RESUMO
Mesangial deposition of aberrantly glycosylated IgA1 (agIgA1) and its immune complexes is a key pathogenic mechanism of IgA nephropathy (IgAN). However, treatment of IgAN remains ineffective. We report here that bacteria-derived IgA proteases are capable of degrading these pathogenic agIgA1 and derived immune complexes in vitro and in vivo. By screening 14 different bacterial strains (6 species), we found that 4 bacterial IgA proteases from H. influenzae, N. gonorrhoeae and N. meningitidis exhibited high cleaving activities on serum agIgA1 and artificial galactose-depleted IgA1 in vitro and the deposited agIgA1-containing immune complexes in the mesangium of renal biopsy from IgAN patients and in a passive mouse model of IgAN in vitro. In the modified mouse model of passive IgAN with abundant in situ mesangial deposition of the agIgA-IgG immune complexes, a single intravenous delivery of IgA protease from H. influenzae was able to effectively degrade the deposited agIgA-IgG immune complexes within the glomerulus, demonstrating a therapeutic potential for IgAN. In conclusion, the bacteria-derived IgA proteases are biologically active enzymes capable of cleaving the circulating agIgA and the deposited agIgA-IgG immune complexes within the kidney of IgAN. Thus, the use of such IgA proteases may represent a novel therapy for IgAN.
Assuntos
Anticorpos Antibacterianos/metabolismo , Complexo Antígeno-Anticorpo/metabolismo , Proteínas de Bactérias/farmacologia , Glomerulonefrite por IGA/tratamento farmacológico , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Imunoglobulina A/metabolismo , Peptídeo Hidrolases/farmacologia , Animais , Glomerulonefrite por IGA/metabolismo , Humanos , Masculino , Camundongos Endogâmicos BALB CRESUMO
Endo-1,4-ß-xylanases are mostly classified into glycoside hydrolase (GH) family 10 or 11. In this study, we examined the catalytic functions of a recombinant endo-1,4-ß-xylanase belonging to GH10 (Xyn10C) from a marine bacterium, Saccharophagus degradans 2-40. Optimal activity of this enzyme was evident at 30 °C and pH 7.0, but activity remained even at low temperatures, indicating its adaptation to cold. With respect to other xylanases known to be active in cold temperatures, Xyn10C is unique in that it showed maximal activity in the presence of 2 M of NaCl. The action patterns of recombinant Xyn10C on xylans from hardwood and softwood differed in part, but the enzyme hydrolyzed polysaccharidic substrates primarily to xylobiose and xylotriose through xylo-oligosaccharides, releasing a small amount of xylose. The K m and V max values on birchwood xylan were 10.4 mg mL(-1) and 253 µmol mg(-1) min(-1), respectively. The efficient catalytic function of Xyn10C on short-length xylo-oligosaccharide chains was similar to the typical function of other known GH10 xylanases.
Assuntos
Organismos Aquáticos/enzimologia , Proteínas de Bactérias/química , Glicosídeo Hidrolases/química , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Xilanos/química , Organismos Aquáticos/genética , Proteínas de Bactérias/genética , Glicosídeo Hidrolases/genética , Bacilos e Cocos Aeróbios Gram-Negativos/genética , Temperatura Alta , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Alkane and aromatic hydroxylase genes in a full-scale aerobic system treating oily produced wastewater under thermophilic condition (45-50 °C) in the Jidong oilfield, China, were investigated using clone library and quantitative polymerase chain reaction methods. Rather than the normally encountered integral-membrane non-haem iron monooxygenase (alkB) genes, only CYP153-type P450 hydroxylase genes were detected for the alkane activation, indicating that the terminal oxidation of alkanes might be mainly mediated by the CYP153-type alkane hydroxylases in the thermophilic aerobic process. Most of the obtained CYP153 gene clones showed distant homology with the reference sequences, which might represent novel alkane hydroxylases. For the aromatic activation, the polycyclic aromatic hydrocarbon-ring hydroxylating dioxygenase (PAH-RHD) gene was derived from Gram-negative PAH-degraders belonging to the Burkholderiales order, with a 0.72% relative abundance of PAH-RHD gene to 16S rRNA gene. This was consistent with the result of 16S rRNA gene analysis, indicating that Burkholderiales bacteria might play a key role in the full-scale process of thermophilic hydrocarbon degradation.
Assuntos
Proteínas de Bactérias/genética , Betaproteobacteria/enzimologia , Betaproteobacteria/genética , Citocromo P-450 CYP4A/genética , Dioxigenases/genética , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Bacilos e Cocos Aeróbios Gram-Negativos/genética , Aerobiose , Proteínas de Bactérias/metabolismo , Reatores Biológicos/microbiologia , Citocromo P-450 CYP4A/metabolismo , Dioxigenases/metabolismo , Temperatura Alta , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Análise de Sequência de DNA , Eliminação de Resíduos LíquidosRESUMO
Direct plating of simulated stool specimens on MacConkey agar (MCA) with 10-µg ertapenem, meropenem, and imipenem disks allowed the establishment of optimal zone diameters for the screening of carbapenem-resistant Gram-negative rods (CRGNR) of ≤ 24 mm (ertapenem), ≤ 34 mm (meropenem), and ≤ 32 mm (imipenem).
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Carbapenêmicos/farmacologia , Bacilos e Cocos Aeróbios Gram-Negativos/efeitos dos fármacos , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Resistência beta-Lactâmica , beta-Lactamases/metabolismo , Ágar , Meios de Cultura/química , Fezes/microbiologia , Bacilos e Cocos Aeróbios Gram-Negativos/isolamento & purificação , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Testes de Sensibilidade Microbiana/métodosAssuntos
Bacteriemia/microbiologia , Infecções Relacionadas a Cateter/microbiologia , Bacilos e Cocos Aeróbios Gram-Negativos/isolamento & purificação , Mieloma Múltiplo/complicações , Idoso , Bacteriemia/complicações , Técnicas Bacteriológicas , Proteína de Bence Jones/urina , Infecções Relacionadas a Cateter/complicações , Exposição Ambiental , Feminino , Bacilos e Cocos Aeróbios Gram-Negativos/classificação , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Bacilos e Cocos Aeróbios Gram-Negativos/crescimento & desenvolvimento , Humanos , Mieloma Múltiplo/urina , Espanha/epidemiologia , Microbiologia da ÁguaRESUMO
SoxAX cytochromes catalyze the formation of heterodisulfide bonds between inorganic sulfur compounds and a carrier protein, SoxYZ. They contain unusual His/Cys-ligated heme groups with complex spectroscopic signatures. The heme-ligating cysteine has been implicated in SoxAX catalysis, but neither the SoxAX spectroscopic properties nor its catalysis are fully understood at present. We have solved the first crystal structure for a group 2 SoxAX protein (SnSoxAX), where an N-terminal extension of SoxX forms a novel structure that supports dimer formation. Crystal structures of SoxAX with a heme ligand substitution (C236M) uncovered an inherent flexibility of this SoxA heme site, with both bonding distances and relative ligand orientation differing between asymmetric units and the new residue, Met(236), representing an unusual rotamer of methionine. The flexibility of the SnSoxAX(C236M) SoxA heme environment is probably the cause of the four distinct, new EPR signals, including a high spin ferric heme form, that were observed for the enzyme. Despite the removal of the catalytically active cysteine heme ligand and drastic changes in the redox potential of the SoxA heme (WT, -479 mV; C236M, +85 mV), the substituted enzyme was catalytically active in glutathione-based assays although with reduced turnover numbers (WT, 3.7 s(-1); C236M, 2.0 s(-1)). SnSoxAX(C236M) was also active in assays using SoxYZ and thiosulfate as the sulfur substrate, suggesting that Cys(236) aids catalysis but is not crucial for it. The SoxYZ-based SoxAX assay is the first assay for an isolated component of the Sox multienzyme system.
Assuntos
Proteínas de Bactérias/química , Grupo dos Citocromos c/química , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Oxirredutases/química , Multimerização Proteica/fisiologia , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Domínio Catalítico/fisiologia , Cristalografia por Raios X , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Bacilos e Cocos Aeróbios Gram-Negativos/genética , Heme/química , Heme/genética , Heme/metabolismo , Mutação de Sentido Incorreto , Oxirredutases/genética , Oxirredutases/metabolismo , Estrutura Quaternária de Proteína , Rhodobacter capsulatus/enzimologia , Rhodobacter capsulatus/genética , Relação Estrutura-AtividadeRESUMO
The biodegradation rate of chlorophenols in the environment seems to be limited by a competitive mechanism of O-methylation which produces chloroanisoles with a high potential of being bioconcentrated in living organisms. In this work we report for the first time the isolation of three soil bacterial strains able to efficiently degrade 2,4,6-trichloroanisole (2,4,6-TCA). These strains were identified as Xanthomonas retroflexus INBB4, Pseudomonas putida INBP1 and Acinetobacter radioresistens INBS1. In these isolates 2,4,6-TCA was efficiently metabolized in a minimal medium containing methanol and 2,4,6-TCA as the only carbon sources, with a concomitant release of 3 mol of chloride ion from 1 mol of 2,4,6-TCA, indicating complete dehalogenation of 2,4,6-TCA. 2,4,6-trichlorophenol (2,4,6-TCP) was identified as a degradative intermediate, indicating that 2,4,6-TCA underwent O-demethylation as the first step in the biodegradation process. 2,4,6-TCP was further transformed into 2,6-dichloro-para-hydroquinone (2,6-DCHQ) and subsequently mineralized. The degradation of chloroanisoles could improve the overall biodegradation of chlorophenols in the environment, because those chlorophenols previously biomethylated might also be later biodegraded. Xanthomonas retroflexus INBB4 has two O-demethylation systems: one is an oxygenase-type demethylase, and the other is a tetrahydrofolate (THF)-dependent O-demethylase. On the contrary O-demethylation of 2,4,6-TCA in P. putida INBP1 is just catalysed by an oxygenase-type NADH/NADPH-dependent O-demethylase, whereas in A. radioresistens INBS1 a THF-dependent O-demethylase activity was detected.
Assuntos
Anisóis/metabolismo , Clorofenóis/metabolismo , Bacilos e Cocos Aeróbios Gram-Negativos/metabolismo , Oxirredutases O-Desmetilantes/metabolismo , Microbiologia do Solo , Anisóis/química , Sequência de Bases , Biodegradação Ambiental , Clorofenóis/química , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Metilação , Oxirredutases O-Desmetilantes/química , RNA Ribossômico 16S/genéticaRESUMO
We characterized 34 endophytic bacterial isolates associated to root nodules collected from spontaneous legumes in the arid zone of Tunisia by 16S rDNA polymerase chain reaction (PCR)-restriction fragment length polymorphism, whole cell protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 16S rDNA and 16S-23S rDNA internal transcribed spacer sequencing. Phylogenetically, these isolates belong to the branches containing the genera Inquilinus, Bosea, Rhodopseudomonas, Paracraurococcus, Phyllobacterium, Ochrobactrum, Starkeya, Sphingomonas, Pseudomonas, Agromyces, Microbacterium, Ornithinicoccus, Bacillus, and Paenibacillus. These strains did not induce any nodule formation when inoculated on the wide host spectrum legume species M. atropurpureum (Siratro) and no nodA gene could be amplified by PCR. However, nifH sequences, most similar to those of Sinorhizobium meliloti, were detected within strains related to the genera Microbacterium, Agromyces, Starkeya and Phyllobacterium.
Assuntos
Fabaceae/microbiologia , Bacilos e Cocos Aeróbios Gram-Negativos/genética , Oxirredutases/genética , Burkholderia/enzimologia , Burkholderia/genética , Burkholderia/isolamento & purificação , Eletroforese em Gel de Poliacrilamida/métodos , Fabaceae/metabolismo , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Bacilos e Cocos Aeróbios Gram-Negativos/isolamento & purificação , Filogenia , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , RNA Ribossômico 16S/genética , Rhizobiaceae/enzimologia , Rhizobiaceae/genética , Rhizobiaceae/isolamento & purificação , Rhizobium/enzimologia , Rhizobium/genética , Rhizobium/isolamento & purificaçãoRESUMO
Seven organophosphorus pesticide-degrading bacteria harboring the methyl parathion degrading (mpd) gene were isolated from a methyl parathion contaminated site. In this study, the 4.7 kb mpd gene cluster, conserved in all seven bacteria capable of degrading methyl parathion, was cloned and further analysis revealed that this cluster contained five ORFs and the mpd gene was associated with a mobile element, IS6100. In addition to mpd gene ORF and tnpA ORF, three other ORFs showed high homology to the permease component of ABC-type transport system, the general secretion pathway protein B, and the RNA polymerase sigma 70 factor, respectively. The mpd genes of these 7 strains were subcloned and expressed in E. coli, SDS-PAGE and zymogram analysis showed that two expression products of mpd genes in E. coli were found, but the one without signal peptide showed the hydrolytic activities. Our evidences collectively suggest that mpd gene cluster may be disseminated through horizontal gene transfer based on phylogenetic analysis of the cluster and their host bacterial strains, and comparisons of GC content of the cluster and respective host's chromosome.
Assuntos
Arildialquilfosfatase/genética , Transferência Genética Horizontal , Genes Bacterianos , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Metil Paration/metabolismo , Praguicidas/metabolismo , Biodegradação Ambiental , Clonagem Molecular , Poluição Ambiental , Escherichia coli/genética , Bacilos e Cocos Aeróbios Gram-Negativos/genética , Bacilos e Cocos Aeróbios Gram-Negativos/isolamento & purificação , Família Multigênica , Fases de Leitura AbertaRESUMO
Subsurface bacteria commonly exist in a starvation state with only periodic exposure to utilizable sources of carbon and energy. In this study, the effect of carbon starvation on aerobic toluene degradation was quantitatively evaluated with a selection of bacteria representing all the known toluene oxygenase enzyme pathways. For all the investigated strains, the rate of toluene biodegradation decreased exponentially with starvation time. First-order deactivation rate constants for TMO-expressing bacteria were approximately an order of magnitude greater than those for other oxygenase-expressing bacteria. When growth conditions (the type of growth substrate and the type and concentration of toluene oxygenase inducer) were varied in the cultures prior to the deactivation experiments, the rate of deactivation was not significantly affected, suggesting that the rate of deactivation is independent of previous substrate/inducer conditions. Because TMO-expressing bacteria are known to efficiently detoxify TCE in subsurface environments, these findings have significant implications for in situ TCE bioremediation, specifically for environments experiencing variable growth-substrate exposure conditions.
Assuntos
Carbono/deficiência , Bacilos e Cocos Aeróbios Gram-Negativos/crescimento & desenvolvimento , Oxigenases de Função Mista/metabolismo , Tolueno/metabolismo , Biodegradação Ambiental , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Tricloroetileno/metabolismoRESUMO
Lipases, triacylglycerol hydrolases, are an important group of biotechnologically relevant enzymes and they find immense applications in food, dairy, detergent and pharmaceutical industries. Lipases are by and large produced from microbes and specifically bacterial lipases play a vital role in commercial ventures. Some important lipase-producing bacterial genera include Bacillus, Pseudomonas and Burkholderia. Lipases are generally produced on lipidic carbon, such as oils, fatty acids, glycerol or tweens in the presence of an organic nitrogen source. Bacterial lipases are mostly extracellular and are produced by submerged fermentation. The enzyme is most commonly purified by hydrophobic interaction chromatography, in addition to some modern approaches such as reverse micellar and aqueous two-phase systems. Most lipases can act in a wide range of pH and temperature, though alkaline bacterial lipases are more common. Lipases are serine hydrolases and have high stability in organic solvents. Besides these, some lipases exhibit chemo-, regio- and enantioselectivity. The latest trend in lipase research is the development of novel and improved lipases through molecular approaches such as directed evolution and exploring natural communities by the metagenomic approach.
Assuntos
Bacillus/enzimologia , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Lipase/biossíntese , Lipase/metabolismo , Bacillus/crescimento & desenvolvimento , Biotecnologia/tendências , Burkholderia/enzimologia , Fermentação , Bacilos e Cocos Aeróbios Gram-Negativos/crescimento & desenvolvimento , Microbiologia Industrial , Lipase/química , Lipase/isolamento & purificação , Pseudomonas/enzimologia , TemperaturaRESUMO
3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase, catalyzes the aldol-type condensation between phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate (A5P) to produce the unusual 8-carbon sugar KDO8P, and inorganic phosphate. A 15.5-kb segment containing the kdsA gene from the hyperthermophilic bacterium Aquifex pyrophilus was cloned from a genomic library and sequenced. The native kdsA gene lacks a typical ribosome binding site, but contains a conserved U,A-rich sequence upstream to the start codon. The purified kdsA gene product catalyzes the formation of KDO8P from its natural substrates, PEP and A5P, as determined by (1)H NMR analysis. KDO8P synthase showed maximum activity at 80 degrees C and pH 5.5-6.0 at 10-min reaction assay. At temperatures of 70, 80, and 90 degrees C, the enzyme exhibited half-lives of 8.0, 2.25, and 0.5 h, respectively. The kinetic constants at 60 degrees C were K(m)(A5P)=70 microM, K(m)(PEP)=290 microM, and k(cat)=4 s(-1). The isolated enzyme contained 0.19 and 0.26 mol iron and zinc, respectively, per mole of enzyme subunit. Treatment with metal chelators eliminated enzyme activity, and by the addition of several divalent metal ions, the activity was restored and even exceeded the original activity. These results indicate that A. pyrophilus KDO8P synthase is a metal-dependent enzyme. A C11A mutant of KDO8P synthase from A. pyrophulis retained less than 1% of the wild-type activity and was shown to be incapable of metal binding.
Assuntos
Aldeído Liases/genética , Clonagem Molecular , Expressão Gênica , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Aldeído Liases/isolamento & purificação , Aldeído Liases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Mapeamento Cromossômico , Escherichia coli/genética , Genes Bacterianos , Bacilos e Cocos Aeróbios Gram-Negativos/genética , Concentração de Íons de Hidrogênio , Cinética , Metais/farmacologia , Modelos Biológicos , Dados de Sequência Molecular , Peso Molecular , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
The enzymatic degradability of chemosynthesized atactic poly([R,S]-3-hydroxybutyrate) [a-P(3HB)] by two types of extracellular poly(3-hydroxyalkanoate) (PHA) depolymerases purified from Ralstonia pickettii T1 (PhaZ(ral)) and Acidovorax Sp. TP4 (PhaZ(aci)), defined respectively as PHA depolymerase types I and II according to the position of the lipase box in the catalytic domain, were studied. The enzymatic degradation of a-P(3HB) by PhaZ(aci) depolymerase was confirmed from the results of weight loss and the scanning electron micrographs. The degradation products were characterized by one- and two-dimension (1)H NMR spectroscopy. It was found that a-P(3HB) could be degraded into monomer, dimer, and trimer by PhaZ(aci) depolymerase at temperatures ranging from 4 to 20 degrees C, while a-P(3HB) could hardly be hydrolyzed by PhaZ(ral) depolymerase in the same temperature range. These results suggested that the chemosynthesized a-P(3HB) could be degraded in the pure state by natural PHA depolymerase.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Biodegradação Ambiental , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Hidrólise , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Varredura , TemperaturaRESUMO
The aim of the study was to isolate extended spectrum beta-laktamases (ESBL) and chromosomal beta-laktamases AmpC producing strains from food products. A total of 739 Gram-negative bacteria were tested with double disc diffusion method using cefotaxim, ceftasidim and amoxycillin with clavulanic acid. One strain producing ESBL and belonging to Stenotrophomonas maltophilia was detected from the ice cream (0.14% of all strains). From different food products a total of 14 microorganisms (1.9%) having AmpC enzymes have been isolated. They belonged to Enterobacter spp, Hafnia spp, Morganella spp, Citrobacter, Acinetobacter, and Cedecea. Appearance of strains producing beta-laktamases ESBL and AmpC among microorganism isolated from food make it necessary to monitor enzymes activity during routine microbiological control of foods.
Assuntos
Proteínas de Bactérias , Microbiologia de Alimentos , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Bacilos e Cocos Aeróbios Gram-Negativos/isolamento & purificação , beta-Lactamases/metabolismo , Bacilos e Cocos Aeróbios Gram-Negativos/classificação , Sorvetes/microbiologia , Especificidade da Espécie , beta-Lactamases/classificaçãoRESUMO
An alpha-N-acetylgalactosaminidase IV able to remove blood type specificity of human A(II)-erythrocytes and not effecting B(III)-erythrocytes was isolated from the marine bacterium Arenibacter latericius KMM 426T. The alpha-N-acetylgalactosaminidase IV preparation exhibits high activity during inhibition of hemagglutination with blood group substance A containing determinants analogous to A-erythrocytes. The enzyme has a pH optimum from 7.0 to 8.0 and completely retains its activity during 30-min heating at 50 degrees C and for a week at 20 degrees C. The enzyme can be stored under the sterile conditions for any length of time at 4 degrees C, but it does not withstand freezing. The alpha-N-acetylgalactosaminidase is resistant to NaCl; for p-nitrophenyl-alpha-N-acetyl-D-galactosaminide, the Km is 0.38 mM. The molecular mass of the enzyme determined by gel filtration is 84 kD.
Assuntos
Sistema ABO de Grupos Sanguíneos/imunologia , Proteínas de Bactérias/metabolismo , Eritrócitos/imunologia , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Hexosaminidases/metabolismo , Sequência de Carboidratos , Hexosaminidases/isolamento & purificação , Cinética , Dados de Sequência Molecular , alfa-N-AcetilgalactosaminidaseRESUMO
Enzymatic degradability has been investigated for a series of bacterial poly(3-hydroxybutyrate-co-3-hydroxypropionate)s (P(3HB-co-3HP)s) with 3-hydroxypropionate (3HP) unit contents from 11 to 86 mol % as well as poly(3-hydroxybutyrate) (P(3HB)) and chemosynthesized poly(3-hydroxypropionate) (P(3HP)). The behavior of degradation by two types of extracellular poly(3-hydroxyalkanoate) (PHA) depolymerases purified from Ralstonia pikettii T1 and Acidovorax Sp. TP4, defined respectively as PHA depolymerase types I and II according to the position of the lipase box in the catalytic domain, were compared in relation to the thermal properties and crystalline structures of the PHA samples elucidated by differential scanning calorimetry and wide-angle X-ray diffraction. The degradation products were characterized by high-performance liquid chromatography and one- (1D) and two-dimension (2D) (1)H NMR spectroscopy. It was found that the PHA depolymerase of Acidovorax Sp. TP4 showed degradation behavior different from that shown by depolymerase of R. pikettii T1. PHA depolymerase from Acidovorax Sp. TP4 degraded the P(3HB-co-3HP) films with lower crystallinity in higher rates than those with higher crystallinity, no matter what kinds of crystalline structures they formed. In contrast, PHA depolymerase from R. pikettii T1 degraded P(3HB-co-3HP) films forming P(3HB) crystalline structure in higher rates than those forming P(3HP)s. The increase in amorphous nature of the P(3HB-co-3HP) films with P(3HB)-homopolymer-like crystalline structure increases and then decreases the rate of degradation by depolymerase from R. pikettii T1. The 3-hydroxybutyrate (3HB) monomer was produced as a major product by the hydrolysis of P(3HB) film by PHA depolymerase from Acidovorax Sp. TP4. The P(3HB-co-3HP) films could be degraded into 3HB and 3-hydroxypropionate (3HP) monomer at last, indicating that the catalytic domain of the enzyme recognized at least two monomeric units as substrates. While the PHA depolymerase from R. pikettii T1 hydrolyzed P(3HB) film into 3HB dimer as a major product, and the catalytic domain recognized at least three monomeric units. The degradation behavior of P(3HB-co-3HP) films by the PHA depolymerase of Acidovorax Sp. TP4 could be distinguished from that by the depolymerase of R. pikettii T1.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Betaproteobacteria/enzimologia , Biodegradação Ambiental , Varredura Diferencial de Calorimetria , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Hidrólise , Hidroxibutiratos/química , Isoenzimas/metabolismo , Espectroscopia de Ressonância Magnética , Poliésteres/química , Difração de Raios XRESUMO
The genes from the oxygenase cluster nagAaGHAbAcAd of naphthalene-degrading Ralstonia sp. strain U2 were cloned and overexpressed. Salicylate 5-hydroxylase (S5H) activity, converting salicylate to gentisate, was present in vitro only in the single extract of cells with overexpressed nagAaGHAb or in a mixture of three cell extracts containing, respectively, NagGH (the oxygenase components), NagAa (ferredoxin reductase), and NagAb (ferredoxin). Each of the three extracts required for S5H activity was rate limiting in the presence of excess of the others but, when in excess, did not affect the rate of catalysis. S5H catalyzed the 5-hydroxylation of the aromatic rings of 3- and 4-substituted salicylates. However, the methyl group of 5-methylsalicylate was hydroxylated to produce the 5-hydroxymethyl derivative and the 6-position on the ring of 5-chlorosalicylate was hydroxylated, producing 5-chloro-2,6-dihydroxybenzoate. In an assay for the nag naphthalene dioxygenase (NDO) based on the indole-linked oxidation of NADH, three extracts were essential for activity (NagAcAd, NagAa, and NagAb). NDO and S5H were assayed in the presence of all possible combinations of the nag proteins and the corresponding nah NDO proteins from the "classical" naphthalene degrader P. putida NCIMB9816. All three oxygenase components functioned with mixed combinations of the electron transport proteins from either strain. The S5H from strain U2 is a unique monooxygenase which shares sequence similarity with dioxygenases such as NDO but is also sufficiently similar in structure to interact with the same electron transport chain and probably does so in vivo during naphthalene catabolism in strain U2.
Assuntos
Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Oxigenases de Função Mista/metabolismo , Dioxigenases , Transporte de Elétrons , Ferredoxina-NADP Redutase/metabolismo , Ferredoxinas/metabolismo , Oxigenases de Função Mista/genética , Complexos Multienzimáticos/metabolismo , Naftalenos/metabolismo , Oxigenases/metabolismo , Proteínas Recombinantes/metabolismo , Salicilatos/metabolismoRESUMO
The in vitro activity of imipenem and meropenem on strains of Gram-negative rods producing extended-spectrum beta-lactamase (ESBL) and inducible beta-lactamase (IBL) was studied using the Etest. In all, 185 strains from the surgical intensive care units of four different hospitals were looked at over 2 years. Of these, 94 were ESBL producers and 91 were IBL positive. The in vitro sensitivities of imipenem and meropenem were 89.7 and 95.1%, respectively, against all strains. The imipenem and meropenem sensitivities of Klebsiella spp. were 98.4 and 100%, respectively, but imipenem resistance (21.6%) in Pseudomonas aeruginosa strains was higher than that of meropenem (10.8%).
Assuntos
Farmacorresistência Bacteriana Múltipla , Bacilos e Cocos Aeróbios Gram-Negativos/efeitos dos fármacos , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Imipenem/farmacologia , Tienamicinas/farmacologia , beta-Lactamases/metabolismo , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Humanos , Meropeném , Testes de Sensibilidade Microbiana , Pseudomonadaceae/efeitos dos fármacos , Pseudomonadaceae/enzimologia , Especificidade da EspécieRESUMO
The structural gene coding for phenylserine dehydratase from Ralstonia pickettii PS22 was cloned into Escherichia coli cells, and the nucleotide sequence was identified. The predicted amino acid sequence had high sequence similarity to biodegradative and biosynthetic threonine dehydratases from E. coli and serine dehydratase from human liver. Transformed E. coli cells overproduced phenylserine dehydratase, and the recombinant enzyme was purified to homogeneity with a high yield and characterized.
Assuntos
Escherichia coli/genética , Bacilos e Cocos Aeróbios Gram-Negativos/enzimologia , Bacilos e Cocos Aeróbios Gram-Negativos/genética , Hidroliases/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Expressão Gênica , Hidroliases/química , Hidroliases/isolamento & purificação , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de DNARESUMO
The gene encoding Aquifex aeolicus (Aae) DNA polymerase was expressed under the control of the trp promoter on a high-copy plasmid, pTRPNS, in Escherichia coli. The expressed enzyme was purified 11-fold with a 13.8% yield and a specific activity of 2268.3 U mg(-1). The optimum pH of the enzyme was 6.8-7.2. The optimal concentrations of KCl and Mg(2+) were 20-30 mM and 4-5 mM, respectively. Aae DNA polymerase contained a double-strand-dependent 3'-->5' proofreading exonuclease activity but lacked any detectable 5'-->3' exonuclease activity.