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1.
Arch Microbiol ; 176(3): 178-86, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11511865

RESUMO

Mycelial cultures of 76 strains of lignocellulose-degrading basidiomycete fungi were screened for the activity of pyranose dehydrogenase, a novel sugar oxidoreductase recently detected in Agaricus bisporus. Of these fungi, 37 strains belonging to seven phylogenetically related genera of mostly litter-decomposing Agaricales were positive for the dehydrogenase, based on activity assays towards D-glucose with 1,4-benzoquinone or ferricenium ion as electron acceptors, and on TLC/HPLC analyses of the reaction products. Lack of activity with O(2) as the oxidant, specificity for C-3 of D-glucose, and active extracellular secretion of the enzyme were used as criteria to differentiate pyranose dehydrogenase from pyranose 2-oxidase (EC 1.1.3.10), known to be produced by numerous wood-rotting fungi. Extracellular pyranose dehydrogenase from Macrolepiota rhacodes was heavily glycosylated. The enzyme was characterized as a 78-kDa flavoprotein under denaturing conditions and a 76-kDa native protein using gel filtration. This enzyme had a maximum extracellular activity of 4.1 U ml(-1) in 39-day liquid cultures. It exhibited broad selectivity for sugar substrates and oxidized D-glucose (K(m)=1.82) exclusively at C-3 to 3-dehydro-D-glucose (D-ribo-hexos-3-ulose), in contrast to pyranose dehydrogenases from Agaricus species, which acted at both C-3 and C-2 of D-glucose. The N-terminal sequence, AVVYRHPDEL, showed significant similarity with that reported for A. bisporus.


Assuntos
Basidiomycota/enzimologia , Desidrogenases de Carboidrato/metabolismo , Metabolismo dos Carboidratos , Flavoproteínas/metabolismo , Oxirredutases/metabolismo , Basidiomycota/crescimento & desenvolvimento , Benzoquinonas/metabolismo , Desidrogenases de Carboidrato/análise , Desidrogenases de Carboidrato/química , Celulose/metabolismo , Flavoproteínas/análise , Flavoproteínas/química , Glucose/metabolismo , Cinética , Lignina/metabolismo , Peso Molecular , Oxirredutases/análise , Oxirredutases/química , Raízes de Plantas/microbiologia
2.
Appl Environ Microbiol ; 67(8): 3636-44, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11472941

RESUMO

We purified an intracellular pyranose oxidase from mycelial extracts of the white rot fungus Trametes multicolor by using ammonium sulfate fractionation, hydrophobic interaction, ion-exchange chromatography, and gel filtration. The native enzyme has a molecular mass of 270 kDa as determined by equilibrium ultracentrifugation and is composed of four identical 68-kDa subunits as determined by matrix-assisted laser desorption ionization mass spectrometry. Each subunit contains one covalently bound flavin adenine dinucleotide as its prosthetic group. The enzyme oxidizes several aldopyranoses specifically at position C-2, and its preferred electron donor substrates are D-glucose, D-xylose, and L-sorbose. During this oxidation reaction electrons are transferred to oxygen, yielding hydrogen peroxide. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones, and 2,6-dichloroindophenol, as well as the one-electron reduction of the ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)] cation radical. As judged by the catalytic efficiencies (k(cat)/K(m)), some of these quinone electron acceptors are much better substrates for pyranose oxidase than oxygen. The optimum pH of the pyranose oxidase-catalyzed reaction depends strongly on the electron acceptor employed and varies from 4 to 8. It has been proposed that the main metabolic function of pyranose oxidase is as a constituent of the ligninolytic system of white rot fungi that provides peroxidases with H(2)O(2). An additional function could be reduction of quinones, key intermediates that are formed during mineralization of lignin.


Assuntos
Basidiomycota/enzimologia , Desidrogenases de Carboidrato , Sequência de Aminoácidos , Basidiomycota/crescimento & desenvolvimento , Desidrogenases de Carboidrato/química , Desidrogenases de Carboidrato/isolamento & purificação , Desidrogenases de Carboidrato/metabolismo , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Temperatura
3.
Carbohydr Res ; 329(1): 219-25, 2000 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-11086703

RESUMO

Pyranose dehydrogenase purified to homogeneity from the mycelia of the basidiomycete fungus Agaricus bisporus catalyzed the oxidation of D-xylose at C-2 to D-threo-pentos-2-ulose (2-keto-D-xylose) and successively at C-3 to D-glycero-pentos-2,3-diulose (2,3-diketo-D-xylose) using 1,4-benzoquinone as an electron acceptor. The sites of oxidation were deduced from the spectroscopic analysis (MS, NMR) of the N,N-diphenylhydrazone derivatives of the reaction products.


Assuntos
Agaricus/enzimologia , Desidrogenases de Carboidrato/metabolismo , Xilose/metabolismo , Desidrogenases de Carboidrato/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Hidrazonas/química , Cetonas/química , Cetonas/metabolismo , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Oxirredução , Xilose/análogos & derivados , Xilose/química
5.
Appl Microbiol Biotechnol ; 47(5): 508-14, 1997 May.
Artigo em Inglês | MEDLINE | ID: mdl-9210340

RESUMO

Pyranose 2-oxidase (P2O) was purified 43-fold to apparent homogeneity from the basidiomycete Phanerochaete chrysosporium using liquid chromatography on phenyl Sepharose, Mono Q (twice) and phenyl Superose. The native enzyme has a molecular mass of about 250 kDa (based on native PAGE) and is composed of four identical subunits of 65 kDa. It contains three isoforms of isoelectric point (pI) 5.0, 5.05 and 5.15 and does not appear to be a glycoprotein. P2O is optimally stable at pH 8.0 and up to 60 degrees C. It is active over a broad pH range (5.0-9.0) with maximum activity at pH 8.0-8.5 and at 55 degrees C, and a broad substrate specificity. D-Glucose is the preferred substrate, but 1-beta-aurothioglucose, 6-deoxy-D-glucose, L-sorbose, D-xylose, 5-thioglucose, D-glucono-1,5-lactone, maltose and 2-deoxy-D-glucose are also oxidised at relatively high rates. A Ping Pong Bi Bi mechanism was demonstrated for the P2O reaction at pH 8.0, with a catalytic constant (kcat) of 111.0 s-1 and an affinity constant (Km) of 1.43 mM for D-glucose and 83.2 microM for oxygen. Whereas the steady-state kinetics for glucose oxidation were unaffected by the medium at pH > or = 7.0, at low pH both pH and buffer composition affected the P2O kinetics with the kcat/K(m) value decreasing with decreasing pH. The greatest effect was observed in acetate buffer (0.1 M, pH 4.5), where the kcat decreased to 60.9 s-1 and the K(m) increased to 240 mM. The activity of P2O was completely inhibited by 10 mM HgCl2, AgNO3 and ZnCl2, and 50% by lead acetate, CuCl2 and MnCl2.


Assuntos
Basidiomycota/enzimologia , Desidrogenases de Carboidrato/metabolismo , Desidrogenases de Carboidrato/isolamento & purificação , Concentração de Íons de Hidrogênio , Cinética , Temperatura
6.
Arch Microbiol ; 167(2-3): 119-25, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9133318

RESUMO

A novel C-2-specific sugar oxidoreductase, tentatively designated as pyranose 2-dehydrogenase, was purified 68-fold to apparent homogeneity (16.4 U/mg protein) from the mycelia of Agaricus bisporus, which expressed maximum activity of the enzyme during idiophasic growth in liquid media. Using 1,4-benzoquinone as an electron acceptor, pyranose 2-dehydrogenase oxidized d-glucose to d-arabino-2-hexosulose (2-dehydroglucose, 2-ketoglucose), which was identified spectroscopically through its N,N-diphenylhydrazone. The enzyme is highly nonspecific. d-,l-Arabinose, d-ribose, d-xylose, d-galactose, and several oligosaccharides and glycopyranosides were all converted to the corresponding 2-aldoketoses (aldosuloses) as indicated by TLC. d-Glucono-1,5-lactone, d-arabino-2-hexosulose, and l-sorbose were also oxidized at significant rates. UV/VIS spectrum of the native enzyme (lambdamax 274, 362, and 465 nm) was consistent with a flavin prosthetic group. In contrast to oligomeric intracellular pyranose 2-oxidase (EC 1.1.3.10), pyranose 2-dehydrogenase is a monomeric glycoprotein (pI 4.2) incapable of reducing O2 to H2O2 (> 5 x 10(4)-fold lower rate using a standard pyranose oxidase assay); pyranose 2-dehydrogenase is actively secreted into the extracellular fluid (up to 0.5 U/ml culture filtrate). The dehydrogenase has a native molecular mass of approximately 79 kDa as determined by gel filtration; its subunit molecular mass is approximately 75 kDa as estimated by SDS-PAGE. Two pH optima of the enzyme were found, one alkaline at pH 9 (phosphate buffer) and the other acidic at pH 4 (acetate buffer). Ag+, Hg2+, Cu2+, and CN- (10 mM) were inhibitory, while 50 mM acetate had an activating effect.


Assuntos
Agaricus/enzimologia , Desidrogenases de Carboidrato/isolamento & purificação , Desidrogenases de Carboidrato/metabolismo , Cetoses , Arabinose/metabolismo , Benzoquinonas/farmacologia , Desidrogenases de Carboidrato/antagonistas & inibidores , Cobre/farmacologia , Cianetos/farmacologia , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos , Galactose/metabolismo , Gluconatos/metabolismo , Glucose/análogos & derivados , Glucose/metabolismo , Glicosídeos/metabolismo , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Lactonas , Mercúrio/farmacologia , Oligossacarídeos/metabolismo , Oxirredução , Oxigênio/metabolismo , Ribose/metabolismo , Prata/farmacologia , Sorbose/metabolismo , Xilose/metabolismo
7.
Arch Microbiol ; 165(6): 421-4, 1996 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8661938

RESUMO

Two d-glucose-oxidizing enzymes, glucose 1-oxidase (G1O) and pyranose 2-oxidase (P2O, glucose 2-oxidase), have been proposed to play an important role in the ligninolytic system of the white rot fungus Phanerochaete chrysosporium by producing hydrogen peroxide. The possible simultaneous expression and metabolic cooperation of the two oxidases was studied in strains ME-446 (reported as G1O positive) and K-3 (P2O positive) grown in liquid media and under near natural conditions on birch wood blocks. The presence of G1O and P2O in extracts from mycelia and decayed wood was determined by chromatographic, electrophoretic, and immunological methods. Attempts to separate these enzymes and to detect G1O and its reaction product, d-glucono-1,5-lactone, failed. Evidence was obtained only for P2O expression in both strains. Accordingly, P2O, rather than G1O, represents a major source of sugar-derived H2O2 under the culture conditions used.


Assuntos
Basidiomycota/metabolismo , Glucose Oxidase/metabolismo , Lignina/metabolismo , Cromatografia Líquida de Alta Pressão , Peróxido de Hidrogênio/metabolismo
8.
Appl Environ Microbiol ; 60(7): 2524-32, 1994 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16349330

RESUMO

The production of the H(2)O(2)-generating enzyme pyranose oxidase (POD) (EC 1.1.3.10) (synonym, glucose 2-oxidase), two ligninolytic peroxidases, and laccase in wood decayed by three white rot fungi was investigated by correlated biochemical, immunological, and transmission electron microscopic techniques. Enzyme activities were assayed in extracts from decayed birch wood blocks obtained by a novel extraction procedure. With the coupled peroxidase-chromogen (3-dimethylaminobenzoic acid plus 3-methyl-2-benzothiazolinone hydrazone hydrochloride) spectrophotometric assay, the highest POD activities were detected in wood blocks degraded for 4 months and were for Phanerochaete chrysosporium (149 mU g [dry weight] of decayed wood), Trametes versicolor (45 mU g), and Oudemansiella mucida (1.2 mU g), corresponding to wood dry weight losses of 74, 58, and 13%, respectively. Mn-dependent peroxidase activities in the same extracts were comparable to those of POD, while lignin peroxidase activity was below the detection limit for all fungi with the veratryl alcohol assay. Laccase activity was high with T. versicolor (422 mU g after 4 months), in trace levels with O. mucida, and undetectable in P. chrysosporium extracts. Evidence for C-2 specificity of POD was shown by thin-layer chromatography detection of 2-keto-d-glucose as the reaction product. By transmission electron microscopy-immunocytochemistry, POD was found to be preferentially localized in the hyphal periplasmic space of P. chrysosporium and O. mucida and associated with membranous materials in hyphae growing within the cell lumina or cell walls of partially and highly degraded birch fibers. An extracellular distribution of POD associated with slime coating wood cell walls was also noted. The periplasmic distribution in hyphae and extracellular location of POD are consistent with the reported ultrastructural distribution of H(2)O(2)-dependent Mn-dependent peroxidases. This fact and the dominant presence of POD and Mn-dependent peroxidase in extracts from degraded wood suggest a cooperative role of the two enzymes during white rot decay by the test fungi.

10.
Arch Microbiol ; 160(1): 27-34, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-8352649

RESUMO

Pyranose oxidase and pyranosone dehydratase (aldos-2-ulose dehydratase), enzymes which convert in coupled reactions D-glucose to beta-pyrone cortalcerone, peaked coincidently during idiophasic growth of Phanerochaete chrysosporium under agitated conditions. The enzymes were purified from mycelial extracts of the fungus and separated from each other by hydrophobic interaction chromatography on Phenyl-Sepharose and Phenyl-Superose. Two pyranosone dehydratase activity peaks, PD I and PD II, were resolved. The major PD I fraction, consisting about 74% of the total dehydratase activity, was further purified by anion exchange chromatography on Mono Q to yield apparently pure enzyme as judged by SDS-PAGE and gel filtration on Superose 12. Isoelectric focusing indicated microheterogeneity of the protein by the presence of at least five protein bands with pI 5.1-5.3. PD II had a pI of 5.75. Overall PD I purification was 60.7-fold with 50% yield. The enzyme acted on several osones (glycosuloses), with the preferred substrate being D-glucosone. D-Xylosone and 6-deoxy-D-glucosone were dehydrated at C-3-C-4 to give the corresponding 5-hydroxy-2,3-dioxoalcanals (4-deoxy-2,3-glycosdiuloses), new enzymatically produced sugar derivatives. The latter labile compounds were trapped as diphenylhydrazine or o-phenylenediamine derivatives and spectroscopically identified. The analogous D-glucosone dehydration product did not accumulate due to its further transformation. pH optimum of PD I activity was 6.0 and its pH stability was optimal at pH 7-11. The enzyme was sensitive to Me2+ chelating agents and some heavy metal ions (Hg2+, Cu2+).


Assuntos
Basidiomycota/enzimologia , Desoxiglucose/análogos & derivados , Hidroliases/isolamento & purificação , Cetoses/metabolismo , Desidrogenases de Carboidrato/biossíntese , Quelantes/farmacologia , Cromatografia por Troca Iônica , Desoxiglucose/biossíntese , Desoxiglucose/metabolismo , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Temperatura Alta , Hidroliases/biossíntese , Hidroliases/química , Hidroliases/metabolismo , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Cetoses/biossíntese , Especificidade por Substrato
11.
Folia Microbiol (Praha) ; 38(1): 40-2, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-8500779

RESUMO

Several dicarbonyl and tricarbonyl sugars were prepared by the use of fungal enzymes and the antimicrobial effects of their N,N-diphenylhydrazine derivatives were tested. G+ bacteria were more sensitive than G- bacteria especially in the group of disubstituted compounds. Peracetyled derivatives were not active. No inhibition of yeast growth was found.


Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Cetoses/farmacologia , Fenil-Hidrazinas/farmacologia , Cetoses/metabolismo , Testes de Sensibilidade Microbiana , Fenil-Hidrazinas/metabolismo
12.
Appl Environ Microbiol ; 58(11): 3667-76, 1992 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16348809

RESUMO

The ultrastructural distribution of the sugar-oxidizing enzyme pyranose 2-oxidase (POD) in hyphae of Phanerochaete chrysosporium K-3 grown under liquid culture conditions optimal for the enzyme's production was studied by transmission electron microscopy immunocytochemistry. Using the 3-dimethylaminobenzoic acid-3-methyl-2-benzothiazolinone hydrazone hydrochloride H(2)O(2) peroxidase spectrophotometric assay, POD was detected in mycelial extracts from days 7 to 18, with maximum activity recorded on day 12. Onset of POD activity occurred in the secondary phase of hyphal development at a time of stationary growth, glucose limitation, and pH increase. POD was also detected extracellularly in the culture fluid from days 7 to 18, with maximum activity recorded on day 13. At early stages of development (3 to 4 days), using anti-POD antibodies and immunogold labeling, POD was localized in multivesicular and electron-dense bodies and in cell membrane regions. After 10 to 12 days of growth, at maximum POD activity, POD was concentrated within the periplasmic space where it was associated with membrane-bound vesicles and other membrane structures. At later stages of development (17 to 18 days), when the majority of hyphae were lysed, POD was observed associated with residual intracellular membrane systems and vesicles. Transmission electron microscopy immunocytochemical studies also demonstrated an extracellular distribution of the enzyme at the stationary growth phase, showing its association with fungal extracellular slime. In studies of ligninolytic cultures of the same fungus, POD was found to have a similar intracellular and extracellular distribution in slime as that recorded for cultures grown with cornsteep. POD's peripheral cytoplasmic distribution shows similarities to the cellular distribution of that reported previously for H(2)O(2)-dependent lignin and manganese peroxidases in P. chrysosporium.

13.
Enzyme Microb Technol ; 13(9): 755-9, 1991 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-1367889

RESUMO

Immobilization of pyranose oxidase (E.C.1.1.3.10) from Phanerochaete chrysosporium is described. The enzyme was bound to a glass-beaded support according to the glutardialdehyde, diazo, and carbodiimide methods with activity yields of 10%-23.3%. Characterization of the enzyme immobilized with the glutardialdehyde showed enhanced operational, storage, and temperature stability. The temperature optimum remained unchanged, but the pH optimum was slightly altered. Kinetic properties and the relative substrate specificities for glucose and xylose showed certain differences.


Assuntos
Agaricales/enzimologia , Desidrogenases de Carboidrato/metabolismo , Enzimas Imobilizadas/metabolismo , Desidrogenases de Carboidrato/isolamento & purificação , Cromatografia em Gel/métodos , Estabilidade Enzimática , Cinética
14.
J Gen Microbiol ; 135(12): 3311-8, 1989 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-2561488

RESUMO

Streptomyces fradiae has two chromatographically distinct forms of glutamate dehydrogenase (GDH): one GDH utilizes NAD as coenzyme, the other uses NADP. The intracellular level of both GDHs is strongly regulated by the nitrogen source in the growth medium. NADP-dependent GDH was purified to homogeneity from crude extracts of S. fradiae. The Mr of the native enzyme was determined to be 200,000 by size-exclusion high-performance liquid chromatography whereas after sodium dodecyl sulphate-polyacrylamide gel electrophoresis one major band of Mr 49,000 was found, suggesting that the enzyme is a tetramer. The enzyme was highly specific for the substrates 2-oxoglutarate and L-glutamate, and required NADP, which could not be replaced by NAD, as a cofactor. The pH optimum was 9.2 for oxidative deamination of glutamate and 8.4 for reductive amination of 2-oxoglutarate. The Michaelis constants (Km) were 28.6 mM for L-glutamate and 0.12 mM for NADP. Km values for reductive amination were 1.54 mM for 2-oxoglutarate, 0.07 mM for NADPH and 30.8 mM for NH+4. The enzyme activity was significantly reduced by adenine nucleotides, particularly ATP.


Assuntos
Glutamato Desidrogenase/isolamento & purificação , Streptomyces/enzimologia , Difosfato de Adenosina/farmacologia , Monofosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Coenzimas/metabolismo , Eletroforese em Gel de Poliacrilamida , Glutamato Desidrogenase/antagonistas & inibidores , Desidrogenase de Glutamato (NADP+) , Peso Molecular , Especificidade por Substrato
15.
J Basic Microbiol ; 29(3): 185-9, 1989.
Artigo em Inglês | MEDLINE | ID: mdl-2501471

RESUMO

Homogeneous alanine dehydrogenase isolated from Streptomyces aureofaciens, a producer of tetracycline, was characterized from the point of its molecular and catalytic properties. Using analytical ultracentrifugation the molecular weight of alanine dehydrogenase was found to be 198,000. The enzyme could use as cofactors apart from NAD+ also 1,N6-etheno-NAD+, 3-acetylpyridine-NAD+, deamino-NAD+ and nicotinamide guanine dinucleotide. The enzyme activity in the direction of oxidative deamination was not affected by the addition of nonsubstrate amino acids, however, it was sensitive to inhibitors of SH-groups. Reductive amination of pyruvate was inhibited by L-alanine, L-serine and D-alanine. The inhibition by L-alanine and L-serine was uncompetitive with respect to NADH and noncompetitive with regard to pyruvate and ammonium ions.


Assuntos
Aminoácido Oxirredutases/análise , Streptomyces aureofaciens/enzimologia , Alanina Desidrogenase , Peso Molecular
16.
Eur J Biochem ; 179(1): 221-7, 1989 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-2917562

RESUMO

Alanine dehydrogenase was purified to homogeneity from a cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1180-fold to give a 21% yield, using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The relative molecular mass of the native enzyme was determined to be 210,000 or 205,000 by equilibrium ultracentrifugation or gel filtration, respectively. The enzyme is composed of four subunits, each of Mr 51,000. Using analytical isoelectric focusing the isoelectric point of alanine dehydrogenase was found to be 6.1. The Km were 10.0 mM for L-alanine and 0.18 mM for NAD+. Km values for reductive amination were 0.23 mM for pyruvate, 11.6 mM for NH4+ and 0.05 mM for NADH. Oxidative deamination of L-alanine proceeds through a sequential-ordered binary-ternary mechanism in which NAD+ binds first to the enzyme, followed by alanine, and products are released in the order ammonia, pyruvate and NADH.


Assuntos
Aminoácido Oxirredutases/isolamento & purificação , Streptomyces/enzimologia , Alanina Desidrogenase , Aminação , Aminoácido Oxirredutases/antagonistas & inibidores , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Cinética , Especificidade por Substrato , Temperatura , Ultracentrifugação
17.
J Gen Microbiol ; 134(12): 3213-9, 1988 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-3269392

RESUMO

Valine dehydrogenase (VDH) was purified to homogeneity from cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1508-fold in a 17.7% yield using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The Mr of the native enzyme was determined to be 218,000 and 215,000, by equilibrium ultracentrifugation and size-exclusion high-performance liquid chromatography, respectively. The enzyme is composed of 12 subunits of Mr 18,000. Using analytical isoelectric focusing the isoelectric point of VDH was found to be 4.7. Oxidative deamination of L-valine was optimal at pH 10.6. Reductive amination of 2-oxoisovalerate was optimal at pH 8.8. The Michaelis constants (Km) were 1 mM for L-valine and 0.029 mM for NAD+. Km values for reductive amination were 0.80 mM for 2-oxoisovalerate, 0.050 mM for NADH and 22 mM for NH4+.


Assuntos
Aminoácido Oxirredutases/isolamento & purificação , Streptomyces/enzimologia , Cromatografia em Agarose , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Coenzimas , Concentração de Íons de Hidrogênio , Peso Molecular , Especificidade por Substrato , Temperatura , Valina Desidrogenase (NADP+)
18.
J Bacteriol ; 170(11): 5192-6, 1988 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-3182727

RESUMO

Valine dehydrogenase was purified to homogeneity from the crude extracts of Streptomyces aureofaciens. The molecular weight of the native enzyme was 116,000 by equilibrium ultracentrifugation and 118,000 by size exclusion high-performance liquid chromatography. The enzyme was composed of four subunits with molecular weights of 29,000. The isoelectric point was 5.1. The enzyme required NAD+ as a cofactor, which could not be replaced by NADP+. Sulfhydryl reagents inhibited the enzyme activity. The pH optimum was 10.7 for oxidative deamination of L-valine and 9.0 for reductive amination of alpha-ketoisovalerate. The Michaelis constants were 2.5 mM for L-valine and 0.10 mM for NAD+. For reductive amination the Km values were 1.25 mM for alpha-ketoisovalerate, 0.023 mM for NADH, and 18.2 mM for NH4Cl.


Assuntos
Aminoácido Oxirredutases/isolamento & purificação , Streptomyces/enzimologia , Aminoácido Oxirredutases/metabolismo , Estabilidade Enzimática , Cinética , Substâncias Macromoleculares , Peso Molecular , Streptomyces/crescimento & desenvolvimento , Especificidade por Substrato
19.
Biochem J ; 253(1): 263-7, 1988 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-3138982

RESUMO

Anhydrotetracycline oxygenase was purified to homogeneity from Streptomyces aureofaciens, a producer of tetracycline. The enzyme was purified 60-fold in a 40% yield by a two-step procedure using a combination of hydrophobic chromatography and ion-exchange h.p.l.c. Purified anhydrotetracycline oxygenase was homogeneous according to SDS/polyacrylamide-gel electrophoresis, isoelectric focusing, ion-exchange h.p.l.c. on a Mono Q HR 5/5 column and size-exclusion h.p.l.c. on a TSK G 3000 SW column. The enzyme consists of two subunits of Mr 57,500, as determined by SDS/polyacrylamide-gel electrophoresis.


Assuntos
Streptomyces aureofaciens/enzimologia , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica
20.
J Basic Microbiol ; 27(9): 529-33, 1987.
Artigo em Inglês | MEDLINE | ID: mdl-3136241

RESUMO

Anhydrotetracycline oxygenase was purified both by affinity chromatography and by hydrophobic interaction chromatography. Molecular weight of anhydrotetracycline oxygenase was determined to be 115,000 by Sephadex G-200 gel filtration. Using preparative isoelectric focusing the isoelectric point of the enzyme was estimated to be 5.3. The enzyme showed a sensitivity to thiol-specific inhibitors. During the hydrophobic interaction purification step, the activity dropped considerably. Reactivation occurred when a heat treated crude extract was added to the reaction mixture.


Assuntos
Oxigenases/isolamento & purificação , Streptomyces aureofaciens/enzimologia , Cromatografia de Afinidade , Cromatografia em Gel , Focalização Isoelétrica , Ponto Isoelétrico , Peso Molecular , Oxigenases/análise
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