RESUMO
Complete hydrolysis of κ-carrageenan produces two sugars, D-galactose and 3,6-anhydro-D-galactose (D-AnG). At present, however, we do not know how carrageenan-degrading microorganisms metabolize D-AnG. In this study, we investigated the metabolic pathway of D-AnG degradation by comparative genomic analysis of Cellulophaga lytica LIM-21, Pseudoalteromonas atlantica T6c, and Epulopiscium sp. N.t. morphotype B, which represent the classes Flavobacteria, Gammaproteobacteria, and Clostridia, respectively. In this bioinformatic analysis, we found candidate common genes that were believed to be involved in D-AnG metabolism. We then experimentally confirmed the enzymatic function of each gene product in the D-AnG cluster. In all three microorganisms, D-AnG metabolizing genes were clustered and organized in operon-like arrangements, which we named as the dan operon (3,6-d-anhydro-galactose). Combining bioinformatic analysis and experimental data, we showed that D-AnG is metabolized to pyruvate and D-glyceraldehyde-3-phosphate via four enzyme-catalyzed reactions in the following route: 3,6-anhydro-D-galactose â 3,6-anhydro-D-galactonate â 2-keto-3-deoxy-D-galactonate (D-KDGal) â 2-keto-3-deoxy-6-phospho-D-galactonate â pyruvate + D-glyceraldehyde-3-phosphate. The pathway of D-AnG degradation is composed of two parts: transformation of D-AnG to D-KDGal using two D-AnG specific enzymes and breakdown of D-KDGal to two glycolysis intermediates using two DeLey-Doudoroff pathway enzymes. To our knowledge, this is the first report on the metabolic pathway of D-AnG degradation.
Assuntos
Carragenina/metabolismo , Clostridiales/metabolismo , Flavobacteriaceae/metabolismo , Galactose/análogos & derivados , Redes e Vias Metabólicas/genética , Pseudoalteromonas/metabolismo , Biotransformação , Clostridiales/genética , Biologia Computacional , Flavobacteriaceae/genética , Galactose/metabolismo , Gliceraldeído 3-Fosfato/metabolismo , Família Multigênica , Óperon , Pseudoalteromonas/genética , Ácido Pirúvico/metabolismoRESUMO
We identified the non-phosphorylated L-rhamnose metabolic pathway (Rha_NMP) genes that are homologous to those in the thermoacidophilic archaeon Thermoplasma acidophilum in the genome of the thermoacidophilic bacterium Sulfobacillus thermosulfidooxidans. However, unlike previously known 2-keto-3-deoxy-L-rhamnonate (L-KDR) dehydrogenase (KDRDH) which belongs to the short chain dehydrogenase/reductase superfamily, the putative KDRDHs in S. thermosulfidooxidans (Sulth_3557) and T. acidophilum (Ta0749) belong to the medium chain dehydrogenase/reductase (MDR) superfamily. We demonstrated that Sulth_3559 and Sulth_3557 proteins from S. thermosulfidooxidans function as L-rhamnose dehydrogenase and KDRDH, respectively. Sulth_3557 protein is an NAD(+)-specific KDRDH with optimal temperature and pH of 50 °C and 9.5, respectively. The K m and V max values for L-KDR were 2.0 mM and 12.8 U/mg, respectively. Sulth_3557 also showed weak 2,3-butanediol dehydrogenase activity. Phylogenetic analysis suggests that Sulth_3557 and its homologs form a new subfamily in the MDR superfamily. The results shown in this study imply that thermoacidophilic archaea metabolize L-rhamnose to pyruvate and L-lactate by using the MDR-family KDRDH similarly to that of the thermoacidophilic bacterium S. thermosulfidooxidans.
Assuntos
Archaea/metabolismo , Bacillales/enzimologia , Proteínas de Bactérias/metabolismo , Desidrogenases de Carboidrato/metabolismo , Ramnose/metabolismo , Archaea/enzimologia , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Bacillales/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Desidrogenases de Carboidrato/química , Desidrogenases de Carboidrato/genética , Filogenia , Ligação ProteicaRESUMO
A novel bacterium, designated F081-2(T), isolated from seawater from Damupo beach in Pohang, Korea, was investigated using a polyphasic taxonomic approach. Cells were yellow-pigmented, strictly aerobic, motile by gliding, Gram-negative-staining and rod-shaped. The temperature, pH and NaCl ranges for growth were 4-35 °C, pH 5.5-9.5 and 1.0-5.0 %, respectively. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain F081-2(T) belonged to a distinct lineage in the genus Winogradskyella of the family Flavobacteriaceae, sharing 93.7-98.1 % similarity with recognized members of the genus. Low levels of DNA-DNA relatedness values were found between strain F081-2(T) and Winogradskyella eximia KCTC 12219(T) (61.1 %), Winogradskyella thalassocola KCTC 12221(T) (47.0 %), Winogradskyella echinorum KCTC 22026(T) (39.3 %), Winogradskyella rapida CCUG 56098(T) (34.3 %) and Winogradskyella arenosi JCM 17633(T) (33.4 %). The major cellular fatty acids were iso-C(15 : 0) (25.3 %), iso-C(15 : 1) G (14.6 %), iso-C(17 : 0) 3-OH (9.3 %), anteiso-C(15 : 0) (7.8 %) and iso-C(15 : 0) 3-OH (7.6 %). The polar lipid profile was composed of phosphatidylethanolamine, one unidentified aminolipid, one unidentified phospholipid, one unidentified aminophospholipid and six unidentified lipids. The major respiratory quinone was menaquinone-6 and the DNA G+C content of the strain was 32.3 mol%. On the basis of phenotypic, phylogenetic and genotypic data, strain F081-2(T) represents a novel species within the genus Winogradskyella, for which the name Winogradskyella damuponensis sp. nov. is proposed. The type strain is F081-2(T) (=KCTC 23552(T) = JCM 17633(T)).
Assuntos
Flavobacteriaceae/classificação , Filogenia , Água do Mar/microbiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/análise , Flavobacteriaceae/genética , Flavobacteriaceae/isolamento & purificação , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Fosfolipídeos/análise , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/análiseRESUMO
A novel bacterium, designated strain F051-1(T), isolated from a seawater sample collected from the coast at Damupo beach in Pohang, Korea, was investigated in a polyphasic taxonomic study. Cells were yellow-pigmented, strictly aerobic, Gram-staining-negative and rod-shaped. The temperature, pH and NaCl ranges for growth were 4-30 °C, pH 6.0-9.0 and 1.0-6.0â% (w/v), respectively. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain F051-1(T) belongs to the genus Psychroserpens in the family Flavobacteriaceae. Its closest relatives were Psychroserpens burtonensis ACAM 188(T) (96.8â% 16S rRNA gene sequence similarity) and Psychroserpens mesophilus KOPRI 13649(T) (95.7â%). The major cellular fatty acids were iso-C(15â:â0), iso-C(15â:â1) G and anteiso-C(15â:â0). The polar lipid profile consisted of a mixture of phosphatidylethanolamine, two unidentified aminolipids, one unidentified phospholipid and eight unidentified lipids. The major respiratory quinone was menaquinone-6 and the genomic DNA G+C content of the strain was 33.5 mol%. On the basis of phenotypic, phylogenetic and genotypic data, strain F051-1(T) represents a novel species within the genus Psychroserpens, for which the name Psychroserpens damuponensis sp. nov. is proposed. The type strain is F051-1(T) (â=âKCTC 23539(T) â=âJCM 17632(T)).
Assuntos
Flavobacteriaceae/classificação , Filogenia , Água do Mar/microbiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/análise , Flavobacteriaceae/genética , Flavobacteriaceae/isolamento & purificação , Dados de Sequência Molecular , Fosfolipídeos/análise , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNARESUMO
A novel bacterium, designated strain F028-2(T), was isolated from seawater at Damupo beach in Pohang, Korea, and investigated in a taxonomic study using a polyphasic approach. This novel strain was strictly aerobic, non-motile, Gram-stain-negative and rod-shaped, and occasionally formed aggregates. The temperature, pH and NaCl ranges for growth were 4-30 °C, pH 6.5-9.0 and 1-7% (w/v), respectively. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain F028-2(T) formed a lineage within the family Rhodobacteraceae of the class Alphaproteobacteria, and was closely related to members of the genera Sagittula and Antarctobacter with 96.3-96.4% sequence similarities. The polar lipid profile of strain F028-2(T) comprised diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids, one unidentified phospholipid and six unidentified lipids. The predominant cellular fatty acids were C18:1ω7c and C12:1 3-OH. The genomic DNA G+C content of strain F028-2(T) was 61.6 mol% and the major respiratory quinone was Q-10. Based on phenotypic, phylogenetic and genotypic data, strain F028-2(T) is considered to represent a novel species in the genus Sagittula, for which the name Sagittula marina sp. nov. is proposed. The type strain is F028-2(T) (=KCTC 23543(T)=JCM 17627(T)). An emended description of the genus Sagittula is also proposed.
Assuntos
Filogenia , Rhodobacteraceae/classificação , Água do Mar/microbiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/análise , Dados de Sequência Molecular , Fosfolipídeos/análise , RNA Ribossômico 16S/genética , República da Coreia , Rhodobacteraceae/genética , Rhodobacteraceae/isolamento & purificação , Análise de Sequência de DNA , Ubiquinona/análiseRESUMO
Volatile organic compounds such as benzene, toluene, ethylbenzene, and isomers of xylenes (BTEX) constitute a group of monoaromatic compounds that are found in petroleum and have been classified as priority pollutants. In this study, based on its newly sequenced genome, we reclassified the previously identified BTEX-degrading thermotolerant strain Ralstonia sp. PHS1 as Cupriavidus cauae PHS1. Also presented are the complete genome sequence of C. cauae PHS1, its annotation, species delineation, and a comparative analysis of the BTEX-degrading gene cluster. Moreover, we cloned and characterized the BTEX-degrading pathway genes in C. cauae PHS1, the BTEX-degrading gene cluster of which consists of two monooxygenases and meta-cleavage genes. A genome-wide investigation of the PHS1 coding sequence and the experimentally confirmed regioselectivity of the toluene monooxygenases and catechol 2,3-dioxygenase allowed us to reconstruct the BTEX degradation pathway. The degradation of BTEX begins with aromatic ring hydroxylation, followed by ring cleavage, and eventually enters the core carbon metabolism. The information provided here on the genome and BTEX-degrading pathway of the thermotolerant strain C. cauae PHS1 could be useful in constructing an efficient production host.
Assuntos
Benzeno , Cupriavidus , Benzeno/metabolismo , Tolueno , Xilenos/metabolismo , Cupriavidus/genética , Cupriavidus/metabolismo , Biodegradação Ambiental , Derivados de Benzeno/metabolismo , GenômicaRESUMO
The unclassified marine gammaproteobacterium BDW918, which utilizes volatile fatty acids but not most common carbohydrates and amino acids, was isolated from Dokdo seawater in South Korea. Here we present a draft genome of the strain BDW918, which encodes many putative genes related to fatty acid metabolism and aromatic hydrocarbon degradation.
Assuntos
Gammaproteobacteria/genética , Genoma Bacteriano , Regulação Bacteriana da Expressão Gênica/fisiologia , Dados de Sequência Molecular , República da Coreia , Água do Mar/microbiologia , Microbiologia da ÁguaRESUMO
A gram-negative, pink-pigmented, non-motile, strictly aerobic rod, designated CNU040(T), was isolated from seawater from the coast of Jeju Island in Korea. The temperature, pH and NaCl ranges for growth were 4-30 °C, pH 5.5-10.0 and 0-5.0â% NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CNU040(T) belonged to a distinct lineage in the genus Algoriphagus and exhibited high sequence similarity with Algoriphagus terrigena DS-44(T) (98.3â%) and Algoriphagus alkaliphilus AC-74(T) (96.6â%) and lower sequence similarity (<96.0â%) with all other members of the genus Algoriphagus. DNA-DNA relatedness between strain CNU040(T) and A. terrigena KCTC 12545(T) was 44.5â%. The DNA G+C content of the isolate was 48.5 mol% and the major respiratory quinone was menaquinone-7. The major cellular fatty acids were iso-C(15â:â0) (28.6â%) and summed feature 3 (consisting of iso-C(15â:â0) 2-OH and/or C(16â:â1)ω7c; 24.0â%). The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, one unknown amino lipid, one unknown aminophospholipid and three unknown polar lipids. On the basis of phenotypic, phylogenetic and genotypic data, strain CNU040(T) represents a novel species within the genus Algoriphagus, for which the name Algoriphagus jejuensis sp. nov. is proposed. The type strain is CNU040(T) (â=âKCTC 22647(T) â=âJCM 16112(T)).
Assuntos
Bacteroidetes/classificação , Bacteroidetes/isolamento & purificação , Água do Mar/microbiologia , Técnicas de Tipagem Bacteriana , Bacteroidetes/química , Bacteroidetes/genética , Composição de Bases , DNA Bacteriano/análise , DNA Ribossômico/análise , Ácidos Graxos/análise , Genes de RNAr , Genótipo , Lipídeos/análise , Dados de Sequência Molecular , Fenótipo , Filogenia , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNA , Especificidade da Espécie , Vitamina K 2/análiseRESUMO
A Gram-staining-negative, strictly aerobic and rod-shaped bacterium, designated strain CNURIC013(T), was isolated from seawater collected on the coast of Jeju Island, South Korea. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain CNURIC013(T) belonged to the genus Tenacibaculum, within the family Flavobacteriaceae. Sequence similarities between the novel strain and the type strains of recognized species of the genus Tenacibaculum were 93.6-96.0â%, the highest value being with Tenacibaculum litopenaei B-I(T) (96â%). The DNA G+C content of the novel strain was 34.5 mol% and the major respiratory quinone was menaquinone-6. The major fatty acids were summed feature 3 (comprising C(16â:â1)ω7c and/or iso-C(15â:â0) 2-OH; 26.0â%), iso-C(15â:â0) (24.4â%), iso-C(15â:â1) G (18.5â%) and iso-C(17â:â0) 3-OH (8.1â%). The polar lipids consisted of phosphatidylethanolamine, one unknown aminophospholipid and nine unknown polar lipids. On the basis of the phenotypic, phylogenetic and genotypic data, strain CNURIC013(T) represents a novel species within the genus Tenacibaculum, for which the name Tenacibaculum jejuense sp. nov. is proposed. The type strain is CNURIC013(T) (â=âKCTC 22618(T)â=âJCM 15975(T)).
Assuntos
Água do Mar/microbiologia , Tenacibaculum/classificação , Tenacibaculum/isolamento & purificação , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/análise , DNA Ribossômico/análise , Ácidos Graxos/análise , Genes de RNAr , Genótipo , Lipídeos/análise , Dados de Sequência Molecular , Fenótipo , Filogenia , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNA , Especificidade da Espécie , Tenacibaculum/química , Tenacibaculum/genética , Vitamina K 2/análiseRESUMO
A Gram-staining-negative, strictly aerobic, non-motile, yellow-pigmented bacterium, designated strain M091(T), was isolated from seawater at Damupo beach in Pohang, Republic of Korea, and investigated using a polyphasic taxonomic approach. The novel strain grew optimally at 25 °C, pH 7.0-8.0, and in the presence of 3% (w/v) NaCl. In a phylogenetic analysis based on 16S rRNA gene sequences, strain M091(T) formed a lineage within the family Flavobacteriaceae that was distinct from the most closely related genera of Flaviramulus (95.1% sequence similarity), Algibacter (94.9-93.9%), Mariniflexile (94.8-94.2%), Winogradskyella (94.8-93.2%), Lacinutrix (94.7-93.8%) and Tamlana (94.7-92.9%). The polar lipid profile of the novel strain comprised phosphatidylethanolamine, two unidentified aminolipids, one unidentified phospholipid and seven unidentified lipids. The predominant cellular fatty acids were iso-C(15:0) (20.5%), iso-C(17:0) 3-OH (15.4%), iso-C(15:0) 3-OH (12.4%), C(15:0) (10.9%) and iso-C(15:1) G (9.9%). The genomic DNA G+C content of strain M091(T) was 34.4 mol% and the major respiratory quinone was MK-6. Based on phenotypic and genotypic data, strain M091(T) represents a new genus and novel species in the family Flavobacteriaceae, for which the name Postechiella marina gen. nov., sp. nov. is proposed. The type strain of the type species is M091(T) (=KCTC 23537(T)=JCM 17630(T)).
Assuntos
Flavobacteriaceae/classificação , Flavobacteriaceae/isolamento & purificação , Água do Mar/microbiologia , Aerobiose , Técnicas de Tipagem Bacteriana , Composição de Bases , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Ácidos Graxos/análise , Flavobacteriaceae/genética , Flavobacteriaceae/fisiologia , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Fosfolipídeos/análise , Filogenia , Pigmentos Biológicos/metabolismo , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNA , Cloreto de Sódio/metabolismo , TemperaturaRESUMO
Thermoplasma acidophilum utilizes L-rhamnose as a sole carbon source. To determine the metabolic pathway of L-rhamnose in Archaea, we identified and characterized L-rhamnose dehydrogenase (RhaD) in T. acidophilum. Ta0747P gene, which encodes the putative T. acidophilum RhaD (Ta_RhaD) enzyme belonging to the short-chain dehydrogenase/reductase family, was expressed in E. coli as an active enzyme catalyzing the oxidation of L-rhamnose to L-rhamnono-1,4-lactone. Analysis of catalytic properties revealed that Ta_RhaD oxidized L-rhamnose, L-lyxose, and L-mannose using only NADP(+) as a cofactor, which is different from NAD(+)/NADP(+)-specific bacterial RhaDs and NAD(+)-specific eukaryal RhaDs. Ta_RhaD showed the highest activity toward L-rhamnose at 60 °C and pH 7. The K (m) and k (cat) values were 0.46 mM, 1,341.3 min(-1) for L-rhamnose and 0.1 mM, 1,027.2 min(-1) for NADP(+), respectively. Phylogenetic analysis indicated that branched lineages of archaeal RhaD are quite distinct from those of Bacteria and Eukarya. This is the first report on the identification and characterization of NADP(+)-specific RhaD.
Assuntos
Proteínas Arqueais/química , Desidrogenases de Carboidrato/química , NADP/química , Thermoplasma/enzimologia , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Desidrogenases de Carboidrato/genética , Desidrogenases de Carboidrato/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , NADP/genética , NADP/metabolismo , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ramnose/química , Ramnose/genética , Ramnose/metabolismo , Thermoplasma/genéticaRESUMO
Glucuronic acid dehydrogenase (GluUADH), the product of the Csal-2474 gene from the halophilic bacterium Chromohalobacter salexigens DSM 3043, is an enzyme with potential use in the conversion of glucuronic acid in seaweed biomass to fuels and chemicals. GluUADH is an enzyme that catalyzes the oxidation of glucuronic acid (GluUA) and galacturonic acid (GalUA) and has a preference for NAD(+) rather than NADP(+) as a cofactor. Recombinant GluUADH was crystallized in the presence of 0.2â M calcium acetate, 0.1â M Tris-HCl pH 7.0 and 20% PEG 3000 at 295â K. X-ray diffraction data were collected to a maximum resolution of 2.1â Å. The GluUADH crystal belonged to space group P6(3), with unit-cell parameters a = b = 122.58, c = 150.49â Å, γ = 120°. With one molecule per asymmetric unit, the crystal volume per unit protein weight (V(M)) is 2.78â Å(3)â Da(-1). The structure was solved by the single anomalous dispersion method and structure refinement is in progress.
Assuntos
Chromohalobacter/enzimologia , Oxirredutases/química , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Expressão Gênica , Oxirredutases/genética , Oxirredutases/isolamento & purificaçãoRESUMO
Expression of archaeal proteins in soluble form is of importance because archaeal proteins are usually produced as insoluble inclusion bodies in Escherichia coli. In this study, we investigated the use of soluble fusion tags to enhance the solubility of two archaeal proteins, d-gluconate dehydratase (GNAD) and 2-keto-3-deoxy-D-gluconate kinase (KDGK), key enzymes in the glycolytic pathway of the thermoacidophilic archaeon Sulfolobus solfataricus. These two proteins were produced as inclusion bodies in E. coli when polyhistidine was used as a fusion tag. To reduce inclusion body formation in E. coli, GNAD and KDGK were fused with three partners, thioredoxin (Trx), glutathione-S-transferase (GST), and N-utilization substance A (NusA). With the use of fusion-partners, the solubility of the archaeal proteins was remarkably enhanced, and the soluble fraction of the recombinant proteins was increased in this order: Trx>GST>NusA. Furthermore, In the case of recombinant KDGKs, the enzyme activity of the Trx-fused proteins was 200-fold higher than that of the polyhistidine-fusion protein. The strategy presented in this work may contribute to the production of other valuable proteins from hyperthermophilic archaea in E. coli.
Assuntos
Proteínas Arqueais/genética , Escherichia coli/genética , Hidroliases/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Arqueais/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Hidroliases/metabolismo , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Solubilidade , Sulfolobus solfataricus/enzimologia , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da TranscriçãoAssuntos
Proteínas de Bactérias/química , Desidrogenases de Carboidrato/química , Chromohalobacter/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Domínio Catalítico , Coenzimas/química , Sequência Conservada , Cristalografia por Raios X , Ácido Glucurônico/química , Modelos Moleculares , Dados de Sequência Molecular , NAD/química , Ligação Proteica , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
Thermoacidophilic archaea such as Thermoplasma acidophilum and Sulfolobus solfataricus are known to metabolize D-glucose via the nED (non-phosphorylated Entner-Doudoroff) pathway. In the present study, we identified and characterized a glyceraldehyde dehydrogenase involved in the downstream portion of the nED pathway. This glyceraldehyde dehydrogenase was purified from T. acidophilum cell extracts by sequential chromatography on DEAE-Sepharose, Q-Sepharose, Phenyl-Sepharose and Affi-Gel Blue columns. SDS/PAGE of the purified enzyme showed a molecular mass of approx. 53 kDa, whereas the molecular mass of the native protein was 215 kDa, indicating that glyceraldehyde dehydrogenase is a tetrameric protein. By MALDI-TOF-MS (matrix-assisted laser-desorption ionization-time-of-flight MS) peptide fingerprinting of the purified protein, it was found that the gene product of Ta0809 in the T. acidophilum genome database corresponds to the purified glyceraldehyde dehydrogenase. The native enzyme showed the highest activity towards glyceraldehyde, but no activity towards aliphatic or aromatic aldehydes, and no activity when NAD+ was substituted for NADP+. Analysis of the amino acid sequence and enzyme inhibition studies indicated that this glyceraldehyde dehydrogenase belongs to the ALDH (aldehyde dehydrogenase) superfamily. BLAST searches showed that homologues of the Ta0809 protein are not present in the Sulfolobus genome. Possible differences between T. acidophilum (Euryarchaeota) and S. solfataricus (Crenarchaeaota) in terms of the glycolytic pathway are thus expected.
Assuntos
Aldeído Desidrogenase/química , Aldeído Desidrogenase/isolamento & purificação , Glucose/metabolismo , Thermoplasma/enzimologia , Aldeído Desidrogenase/metabolismo , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Cinética , Dados de Sequência Molecular , Mapeamento de Peptídeos , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , Sulfolobus/enzimologiaRESUMO
Dihydroxy-acid dehydratase (DHAD) is one of the key enzymes involved in the biosynthetic pathway of the branched chain amino acids. Although the enzyme has been purified and characterized in various mesophiles, including bacteria and eukarya, the biochemical properties of DHAD from hyperthermophilic archaea have not yet been reported. In this study we cloned, expressed in Escherichia coli, and purified a DHAD homologue from the thermoacidophilic archaeon Sulfolobus solfataricus, which grows optimally at 80 degrees C and pH 3. The recombinant S. solfataricus DHAD (rSso_DHAD) showed the highest activity on 2,3-dihydroxyisovalerate among 17 aldonic acids tested. Interestingly, this enzyme also displayed high activity toward d-gluconate and some other pentonic and hexonic sugar acids. The k(cat)/K(m) values were 140.3 mM(-1) s(-1) for 2,3-dihydroxyisovalerate and 20.0 mM(-1) s(-1) for d-gluconate, respectively. A possible evolutionary explanation for substrate promiscuity was provided through amino acid sequence alignments of DHADs and 6-phosphogluconate dehydratases from archaea, bacteria and eukarya.
Assuntos
Hidroliases/química , Hidroliases/metabolismo , Sulfolobus solfataricus/enzimologia , Catálise , Evolução Molecular , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Light intensity is a very important factor that determines the growth of photosynthetic cells. In this study, the light distribution in a photobioreactor was analyzed by processing the images captured with a digital camera. The contour images obtained by filtering the original images clearly showed the effects of the cell concentration and external light intensity on the light distribution. Image-processing techniques were then applied to predict the cell density in the photobioreactor. To correlate the cell concentration with the light intensity in the photobioreactor, the captured images were processed using two different approaches. The first method involved the use of an average gray value after deriving a simplified model equation that could be related to the cell density. The second method involved the use of local points instead of a representative value. In this case, an artificial neural network model was adopted to infer the cell density from the information of the local points. By using these two methods, it was possible to relate the image data to the cell concentration. Finally, we compared these two methods with regard to their accuracy, easiness, and effectiveness.
Assuntos
Reatores Biológicos , Biotecnologia/métodos , Processamento de Imagem Assistida por Computador/métodos , Luz , Fotobiologia/instrumentação , Desenho de Equipamento , Modelos Biológicos , Modelos Estatísticos , Modelos Teóricos , Redes Neurais de Computação , Fotossíntese , Synechococcus/metabolismo , Fatores de TempoRESUMO
The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner-Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 degrees C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 degrees C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with K(m)=0.45 mM and V(max)=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation-dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner-Doudoroff pathway.
Assuntos
Hidroliases/química , Hidroliases/genética , Sulfolobus solfataricus/enzimologia , Sequência de Aminoácidos , Dissacarídeos/metabolismo , Hexoses/metabolismo , Hidroliases/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Pentoses/metabolismo , Fosforilação , Açúcares Ácidos/metabolismoRESUMO
The Hildebrand solubility parameters, cohesive energy densities and internal energies of 1-alkyl-3-methylimidazolium-based room temperature ionic liquids were determined by the intrinsic viscosity method and their dependencies on the length of the alkyl group analyzed.
RESUMO
Most of digital subtraction methods in dental radiography are based on registration using manual landmarks. We have developed an automatic registration method without using the manual selection of landmarks. By restricting a geometrical matching of images to a region of interest (ROI), we compare the cross-correlation coefficient only between the ROIs. The affine or perspective transform parameters satisfying maximum of cross-correlation between the local regions are searched iteratively by a fast searching strategy. The parameters are searched on the 14 scale image coarsely and then, the fine registration is performed on the original scale image. The developed method can match the images corrupted by Gaussian noise with the same accuracy for the images without any transform simulation. The registration accuracy of the perspective method shows a 17% improvement over the manual method. The application of the developed method to radiographs of dental implants provides an automatic noise robust registration with high accuracy in almost real time.