V. Musílek

Formation and partial characterization of glucose-2-oxidase, a H2O2 producing enzyme in Phanerochaete chrysosporium

SummaryA sugar oxidizing enzyme which produces H2O2 during glucose starvation in the white-rot fungus Phanerochaete chrysosporium has been purified from mycelial extracts and somewhat characterized. Enzyme purity was confirmed by analytical isoelectric focusing and by dodecylsulfate/polyacrylamide gel electrophoresis, both techniques revealing a homogeneous protein. The enzyme is active over a broad pH range with maximum activity at pH 7.5. Of several sugars tested, glucose was the preferred substrate although δ-d-gluconolactone and d-xylose were also oxidized at significant rates (at 60% and 37%, respectively, of the rate observed with glucose). Km-values for glucose and xylose are 1.03 and 20 mM respectively and the glucose oxidation product was idenitified as d-arabino-2-hexosulose. The possible importance of glucose-2-oxidase in lignin degradation is discussed.

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.

Antifungal antibiotic of the basidiomyceteOudemansiella mucida

The developmental cycle of the fungusOudemansiella mucida, the producer of a new antifungal antibiotic, was found to be controlled by the mechanism of homogenic tetrapolar incompatibility; under our conditions, the cycle took about 12 weeks to completion. Optimum conditions for a laboratoryscale fructification were investigated. Normal fruiting body formation required sufficient illumination, temperatures below 20 °C, and relative humidity in excess of 70%. Flask-grown fruiting bodies did not differ from the naturally occurring ones. The basidiospores of the produced fruiting bodies yielded the reference monokaryons necessary for physiological, cytological and genetical studies.

Antibiotic mucidin, a new antimycin A-like inhibitor of electron transport in rat liver mitochondria.

Summary Mucidin at concentration 1 μg/mg mitochondrial protein completely inhibited the oxidation of succinate and NADH-linked substrates in rat liver mitochondria. In succinate oxidizing mitochondria mucidin induced a crossover point between cytochromes b and c + c 1 . Under these conditions mucidin had no effect on the ATPase activity as well as on the phosphorylation efficiency of rat liver mitochondria measured with TMPD plus ascorbate as substrate. These properties of mucidin resemble those of other inhibitors of mitochondrial electron transport such as antimycin A and HQNO.

Mode of action of mucidin, a new antifungal antibiotic produced by the basidiomycete Oudemansiella mucida

Abstract 1. 1. Antibiotic mucidin inhibited the growth and germination of conidia of Aspergillus niger . Under these conditions, both the respiration and the incorporation of [ 14 C] leucine and [ 14 C] uracil into the HClO 4 -insoluble fraction of conidia were also inhibited. 2. 2. In yeast Saccharomyces cerevisiae , mucidin inhibited the oxidation of glucose and ethanol, while the anaerobic metabolism of glucose was not affected. In intact cells under aerobic conditions in the presence of mucidin and glucose the cytochrome b was fully reduced, while cytochromes c and a were completely oxidized. The reduction of cytochromes c and a by lactate was not inhibited by mucidin. 3. 3. Mucidin inhibited the growth of yeast on glycerol and ethanol completely. Mucidin reduced the aerobic growth yields of wild-type yeast to the level of the anaerobic one. The growth yields of the corresponding cytoplasmic respiratory-deficient mutant of Saccharomyces cerevisiae were not affected by mucidin. 4. 4. The results indicate that the specific inhibition of mitochondrial electron transport between cytochromes b and c is the primary site of action of mucidin. This effect is responsible for the antifungal activity of the new antibiotic.

Cultivation and antibiotic activity of mycorrhizal basidiomycetes

In 17 species (35 strains) of basidiomycetes which form mycorrhizas with Scotch pine (Pinus silvestris L.) the influence of media and method of cultivation on growth and manifestation of antibiotic activity were investigated under laboratory conditions. The antibiotic activity was influenced by the composition of the nutrient media as well as by mode of cultivation, while the ability and intensity of growth depended first of all on the composition of the media.Amanita citrina, Clitopilus prunulus, Lactarius helvus, Rhizopogon roseolus, Russula fragilis, Tricholoma albobrunneum, Tricholoma imbricatum, andTricholoma saponaceum exhibited antibiotic activity. The intracellular antibiotics ofTricholoma saponaceum andRhizopogon roseolus exerted the highest activity. It has been confirmed that the antibiotic activity of the cultures corresponds in most cases to the activity of the fruit-bodies grown in nature.

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.

Laboratory fermentation of gibberellic acid

SummaryIn studying the fermentation conditions suitable for gibberellic acid production, 6 collection strains ofFusarium moniliforme andGibberella fujikuroi were used. The strain used was of decisive importance for the yield and composition of the effective metabolite.After verification of methods described in the literature a new medium for gibberellic acid production was developed to be used for fermentation on a shaker and in laboratory fermentors, the medium containing sucrose and corn steep liquor. By using a reciprocal shaker, maximum production of gibberellic acid was obtained after 14 days, by using three-stage propagations after 12 days of fermentation.Maximum values achieved during fermentation of the most suitable strains amounted to 150–240 μg./ml. Maximum production in laboratory fermentors reached 212 μg./ml.The metabolite isolated after fermentation under the described conditions was identified as gibberellic acid.AbstractПри изучении условий ферментации пригодные для gibberellic кислоты 6 коллекция штаммов Fusarium moniliforme и Gibberella fujikuroi были использованы Используется штамм имеет решающее значение за урожайностью и состав эффективный метаболит. После проверки методов, описанных в литературе нового средства массовой информации для gibberellic кислоты был разработан, которые будут использоваться для брожения на шейкер и в лабораторных fermentors, средних содержащие сахарозы и кукуруза крутых спиртными напитками. С помощью взаимных шейкер, максимум производство gibberellic кислота была получена после 14 дней, используя три этапа propagations по истечении 12 дней после брожения Максимальные значения, достигнутые в ходе ферментации из наиболее подходящие штаммы составил 150-240 μ g. / мл. Максимум производства в лаборатории fermentors достигло 212 μ g. / мл Метаболит изолированных после брожения в рамках описанных условиях определены в качестве gibberellic кислота

α-mannosidase and mannanase of some wood-rotting fungi

Cultivation media from 11 wood-rotting fungi contained α-mannosidase and mannanase activity. α-Mannosidase was studied in more detail inPhellinus abietis and mannanase was studied more intimately in basidiomycetesPhellinus abietis, Trametes sanguinea andPholiota aurivella. Suitable cultivation conditions and optimum conditions for the production of α-mannosidase and mannanase were determined. Both enzymes are constitutive; mannanase is extracellular, α-mannosidase was found in both mycelium and cultivation medium.

Dynamics of enzymes generating hydrogen peroxide in solid-state fermentation ofPanus tigrinus on wheat straw

The relationship between the production of extracellular H2O2, hydrogen peroxide-producing enzymes and ligninolytic peroxidase was examined during solid-state cultivation ofPanus tigrinus on wheat straw. Glyoxal oxidase, Mn2+-dependent peroxidase and glucose oxidase, capable of H2O2 generation, were found in the extracellular enzyme preparation. The production of H2O2 has two maxima: the maximal production correlates well with the maximal activities of glyoxal oxidase and Mn2+-dependent peroxidase, while another, lower peak of H2O2 generation is related to the second peak of Mn2+-dependent peroxidase activity. The contribution of glucose oxidase to the production of hydrogen peroxide is probably only marginal. Comparison of the dynamics of these extracellular activities and the ligninolytic peroxidase showed good temporal correlation indicating an interrelation of the two processes.