Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.220
Filtrar
Mais filtros










Intervalo de ano de publicação
1.
Mol Biol Rep ; 46(1): 505-510, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30498881

RESUMO

NdmB genes from Pseudomonas putida CBB5 and GO genes from spinach, which encode N-demethylase B (NdmB) and Glycolate oxidase (GO) respectively, were separately ligated into expression vectors of pACYCDuet-1 and pET32a to construct recombinant plasmids of pACYCDuet-1-ndmBHis (pBH) and pET32a-GOHis (pGOH). Then the two plasmids were both transformed in Escherichia coli (E. coli) strain BL21 (DE3) and screening the recombinants (pBHGOH) using ampicillin and chloramphonicol as two antibiotics in Luria-Bertani medium. After induction with IPTG, both recombinant ndmB and GO genes were coexpressed in E. coli. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the estimated molecular weight of NdmB and GO was 35 kDa and 40 kDa, respectively. By two-step purification of Ni affinity chromatography and Q-Sepharose chromatography, the coexpressed NdmB and GO were separated and resulted in a 15.8-fold purification with 8.7% yield and 12.8-fold purification with 7.2% yield, respectively.


Assuntos
Oxirredutases do Álcool/genética , Oxirredutases N-Desmetilantes/genética , Oxirredutases do Álcool/isolamento & purificação , Oxirredutases do Álcool/metabolismo , Cromatografia de Afinidade/métodos , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida/métodos , Escherichia coli/genética , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos , Oxirredutases N-Desmetilantes/isolamento & purificação , Oxirredutases N-Desmetilantes/metabolismo , Plasmídeos , Pseudomonas putida/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes/genética
2.
Methods Mol Biol ; 1876: 125-140, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30317478

RESUMO

Nitrogenase-like enzymes play a vital role in the reduction of the conjugated ring systems of diverse tetrapyrrole molecules. The biosynthesis of all bacteriochlorophylls involves the two-electron reduction of the C7-C8 double bond of the green pigment chlorophyllide, which is catalyzed by the nitrogenase-like two-component metalloenzyme chlorophyllide oxidoreductase (COR); whereas in all methanogenic microbes, another nitrogenase-like system, CfbC/D, is responsible for the sophisticated six-electron reduction of Ni2+-sirohydrochlorin a,c-diamide in the course of coenzyme F430 biosynthesis. The first part of this chapter describes the production and purification of the COR components (BchY/BchZ)2 and BchX2, the measurement of COR activity, and the trapping of the ternary COR complex; and the second part describes the strategy for obtaining homogenous and catalytically active preparations of CfbC2 and CfbD2 and a suitable method for extracting the reaction product Ni2+-hexahydrosirohydrochlorin a,c-diamide.


Assuntos
Metaloproteínas/isolamento & purificação , Metaloproteínas/metabolismo , Uroporfirinas/química , Oxirredutases do Álcool/química , Oxirredutases do Álcool/isolamento & purificação , Oxirredutases do Álcool/metabolismo , Domínio Catalítico , Clorofila/biossíntese , Metaloporfirinas/metabolismo , Metaloproteínas/química , Complexos Multienzimáticos , Níquel/química , Oxirredução
3.
J Biosci Bioeng ; 127(2): 145-149, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30075940

RESUMO

The carbonyl reductase from the methylotrophic yeast Ogataea minuta can catalyze the regio- and enantio-selective reduction of prochiral ketones to chiral alcohols, and is available for industrial manufacturing of statin drugs. We previously conducted a directed evolution experiment of the enzyme, and obtained a mutant (OCR_V166A) with improved tolerance to organic solvents. This expanded the applicability of the enzyme to the bioconversion of water-insoluble compounds (Honda et al., J. Biosci. Bioeng., 123, 673-678, 2017). In the present study, we expressed OCR_V166A in Rhodococcus opacus cells, which have a highly lipophilic surface structure and are dispersible in anhydrous organic solvents, and developed a whole-cell biocatalyst which can function in an organic-solvent-based reaction medium. The secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (TeADH) was employed as an NADPH-regenerating enzyme and co-expressed with OCR_V166A in R. opacus. The whole-cell bioconversion of 2,2,2-trifluoroacetophenone to α-(trifluoromethyl)benzyl alcohol was performed in organic solvents, including isopropanol, isobutanol, and cyclohexanol, which served both as reaction media and as substrates for TeADH. The type of organic solvents markedly affected not only the product titer but also the enantio-purity of the product. When isobutanol was used as the reaction medium, the whole-cell biocatalyst showed higher stability than the isolated enzyme. Consequently, a high concentration (1 M) of the substrate was converted to the product with an overall conversion yield of 81% (mol/mol) in 24 h.


Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Biocatálise , Rhodococcus/genética , Rhodococcus/metabolismo , Leveduras/enzimologia , Oxirredutases do Álcool/isolamento & purificação , Álcoois/metabolismo , Catálise , Regulação Enzimológica da Expressão Gênica , Oxirredução , Engenharia de Proteínas , Solventes/química , Thermoanaerobacter/metabolismo , Água/química , Leveduras/genética
4.
Med Mycol J ; 59(3): E47-E52, 2018.
Artigo em Japonês | MEDLINE | ID: mdl-30175812

RESUMO

Interactions between virulence factors of pathogens and host responses play an important role in the establishment of infection by microbes. We focused on interactions between Cryptococcus neoformans proteins and heparin, which is abundant on host epithelial cells. Surface proteins were extracted and analyzed. Fractions from anion-exchange column chromatography interacted with heparin in surface plasmon resonance analyses. Heparin-binding proteins were purified and then separated by gel electrophoresis; and were identified as transaldolase, glutathione-disulfide reductase, and glyoxal oxidase. These results imply that multifunctional molecules on C. neoformans cells, such as those involved in heparin binding, may play roles in adhesion that trigger responses in the host.


Assuntos
Oxirredutases do Álcool/isolamento & purificação , Oxirredutases do Álcool/metabolismo , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/metabolismo , Cryptococcus neoformans/citologia , Cryptococcus neoformans/metabolismo , Glutationa Redutase/isolamento & purificação , Glutationa Redutase/metabolismo , Heparina/metabolismo , Transaldolase/isolamento & purificação , Transaldolase/metabolismo , Fatores de Virulência/isolamento & purificação , Fatores de Virulência/metabolismo , Cryptococcus neoformans/patogenicidade , Ligação Proteica
5.
Dalton Trans ; 47(31): 10463-10472, 2018 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-30020281

RESUMO

Interest in the bioinorganic chemistry of lanthanides is growing rapidly as more and more lanthanide-dependent bacteria are being discovered. Especially the earlier lanthanides have been shown to be preferentially utilized by bacteria that need these Lewis acids as cofactors in their alcohol dehydrogenase enzymes. Here, we investigate the impact of the lanthanide ions lanthanum(iii) to lutetium(iii) (excluding Pm) on the catalytic parameters (vmax, KM, kcat/KM) of a methanol dehydrogenase (MDH) isolated from Methylacidiphilum fumariolicum SolV. Kinetic experiments and DFT calculations were used to discuss why only the earlier lanthanides (La-Gd) promote high MDH activity. Impact of Lewis acidity, coordination number preferences, stability constants and other properties that are a direct result of the lanthanide contraction are discussed in light of the two proposed mechanisms for MDH.


Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/isolamento & purificação , Complexos de Coordenação/química , Elementos da Série dos Lantanídeos/química , Metanol/metabolismo , Catálise , Domínio Catalítico , Cinética , Estrutura Molecular , Polietilenoglicóis/química , Verrucomicrobia/metabolismo , Água/química
6.
Protein Expr Purif ; 150: 26-32, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29738827

RESUMO

Alcohol oxidase (AOX) functions in oxidation of primary alcohols into the corresponding aldehydes with potential on catalyzing synthesis reactions in chemical industry. In this study, AOX from a thermotolerant methylotrophic yeast, Ogataea thermomethanolica (OthAOX) was purified to high homogeneity using a single step chromatographic separation on a DEAE-Sepharose column. The purified OthAOX had a specific activity of 15.34 U/mg with 77.5% recovery yield. The enzyme worked optimally at 50 °C in an alkaline range (pH 9.0). According to kinetic analysis, OthAOX showed a higher affinity toward short-chain aliphatic primary alcohol with the Vmax, Km, and kcat of 0.24 nmol/min, 0.27 mM, and 3628.8 min-1, respectively against methanol. Addition of alginic acid (0.35%) showed a protective effect on enhancing thermal stability of the enzyme, resulting in 72% increase in its half-life at 40 °C under the operational conditions. This enzyme represents a promising candidate for conversion of bioethanol to acetaldehyde as secondary chemical in biorefinery.


Assuntos
Oxirredutases do Álcool , Proteínas Fúngicas , Saccharomycetales/enzimologia , Oxirredutases do Álcool/química , Oxirredutases do Álcool/isolamento & purificação , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação
7.
J Microbiol ; 56(4): 246-254, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29492864

RESUMO

The first crystal structure of a pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH) from a marine methylotrophic bacterium, Methylophaga aminisulfidivorans MPT (MDH Mas ), was determined at 1.7 Å resolution. The active form of MDH Mas (or MDHI Mas ) is a heterotetrameric α2ß2, where each ß-subunit assembles on one side of each of the α-subunits, in a symmetrical fashion, so that two ß-subunits surround the two PQQ-binding pockets on the α-subunits. The active site consists of a PQQ molecule surrounded by a ß-propeller fold for each α-subunit. Interestingly, the PQQ molecules are coordinated by a Mg2+ ion, instead of the Ca2+ ion that is commonly found in the terrestrial MDHI, indicating the efficiency of osmotic balance regulation in the high salt environment. The overall interaction of the ß-subunits with the α-subunits appears tighter than that of terrestrial homologues, suggesting the efficient maintenance of MDHI Mas integrity in the sea water environment to provide a firm basis for complex formation with MxaJ Mas or Cyt cL. With the help of the features mentioned above, our research may enable the elucidation of the full molecular mechanism of methanol oxidation by taking advantage of marine bacterium-originated proteins in the methanol oxidizing system (mox), including MxaJ, as the attainment of these proteins from terrestrial bacteria for structural studies has not been successful.


Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/isolamento & purificação , Piscirickettsiaceae/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Magnésio/metabolismo , Modelos Moleculares , Oxirredução , Cofator PQQ/metabolismo , Piscirickettsiaceae/metabolismo
8.
Molecules ; 23(1)2018 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-29342886

RESUMO

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para-substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para-phenol oxidases, facilitating the enzyme engineering of known para-phenol oxidases and the evaluation of the substrate specificity of novel para-phenol oxidases.


Assuntos
Flavinas/química , Monofenol Mono-Oxigenase/química , Fenóis/química , Sulfóxidos/química , Oxirredutases do Álcool/química , Oxirredutases do Álcool/isolamento & purificação , Ativação Enzimática , Cinética , Monofenol Mono-Oxigenase/isolamento & purificação , Proteínas Recombinantes de Fusão , Especificidade por Substrato
9.
Appl Microbiol Biotechnol ; 102(3): 1307-1316, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29238872

RESUMO

The enzyme responsible for the enantioselective production of (S)-1,1,1-trifluoro-2-propanol ((S)-TFP) from 1,1,1-trifluoroacetone (TFA) has been identified in Ogataea polymorpha NBRC 0799. We purified two carbonyl reductases, OpCRD-A and OpCRD-B from this strain, and revealed their characteristics. Both enzymes were specific to NADH, but the following characteristics were different: The molecular mass of subunit OpCRD-A was 40 kDa and that of OpCRD-B was 43 kDa. Amino acid sequences of both enzymes were only 21% identical. OpCRD-B contained 4 mol of zinc per mole of enzyme, but OpCRD-A did not. The optimal pH, temperature, pH stability, thermostability, and inhibitor specificity were also remarkably different. With regard to substrate specificity, both enzymes exhibited high reductase activity toward a wide variety of ketones, aldehydes and fluoroketones, and dehydrogenase activity toward 2-propanol and 2-butanol. The reductase activity was much higher than the dehydrogenase activity at acidic pH. OpCRD-A enantioselectively produced (S)-TFP from TFA, but OpCRD-B preferentially produced (R)-TFP. Thus, we concluded that OpCRD-A plays the main role in the production of (S)-TFP by a reaction of O. polymorpha NBRC 0799 cells and that OpCRD-A has great potential for efficient production of (S)-TFP, as it is an S-specific enzyme and does not catalyze the dehydrogenation of (S)-TFP.


Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas Fúngicas/metabolismo , Saccharomycetales/enzimologia , 2-Propanol/metabolismo , Oxirredutases do Álcool/isolamento & purificação , Proteínas Fúngicas/isolamento & purificação , Concentração de Íons de Hidrogênio , Cetonas/metabolismo , Cinética , Peso Molecular , Oxirredução , Especificidade por Substrato , Temperatura , Ácido Trifluoracético/metabolismo
10.
Plant Physiol ; 175(1): 51-61, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28705827

RESUMO

In plants, amino acid catabolism is especially relevant in metabolic stress situations (e.g. limited carbohydrate availability during extended darkness). Under these conditions, amino acids are used as alternative substrates for respiration. Complete oxidation of the branched-chain amino acids (BCAAs) leucine, isoleucine (Ile), and valine (Val) in the mitochondria efficiently allows the formation of ATP by oxidative phosphorylation. However, the metabolic pathways for BCAA breakdown are largely unknown so far in plants. A systematic search for Arabidopsis (Arabidopsis thaliana) genes encoding proteins resembling enzymes involved in BCAA catabolism in animals, fungi, and bacteria as well as proteomic analyses of mitochondrial fractions from Arabidopsis allowed the identification of a putative 3-hydroxyisobutyrate dehydrogenase, AtHDH1 (At4g20930), involved in Val degradation. Systematic substrate screening analyses revealed that the protein uses 3-hydroxyisobutyrate but additionally 3-hydroxypropionate as substrates. This points to a role of the enzyme not only in Val but possibly also in Ile metabolism. At4g20930 knockdown plants were characterized to test this conclusion. Root toxicity assays revealed increased root growth inhibition of the mutants if cultivated in the presence of Val or Ile but not in the presence of leucine. We conclude that AtHDH1 has a dual role in BCAA metabolism in plants.


Assuntos
Oxirredutases do Álcool/metabolismo , Arabidopsis/enzimologia , Isoleucina/metabolismo , Valina/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/isolamento & purificação , Escherichia coli , Técnicas de Silenciamento de Genes , Proteínas de Plantas/metabolismo
11.
Bioorg Med Chem ; 25(1): 116-125, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28340986

RESUMO

Arachidonic acid (AA) is converted to biologically active metabolites by different pathways, one of the most important of which is initiated by 5-lipoxygenase (5-LO). 5-Hydroxyeicosatetraenoic acid (5-HETE), although possessing only weak biological activity itself, is oxidized to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent chemoattractant for eosinophils and neutrophils. Our main goal is to determine how the biosynthesis of 5-oxo-ETE is regulated and to determine its pathophysiological roles. To achieve this task, we designed and synthesized affinity chromatography ligands for the purification of 5-hydroxyeicosanoid dehydrogenase (5-HEDH), the enzyme responsible for the formation of 5-oxo-ETE.


Assuntos
Oxirredutases do Álcool/isolamento & purificação , Cromatografia de Afinidade/métodos , Oxirredutases do Álcool/metabolismo , Ácidos Araquidônicos/metabolismo , Linhagem Celular , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ligantes , Neutrófilos/metabolismo
12.
J Biotechnol ; 241: 69-75, 2017 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-27836796

RESUMO

Formaldehyde dismutase (FDM) is a very interesting enzyme, due to the fact that it comprises an internal cofactor regeneration mechanism. The FDM, therefore, is able to catalyze redox reactions independent of exogenous cofactor addition, rendering the enzyme powerful for industrial applications. Currently, only one enzyme of this type has been characterized enzymatically. Furthermore, only one additional DNA-sequence with high homology to FDM has been published. In this work, we identified a new variant of a formaldehyde dismutase gene (fdm) in the Pseudomonas putida J3 strain. To isolate and characterize the enzyme, we developed a simplified method for its purification. This purification is based on a C-terminal 6xHis-tag, which enables functional expression of the enzyme in E. coli and a one-step purification method. In addition, we tested several expression systems for optimal yields and combined this with co-expression of the chaperonins GroESL. Using this simplified and rapid method, we are now able to produce sufficient material in reproducible quality and quantity for application tests with the enzyme. The newly identified enzyme will be applied in a redox cascade for biomethanol production from biogas and shows potential for further industrial biotransformation with integrated cofactor recycling.


Assuntos
Oxirredutases do Álcool/isolamento & purificação , Escherichia coli/genética , Pseudomonas putida/enzimologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Clonagem Molecular , Pseudomonas putida/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
13.
Sci Rep ; 6: 37356, 2016 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-27869125

RESUMO

Glucose oxidase (GO) activity is generally restricted to glucose and is susceptible to inactivation by H2O2. By comparison, the Y300A variant of gluco-oligosaccharide oxidase (GOOX) from Sarocladium strictum showed broader substrate range and higher H2O2 stability. Specifically, Y300A exhibited up to 40 times higher activity on all tested sugars except glucose, compared to GO. Moreover, fusion of the Y300A variant to a family 22 carbohydrate binding module from Clostridium thermocellum (CtCBM22A) nearly doubled its catalytic efficiency on glucose, while retaining significant activity on oligosaccharides. In the presence of 200 mM of H2O2, the recombinant CtCBM22A_Y300A retained 80% of activity on glucose and 100% of activity on cellobiose, the preferred substrate for this enzyme. By contrast, a commercial glucose oxidase reported to contain ≤0.1 units catalase/ mg protein, retained 60% activity on glucose under the same conditions. GOOX variants appear to undergo a different mechanism of inactivation, as a loss of histidine instead of methionine was observed after H2O2 incubation. The addition of CtCBM22A also promoted functional binding of the fusion enzyme to xylan, facilitating its simultaneous purification and immobilization using edible oat spelt xylan, which might benefit the usage of this enzyme preparation in food and baking applications.


Assuntos
Oxirredutases do Álcool/química , Proteínas Fúngicas/química , Glucose Oxidase/química , Peróxido de Hidrogênio/química , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/isolamento & purificação , Substituição de Aminoácidos , Ascomicetos/enzimologia , Domínio Catalítico , Estabilidade Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Glucose Oxidase/genética , Glucose Oxidase/isolamento & purificação , Cinética , Modelos Moleculares , Oligossacarídeos/química , Oxirredução , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Especificidade por Substrato
14.
Appl Microbiol Biotechnol ; 100(24): 10573-10583, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27687994

RESUMO

The stereochemistry of 2,3-butanediol (2,3-BD) synthesis in microbial fermentations is important for many applications. In this work, we showed that Corynebacterium glutamicum endowed with the Lactococcus lactis genes encoding α-acetolactate synthase and decarboxylase activities produced meso-2,3-BD as the major end product, meaning that (R)-acetoin is a substrate for endogenous 2,3-butanediol dehydrogenase (BDH) activity. This is curious in view of the reported absolute stereospecificity of C. glutamicum BDH for (S)-acetoin (Takusagawa et al. Biosc Biotechnol Biochem 65:1876-1878, 2001). To resolve this discrepancy, the enzyme encoded by butA Cg was produced in Escherichia coli and purified, and the stereospecific properties of the pure protein were examined. Activity assays monitored online by 1H-NMR using racemic acetoin and an excess of NADH showed an initial, fast production of (2S,3S)-2,3-BD, followed by a slow (∼20-fold lower apparent rate) formation of meso-2,3-BD. Kinetic parameters for (S)-acetoin, (R)-acetoin, meso-2,3-BD and (2S,3S)-BD were determined by spectrophotometric assays. V max values for (S)-acetoin and (R)-acetoin were 119 ± 15 and 5.23 ± 0.06 µmol min-1 mg protein-1, and K m values were 0.23 ± 0.02 and 1.49 ± 0.07 mM, respectively. We conclude that C. glutamicum BDH is not absolutely specific for (S)-acetoin, though this is the preferred substrate. Importantly, the low activity of BDH with (R)-acetoin was sufficient to support high yields of meso-2,3-BD in the engineered strain C. glutamicum ΔaceEΔpqoΔldhA(pEKEx2-als,aldB,butA Cg ). Additionally, we found that the BDH activity was nearly abolished upon inactivation of butA Cg (from 0.30 ± 0.03 to 0.004 ± 0.001 µmol min-1 mg protein-1), indicating that C. glutamicum expresses a single BDH under the experimental conditions examined.


Assuntos
Oxirredutases do Álcool/metabolismo , Butileno Glicóis/metabolismo , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/metabolismo , Engenharia Metabólica , Acetoína/metabolismo , Acetolactato Sintase/genética , Acetolactato Sintase/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/isolamento & purificação , Carboxiliases/genética , Carboxiliases/metabolismo , Corynebacterium glutamicum/genética , Escherichia coli/genética , Lactococcus lactis/enzimologia , Lactococcus lactis/genética , Espectroscopia de Ressonância Magnética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
15.
Appl Environ Microbiol ; 82(21): 6378-6385, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27542933

RESUMO

Most plant-produced monoterpenes can be degraded by soil microorganisms. Borneol is a plant terpene that is widely used in traditional Chinese medicine. Neither microbial borneol dehydrogenase (BDH) nor a microbial borneol degradation pathway has been reported previously. One borneol-degrading strain, Pseudomonas sp. strain TCU-HL1, was isolated by our group. Its genome was sequenced and annotated. The genome of TCU-HL1 consists of a 6.2-Mbp circular chromosome and one circular plasmid, pTHL1 (12.6 kbp). Our results suggest that borneol is first converted into camphor by BDH in TCU-HL1 and is further decomposed through a camphor degradation pathway. The recombinant BDH was produced in the form of inclusion bodies. The apparent Km values of refolded recombinant BDH for (+)-borneol and (-)-borneol were 0.20 ± 0.01 and 0.16 ± 0.01 mM, respectively, and the kcat values for (+)-borneol and (-)-borneol were 0.75 ± 0.01 and 0.53 ± 0.01 s-1, respectively. Two plant BDH genes have been reported previously. The kcat and kcat/Km values of lavender BDH are about 1,800-fold and 500-fold lower, respectively, than those of TCU-HL1 BDH. IMPORTANCE: The degradation of borneol in a soil microorganism through a camphor degradation pathway is reported in this study. We also report a microbial borneol dehydrogenase. The kcat and kcat/Km values of lavender BDH are about 1,800-fold and 500-fold lower, respectively, than those of TCU-HL1 BDH. The indigenous borneol- and camphor-degrading strain isolated, Pseudomonas sp. strain TCU-HL1, reminds us of the time 100 years ago when Taiwan was the major producer of natural camphor in the world.


Assuntos
Oxirredutases do Álcool/metabolismo , Cânfora/metabolismo , Pseudomonas/enzimologia , Oxirredutases do Álcool/isolamento & purificação , Biocatálise , Biodegradação Ambiental , Isomerismo , Cinética , Oxirredução , Extratos Vegetais , Pseudomonas/metabolismo
16.
J Biotechnol ; 234: 50-57, 2016 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-27480343

RESUMO

The gene xylB from Caulobacter crescentus has been cloned and expressed in Escherichia coli providing a high yield of xylose dehydrogenase (XylB) production and excellent purity (97%). Purified recombinant XylB showed an absolute dependence on the cofactor NAD(+) and a strong preference for d-xylose against other assayed mono and disaccharides. Additionally, XylB showed strong stability when stored as freeze-dried powder at least 250days both at 4°C and room temperature. In addition, more than 80% of the initial activity of rehydrated freeze-dried enzyme remained after 150days of incubation at 4°C. Based on these characteristics, the capability of XylB in d-xylose detection and quantification was studied. The linearity of the method was maintained up to concentrations of d-xylose of 10mg/dL and the calculated limits of detection (LoD) and quantification (LoQ) of xylose in buffer were 0.568mg/dL and 1.89mg/dL respectively. Thus, enzymatic detection was found to be an excellent method for quantification of d-xylose in both buffer and urine samples. This method can easily be incorporated in a new test for the diagnosis of hypolactasia through the measurement of intestinal lactase activity.


Assuntos
Oxirredutases do Álcool/metabolismo , Caulobacter crescentus/enzimologia , Xilose/urina , Oxirredutases do Álcool/biossíntese , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/isolamento & purificação , Caulobacter crescentus/genética , Ativação Enzimática , Estabilidade Enzimática , Escherichia coli/genética , Humanos , Cinética , Limite de Detecção , Espectrometria de Massas , NAD/metabolismo , Oligossacarídeos/análise , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação
17.
Appl Microbiol Biotechnol ; 100(18): 8021-30, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27138199

RESUMO

Aryl-alcohol oxidases (AAOs) are enzymes supporting the degradation of lignin by fungal derived class II peroxidases produced by white-rot fungi. AAOs are able to generate H2O2 as a by-product via oxidation of an aryl-alcohol into its correspondent aldehyde. In this study, an AAO was heterologously expressed in a basidiomycete host for the first time. The gene for an AAO of the white-rot fungus Pleurotus sapidus, a close relative to the oyster mushroom Pleurotus ostreatus, was cloned into an expression vector and put under control of the promotor of the glyceraldehyde-3-phosphate dehydrogenase gene 2 (gpdII) of the button mushroom Agaricus bisporus. The expression vector was transformed into the model basidiomycete Coprinopsis cinerea, and several positive transformants were obtained. The best producing transformants were grown in shake-flasks and in a stirred tank reactor reaching enzymatic activities of up to 125 U L(-1) using veratryl alcohol as a substrate. The purified AAO was biochemically characterized and compared to the previously described native and recombinant AAOs from other Pleurotus species. In addition, a two-enzyme system comprising a dye-decolorizing peroxidase (DyP) from Mycetinis scorodonius and the P. sapidus AAO was successfully employed to bleach the anthraquinone dye Reactive Blue 5.


Assuntos
Oxirredutases do Álcool/metabolismo , Pleurotus/enzimologia , Proteínas Recombinantes/metabolismo , Agaricales/genética , Agaricales/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/isolamento & purificação , Antraquinonas/metabolismo , Clonagem Molecular , Expressão Gênica , Peroxidase/metabolismo , Pleurotus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
18.
Biosci Biotechnol Biochem ; 80(9): 1753-8, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27121905

RESUMO

From investigation of 60 filamentous fungi, we identified Fusarium merismoides var. acetilereum, which uses 4-N-trimethylamino-1-butanol (TMA-butanol) as the sole source of carbon and nitrogen. The fungus produced NAD(+)-dependent TMA-butanol dehydrogenase (DH) when it was cultivated in medium containing TMA-butanol. The enzyme showed molecular mass of 40 kDa by SDS-PAGE and 160 kDa by gel filtration, suggesting that it is a homotetramer. TMA-butanol DH is stable at pH 7.5-9.0. It exhibits moderate stability with respect to temperature (up to 30 °C). Additionally, it has optimum activity at 45 °C and at pH 9.5. The enzyme has broad specificity to various alkyl alcohols and amino alkyl alcohols, and the carbon chains of which are longer than butanol. Moreover, the activity is strongly inhibited by oxidizing agents, carbonyl and thiol modulators, and chelating agents. This report is the first study examining TMA-butanol DH from eukaryotic microbes.


Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/isolamento & purificação , Fusarium/enzimologia , Oxirredutases do Álcool/genética , Amino Álcoois/química , Carbono/química , Fusarium/química , Concentração de Íons de Hidrogênio , Cinética , Especificidade por Substrato , Temperatura
19.
Lett Appl Microbiol ; 61(6): 573-9, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26393961

RESUMO

UNLABELLED: Acetoin and 2,3-butanediol are widely used in the chemical and pharmaceutical industries. The enzyme, 2,3-butanediol dehydrogenase/acetoin reductase (2,3-BDH/AR), plays a significant role in the microbial production of acetoin and 2,3-butanediol by catalysing a reversible reaction between acetoin and 2,3-butanediol. To date, a 2,3-BDH has not been characterized from Corynebacterium crenatum. 2,3-BDH was cloned from Coryne. crenatum SYPA5-5 and expressed in Escherichia coli BL21. Sequence analysis suggested that the 2,3-BDH from Coryne. crenatum SYPA5-5 belongs to the short-chain dehydrogenase/reductase superfamily. Its maximum specific activity was obtained at 35°C, however, it became very unstable when the temperature was above 35°C. Its optimal pH was 4·0 for reduction reaction and 10·0 for oxidation reaction. The 2,3-BDH activity was increased to some extent by Ca(2+) , Mg(2+) , Zn(2+) and Mn(2+) ions. In particular, Ca(2+) induced about 1·5-fold increase. The value of kcat /Km for diacetyl and acetoin are higher than for 2,3-butanediol indicating that 2,3-BDH can easily reduce diacetyl or acetoin to 2,3-butanediol under lower pH conditions. The characteristics of 2,3-BDH from Coryne. crenatum SYPA5-5 will give guide to further studies for the production of acetoin and 2,3-butanediol with engineered Coryne. crenatum SYPA5-5. SIGNIFICANCE AND IMPACT OF THE STUDY: Acetoin and 2,3-butanediol are commonly used as platform chemicals and widely used in pharmaceutical industries. 2,3-butanediol dehydrogenase/acetoin reductase (2,3-BDH/AR) plays a significant role in the microbial production of acetoin and 2,3-butanediol. In this study, 2,3-BDH was cloned from Corynebacterium crenatum SYPA5-5, was expressed in Escherichia coli BL21 and characterized with respect to the optimal temperature, pH, substrate specificity and kinetics. The results will guide further studies in Coryne. crenatum SYPA5-5 for the production of acetoin and 2,3-butanediol.


Assuntos
Acetoína/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/isolamento & purificação , Butileno Glicóis/metabolismo , Corynebacterium/enzimologia , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Corynebacterium/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Dados de Sequência Molecular , Alinhamento de Sequência , Especificidade por Substrato , Temperatura
20.
Free Radic Biol Med ; 83: 66-76, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25680283

RESUMO

An NADP(+)-dependent dehydrogenase activity on 3-glutathionyl-4-hydroxynonanal (GSHNE) was purified to electrophoretic homogeneity from a line of human astrocytoma cells (ADF). Proteomic analysis identified this enzymatic activity as associated with carbonyl reductase 1 (EC 1.1.1.184). The enzyme is highly efficient at catalyzing the oxidation of GSHNE (KM 33 µM, kcat 405 min(-1)), as it is practically inactive toward trans-4-hydroxy-2-nonenal (HNE) and other HNE-adducted thiol-containing amino acid derivatives. Combined mass spectrometry and nuclear magnetic resonance spectroscopy analysis of the reaction products revealed that carbonyl reductase oxidizes the hydroxyl group of GSHNE in its hemiacetal form, with the formation of the corresponding 3-glutathionylnonanoic-δ-lactone. The relevance of this new reaction catalyzed by carbonyl reductase 1 is discussed in terms of HNE detoxification and the recovery of reducing power.


Assuntos
Oxirredutases do Álcool/metabolismo , Aldeídos/metabolismo , Astrocitoma/metabolismo , Glutationa/análogos & derivados , Glutationa/metabolismo , Inativação Metabólica , NADPH Desidrogenase/metabolismo , NADP/metabolismo , Oxirredutases do Álcool/isolamento & purificação , Aldeído Redutase/metabolismo , Astrocitoma/patologia , Humanos , Lactonas/metabolismo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Oxirredução , Proteômica , Especificidade por Substrato , Compostos de Sulfidrila/metabolismo , Células Tumorais Cultivadas
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA