Jindřich Volc

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 sugarD-arabino-2-hexosulose was demonstrated in mycelial extracts of 9 species ofAphyllophorales and 6 species ofAgaricales 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 inOudemansiella mucida, Coriolopsis occidentalis, Fomes fomentarius, Trametes versicolor and a not-yet-classified species of the genusTrametes.

Pyranose oxidase and pyranosone dehydratase: enzymes responsible for conversion of d-glucose to cortalcerone by the basidiomycete Phanerochaete chrysosporium

Pyranose oxidase (glucose 2-oxidase) and pyranosone dehydratase were purified 27.6- and 43.9-fold respectively from mycelial extracts of the fungus Phanerochaete chrysosporium using hydrophobic interaction, anion exchange and gel filtration chromatography. The enzymes appeared substantially homogeneous on SDS-PAGE and were comprised of identical subuntis with apparent Mr values of 69 000 and 99 000 for pyranose oxidase and pyranosone dehydratase, respectively. The apparent Mrs of the native enzymes, based on equilibrium ultracentrifugation, were 308 000 and 221 000. In coupled reactions, the enzymes catalyzed conversion of d-glucose via d-glucosone (d-arabino-2-hexosulose) to the antibiotic β-pyrone, cortalcerone. The latter compound was isolated as a diphenylhydrazone derivative and spectroscopically identified.

Valine Dehydrogenase from Streptomyces fradiae: Purification and Properties

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+.

Purification and partial characterization of alanine dehydrogenase from Streptomyces aureofaciens

Alanine dehydrogenase was purified to near homogeneity from cell-free extract of Streptomyces aureofaciens, which produces tetracycline. The molecular weight of the enzyme determined by size-exclusion high-performance liquid chromatography was 395 000. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis was 48 000, indicating that the enzyme consists of eight subunits with similar molecular weight. The isoelectric point of alanine dehydrogenase is 6.7. The pH optimum is 10.0 for oxidative deamination of L-alanine and 8.5 for reductive amination of pyruvate. KM values were 5.0 mM for L-alanine and 0.11 mM for NAD+. KM values for reductive amination were 0.56 mM for pyruvate, 0.029 mM for NADH and 6.67 mM for NH4Cl.

Glucose-2-oxidase activity and accumulation of D-arabino-2-hexosulose in cultures of the basidiomyceteOudemansiella mucida

Submerged cultures of the basidiomyceteOudemansiella mucidd, 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 ofd-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 fungiPleurotus ostreatus, Laetiporus sulphureus, andPhellinus abietis.

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.

Soluble sugars in the mycelium of the basidiomyceteOudemansiella mucida

Quantitative gas chromatography was used to determine soluble neutral sugars in an extract of the fungusOudemansiella 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 containedd-arabino-2-hexosulose (d-glucosone). In the period of maximum growth, this aldoketose was the predominant monosaccharide (3.4 % mycelial dry weight).

High performance liquid chromatographic determination of the antibiotic cortalcerone

Abstract Cortalcerone (2-hydroxy-6 H -3-pyrone-2-carboxaldehyde hydrate) is an antibacterial antibiotic produced by a number of basidiomycetous fungi. Its biosynthesis is connected with C-2 oxidation of d -glucose. A method for the reliable determination of this antibiotic is required for the study of the mechanism of its biosynthesis and for the determination of its pharmacological properties. The high-performance liquid chromatographic determination of cortalcerone in the presence of d -glucose, d -fructose, d - arabino -2-hexosulose, d -gluconic acid and d - arabino -2-hexulosonic acid on an amino-phase column with 70% aqueous acetonitrile (pH 5.00) and UV detection at 195 nm is reported. A linear relationship between response and cortalcerone concentration was found in the range 0.25–10.00 mg/ml. The detection limit is 6.5 μg/ml.