, and the enzyme was purified. The molecular weight of the enzyme was estimated to be 139,000, and that of the subunit was determined to be 35,000. The enzyme is a homotetramer. Atomic absorption analysis showed that the enzyme contains no metals. In these respects, the Cytophaga enzyme is distinct from other l-threonine dehydrogenases that have thus far been studied. l-Threonine and dl-threo-3-hydroxynorvaline were the substrates, and NAD+ and some of its analogs served as coenzymes. The enzyme showed maximum activity at pH 9.5 and at 45°C. The kinetic parameters of the enzyme are highly influenced by temperatures. The Km for l-threonine was lowest at 20°C. Dead-end inhibition studies with pyruvate and adenosine-5′-diphosphoribose showed that the enzyme reaction proceeds via the ordered Bi Bi mechanism in which NAD+ binds to an enzyme prior to l-threonine and 2-amino-3-oxobutyrate is released from the enzyme prior to NADH. The enzyme gene was cloned into Escherichia coli, and its nucleotides were sequenced. The enzyme gene contains an open reading frame of 939 bp encoding a protein of 312 amino acid residues. The amino acid sequence of the enzyme showed a significant similarity to that of UDP-glucose 4-epimerase from Staphylococcus aureus and belongs to the short-chain dehydrogenase-reductase superfamily. In contrast, l-threonine dehydrogenase from E. coli belongs to the medium-chain alcohol dehydrogenase family, and its amino acid sequence is not at all similar to that of the Cytophaga enzyme. l-Threonine dehydrogenase is significantly similar to an epimerase, which was shown for the first time. The amino acid residues playing an important role in the catalysis of the E. coli and human UDP-glucose 4-epimerases are highly conserved in the Cytophaga enzyme, except for the residues participating in the substrate binding.
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Thermostable aldehyde dehydrogenase from psychrophile, Cytophaga sp. KUC-1: enzymological characteristics and functional properties.
Biochemical and Biophysical Research Communications, 2002Co-Authors: Yuko Yamanaka, Takayuki Kazuoka, Kazuya Yamanaka, Tadao Oikawa, Masahiro Yoshida, Kenji SodaAbstract:Abstract We found the occurrence of NAD(P) + -dependent aldehyde dehydrogenase (EC1.2.1.5) in the cells of a psychrophile from Antarctic seawater, Cytophaga sp. KUC-1, and purified to homogeneity. About 50% of the enzyme activity remained even after heating at 50 °C for 65 min and the highest activity was observed in the range of 55–60 °C. The enzyme was thermostable and thermophilic, although it was derived from a psychrophile. The circular dichroism at 222 nm of the enzyme showed a peak at 32 °C. This temperature was closely similar to the transition temperature in the Arrhenius plots. The stereospecificity for the hydride transfer at C4-site of nicotinamide moiety of NADH was pro-R . The gene encoding the enzyme consisted of an open reading frame of 1506-bp encoding a protein of 501 amino acid residues. The significant sequence identity (61%) was found between the Cytophaga and the Pseudomonas aeruginosa enzymes, although their thermostabilities are completely different.
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psychrophilic valine dehydrogenase of the antarctic psychrophile Cytophaga sp kuc 1 purification molecular characterization and expression
FEBS Journal, 2001Co-Authors: Tadao Oikawa, Takayuki Kazuoka, Kazuya Yamanaka, Noriyuki Kanzawa, Kenji SodaAbstract:1We found the occurrence of valine dehydrogenase in the cell extract of a psychrophilic bacterium, Cytophaga sp. KUC-1, isolated from Antarctic seawater and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be ≈ 154 kDa by gel filtration and that of the subunit was 43 kDa by SDS/PAGE: the enzyme was a homotetramer. The enzyme required NAD+ as a coenzyme, and catalyzed the oxidative deamination of l-valine, l-isoleucine, l-leucine and the reductive amination of α-ketoisovalerate, α-ketovalerate, α-ketoisocaproate, and α-ketocaproate. The reaction proceeds through an iso-ordered bi–bi mechanism. The enzyme was highly susceptible to heat treatment and the half-life at 45 °C was estimated to be 2.4 min. The kcat/Km (µm−1·s−1) values for l-valine and NAD+ at 20 °C were 27.48 and 421.6, respectively. The enzyme showed pro-S stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of coenzyme. The gene encoding valine dehydrogenase was cloned into Escherichia coli (Novablue), and the primary structure of the enzyme was deduced on the basis of the nucleotide sequence of the gene encoding the enzyme. The enzyme contains 370 amino-acid residues, and is highly homologous with S. coelicolor ValDH (identity, 46.7%) and S. fradiae ValDH (43.1%). Cytophaga sp. KUC-1 ValDH contains much lower numbers of proline and arginine residues than those of other ValDHs. The changes probably lead to an increase in conformational flexibility of the Cytophaga enzyme molecule to enhance the catalytic activity at low temperatures.
P Potin - One of the best experts on this subject based on the ideXlab platform.
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Zobellia galactanovorans gen. nov., sp nov., a marine species of Flavobacteriaceae isolated from a red alga, and classification of [Cytophaga] uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Zobellia uliginosa gen. nov., comb. nov.
International Journal of Systematic and Evolutionary Microbiology, 2001Co-Authors: T Barbeyron, Stéphane L'haridon, E Corre, B Kloareg, P PotinAbstract:A mesophilic, aerobic, non-flagellated, gliding bacterium, forming yellow colonies and designated Dsij(T), was isolated from a red alga on the sea-shore of Roscoff, Brittany, France. Dsij(T) was selected for its ability to actively degrade both agars and carrageenans. The Cram-negative cells occurred singly or in pairs as long rods. The temperature range for growth was 13-45 degreesC, with an optimum at 35 degreesC. The ph range for growth at 35 degreesC was from 6.0 to 8.5, with an optimum around ph 7.0. The NaCl concentrations required for growth at 35 degreesC and ph 7.0 ranged from 5 to 60 g l(-1), with an optimum around 25 g l(-1). The G+C content of the genomic DNA was 42-43 mol%. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain Dsij(T) is closely related to [Cytophaga] uliginosa DSM 2061(T). Phenotypic features, however, allowed Dsij(T) and [Cytophaga] uliginosa strains to be distinguished on the basis of ten traits (spreading behaviour, assimilation of eight compounds and amylase production). Their total protein profiles were also different and DNA-DNA hybridization experiments confirmed that Dsij(T) constitutes a new species, distinct from [Cytophaga] uliginosa. Based on the phenotypic features and the phylogenetic relationships of the Flavobacteriaceae, a new genus designated Zobellia gen, nov, is proposed to include Zobellia galactanovorans gen. nov., sp. nov., while [Cytophaga] uliginosa becomes Zobellia uliginosa comb, nov. The type strain of Zobellia galactanovorans is Dsij(T) (= DSM 12802(T) = CIP 106680(T)).
