Double oxidation of D-xylose to D-glycero -pentos-2,3-diulose (2,3-diketo-D-xylose) by pyranose dehydrogenase from the mushroom Agaricus bisporus.

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.

Immobilization of pyranose oxidase (Phanerochaete chrysosporium): characterization of the enzymic properties.

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.

Glucose-2-oxidase activity and accumulation of D-arabino-2-hexosulose in cultures of the basidiomycete Oudemansiella mucida.

Submerged cultures of the basidiomycete Oudemansiella mucida, strain III, accumulate D-arabino-2-hexosulose. The maximum yields during cultivations in shaker flasks or in a laboratory fermentor are 6--12 and 15 mg/ml, respectively (20--50% conversion of substrate glucose). The accumulation is transient, the aldoketose being again utilized after glucose exhaustion. Its production is stimulated by fluoride ions. The enzyme responsible for the C(2)-specific oxidation of D-glucose acts as an intracellular oxidase with a maximum activity in the exponential phase of growth. D-arabino-2-Hexosulose was also detected in the cultivation medium of the wood-rotting fungi Pleurotus ostreatus, Laetiporus sulphureus, and Phellinus abietis.

Soluble sugars in the mycelium of the basidiomycete Oudemansiella mucida.

Quantitative gas chromatography was used to determine soluble neutral sugars in an extract of the fungus Oudemansiella mucida grown on a synthetic glucose medium. Apart from the usual fungal sugar components, viz. trehalose, D-glucose, D-mannitol, D-arabinitol, glycerol and inositol, the 6-day-old mycelium contained D-arabino-2-hexosulose (D-glucosone). In the period of maximum growth, this aldoketose was the predominant monosaccharide (3.4% mycelial dry weight).

Antimicrobial effects of some dicarbonyl and tricarbonyl sugar hydrazone derivatives.

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.

Glucose-2-oxidase activity in mycelial cultures of basidiomycetes.

The activity of intracellular glucose-2-oxidase was tested in 40 species of 26 basidiomycete genera. The enzyme catalyzing the oxidation of D-glucose to the dicarbonyl sugar D-arabino-2-hexosulose was demonstrated in mycelial extracts of 9 species of Aphyllophorales and 6 species of Agaricales cultivated in liquid media. In the majority of species exhibiting this activity hexosulose was detected in the cultivation medium. The highest enzyme activity was detected in Oudemansiella mucida, Coriolopsis occidentalis, Fomes fomentarius, Trametes versicolor and a not-yet-classified species of the genus Trametes.

Pyranose Oxidase, a Major Source of H(2)O(2) during Wood Degradation by Phanerochaete chrysosporium, Trametes versicolor, and Oudemansiella mucida.

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.

Purification and characterization of pyranose oxidase from the white rot fungus Trametes multicolor.

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.

Ultrastructural and Immunocytochemical Studies on the H(2)O(2)-Producing Enzyme Pyranose Oxidase in Phanerochaete chrysosporium Grown under Liquid Culture Conditions.

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.

Pyranose 2-dehydrogenase, a novel sugar oxidoreductase from the basidiomycete fungus Agaricus bisporus.

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.

Only C-2 specific glucose oxidase activity is expressed in ligninolytic cultures of the white rot fungus Phanerochaete chrysosporium.

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.

Purification and properties of NADP-dependent glutamate dehydrogenase from Streptomyces fradiae.

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.

Pyranose 2-oxidase from Phanerochaete chrysosporium--further biochemical characterisation.

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.

Isolation of pure anhydrotetracycline oxygenase from Streptomyces aureofaciens.

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.

A further characterization of alanine dehydrogenase from Streptomyces aureofaciens.

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.

Pyranosone dehydratase from the basidiomycete Phanerochaete chrysosporium: improved purification, and identification of 6-deoxy-D-glucosone and D-xylosone reaction products.

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+).

Screening of basidiomycete fungi for the quinone-dependent sugar C-2/C-3 oxidoreductase, pyranose dehydrogenase, and properties of the enzyme from Macrolepiota rhacodes.

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.

Isolation and characterization of valine dehydrogenase from Streptomyces aureofaciens.

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.