Mycelium cultivation, chemical composition and antitumour activity of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis

AIMS: To examine and illustrate the morphological characteristics and growth kinetics of Cs-HK1, a Tolypocladium fungus, isolated from wild Cordyceps sinensis in solid and liquid cultures, and the major chemical constituents and antitumour effects of Cs-HK1 mycelium. METHODS AND RESULTS: The Cs-HK1 fungus was isolated from the fruiting body of a wild C. sinensis and identified as a Tolypocladium sp. fungus. It grew rapidly at 22-25 degrees C on a liquid medium containing glucose, yeast extract, peptone and major inorganic salts, with a specific growth rate of 1.1 day(-1), reaching a cell density of 23.0 g dw l(-1) in 7-9 days. Exopolysaccharides accumulated in the liquid culture to about 0.3 g l(-1) glucose equivalent. In comparison with natural C. sinensis, the fungal mycelium had similar contents of protein (11.7-microg) and carbohydrate (654.6-microg) but much higher contents of polysaccharide (244.2 mg vs 129.5 mg), adenosine (1116.8-microg vs 264.6 microg) and cordycepin (65.7 microg vs 20.8 microg) (per gram dry weight). Cyclosporin A, an antibiotic commonly produced by Tolypocladium sp., was also detected from the mycelium extract. The hot water extract of mycelium showed low cytotoxic effect on B16 melanoma cells in culture (about 25% inhibition) but significant antitumour effect in animal tests, causing 50% inhibition of B16 cell-induced tumour growth in mice. CONCLUSIONS: The Tolypocladium sp. fungus, Cs-HK1, can be easily cultivated by liquid fermentation. The mycelium biomass contained the major bioactive compounds of C. sinensis, and the mycelium extract had significant antitumour activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The Cs-HK1 fungus may be a new and promising medicinal fungus and an effective and economical substitute of the wild C. sinensis for health care.     

Study of certain carbohydrate metabolism enzymes in an active oxytetracycline producer strain and in an inactive mutant in relation to antibiotic biosynthesis]

The activity of the glycolysis enzymes, i.e. aldolase and pyruvate decarboxylase and the enzymes of the pentose cycle, i.e. transketolase were investigated in the process of cultivation of an active strain and inactive mutant of Act. rimosus under conditions favourable for oxytetracycline biosynthesis on starch medium and under unfavourable conditions on glucose medium. It was shown that the aldolase and transketolase activity in the inactive mutant was higher on the starch medium as compared to the active strain, while the activity of pyruvate dekarboxylase was lower. The above difference between the both strains was preserved on the glucose medium and the activity of aldolase and transketolase in both strains increased, while the activity of pyruvate dekarboxylase remained at the same level.     

Study of the carbohydrate metabolic enzyme activity of Act. noursei]

Activity of aldolase and threosophosphate dehydrogenase, transketolase and phosphogluconate dehydrogenase in Act. noursei, strain 153 and its inactive mutant 149 was studied comparatively. The enzyme activity of the inactive mutant was investigated in the absence of the antibiotic production and under conditions of reduced biosynthesis of nystatin in this strain after addition of the fermentation broth filtrate of the inactive mutant 369 to the medium. The activity of the enzymes of the hexosomonophosphate metabolic pathway in the active strain 153 of Act. noursei was 2-4 times higher than that of the inactive mutant 149. The activity of the enzymes of the hexosomonophosphate metabolic pathways increased and reached the level of the enzyme of the active mutant. The high level of the enzyme activity of the hexosomonophosphate glycolysis pathway is probably one of the necessary conditions for nystatin production.     

Characteristics of obtaining protoplasts from 2 strains of Tolypocladium inflatum subsp. Blastosporum]

The optimal conditions for preparing protoplasts with high yields by using the cells of two (low and high potent) isogenic cyclosporine-producing Tolypocladium strains were developed. A specific medium containing 0.5 per cent yeast autolysate (by dry weight) and 3 per cent glucose was used. When grown on this medium the cells of the highly potent strain 847 acquired a yeast-like shape. High yields of protoplasts prepared from the low potent strain 43 mycelium were obtained via prior incubation with 0.01 M dithiothreitol followed by treatment with a complex of enzymes from Helix pomatia for 1.5 to 2 hours was used. For preparation of the protoplasts with employing the highly potent strain 847 cells the prior incubation with dithiothreitol was not required, but it was necessary to employ a mixture of the enzyme complex (Helix pomatia), drizilase (Irpex lacteus) and chitinase (Streptomyces griseus) for 18 hours. The electron microscopic data on the two isogenic strains and their protoplasts are presented. The protoplasts proved to be a suitable initial material for investigating bioenergetic processes at the subcellular level and further genetic improvement of the strains.     

Rhythms of Enzyme Activity Associated with Circadian Conidiation in Neurospora crassa

The mycelial growth front of the band strain of Neurospora grown on a solid surface exhibits a circadian rhythm of conidiation. Enzyme assays on extracts from that mycelium have shown that the activities of 6 of 13 enzymes (nicotinamide adenine dinucleotide nucleosidase, isocitrate lyase, citrate synthase, glyceraldehydephosphate dehydrogenase, phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase) and soluble-protein content oscillate with the visible morphological change. The rhythmic enzymes associated with the Krebs and glyoxylate cycles are more active during conidiogenesis, whereas the activities of the rhythmic enzymes of glycolysis and the hexose monophosphate shunt are reduced during that phase. The absence of enzyme oscillations in wild-type and fluffy strains which do not form conidia under the conditions employed suggests that the enzyme fluctuations are associated with conidiogenesis itself. Oscillations of enzyme activity as a function of time are restricted to the growth front. A permanent record of rhythmicity associated with conidial and nonconidial regions does, however, exist in the mycelial mat behind the growth front. The activities of three enzymes (nicotinamide adenine dinucleotide nucleosidase, glucose-6-phosphate dehydrogenase, and phosphogluconate dehydrogenase) are not directly influenced by CO(2) concentration, but are correlated with the prescence or absence of conidiation which is controlled by CO(2) concentration. In contrast, citrate synthase and malate dehydrogenase activities are correlated with changes in CO(2) concentration.     

Tolypocladin — a new metal-chelating 2-aza-anthraquinone fromTolypocladium inflatum

Summary A new chelator of di- and trivalent cations (tolypocladin) was isolated from the mycelium ofTolypocladium inflatum DSM 915. The structure has been determined by NMR methods as 3-methyl-5,6(7),8-trihydroxy-2-aza-anthraquinone. The ultraviolet, visible and fluorescence spectral properties of some metal complexes (in methanol) are described. The compound forms water-soluble fluorescent aluminium complexes. Its production is dependent on zinc ions in the medium. It serves as an endogenous hydrogen acceptor under oxygen limitation in the producing strain,T. inflatum.     

Staphylococcus aureus physiological growth limitations: insights from flux calculations built on proteomics and external metabolite data

Comparing proteomics and metabolomics allows insights into Staphylococcus aureus physiological growth. We update genome and proteome information and deliver strain-specific metabolic models for three S. aureus strains (COL, N315, and Newman). We find a number of differences in metabolism and enzymes. Growth experiments (glucose or combined with oxygen limitation) were conducted to measure external metabolites. Fluxes of the central metabolism were calculated from these data with low error. In exponential phase, glycolysis is active and amino acids are used for growth. In later phases, dehydroquinate synthetase is suppressed and acetate metabolism starts. There are strain-specific differences for these phases. A time series of 2-D gel protein expression data on COL strain delivered a second data set (glucose limitation) on which fluxes were calculated. The comparison with the metabolite-predicted fluxes shows, in general, good correlation. Outliers point to different regulated enzymes for S. aureus COL under these limitations. In exponential growth, there is lower activity for some enzymes in upper glycolysis and pentose phosphate pathway and stronger activity for some in lower glycolysis. In transition phase, aspartate kinase is expressed to meet amino acid requirements and in later phases there is high expression of glyceraldehyde-3-phosphate dehydrogenase and lysine synthetase. Central metabolite fluxes and protein expression of their enzymes correlate in S. aureus.     

Evidence for the activation of 6-phosphofructo-1-kinase by cAMP-dependent protein kinase in Aspergillus niger

Abstract The change from pentose phosphate pathway to glycolysis plays a significant role in the physiology of Aspergillus niger during the induction of citric acid accumulation. Evidence is shown for the importance of 6-phophofructo-1-kinase in this process since it is activated by phosphorylation. By incubating a purified active form of enzyme together with commercially available alkaline phosphatase, 6-phosphofructo-1-kinase activity was lost after a certain time suggesting that the enzyme was dephosphorylated. Inactive 6-phosphofructo-1-kinase could be isolated from the cells in the early stage of growth in a high citric acid yielding medium. The enzyme was 'in vitro' activated by isolated protein kinase in the presence of cAMP, ATP and Mg²⁺ ions. Additional evidence for covalent phosphorylation of inactive 6-phosphofructo-1-kinase was obtained by incubating both enzymes together with labelled [ γ −³²P]ATP. The activating enzyme was partially purified from A. niger mycelium.     

Carbohydrate and pyruvic acid degradation pathways in Fusidium coccineum strains with varying levels of antibiotic synthesis]

A number of enzymes and reactions of glycolysis, pentose-phosphate cycle and degradation of pyruvic acid in strains of F. coccineum with various levels of antibiotic production was studied comparatively. The experiments showed that highly productive strains were characterized by higher activity of the NADP-deficient enzymes of the pentoze-phosphate cycle as compared to the low active strains. The activity levels of glycolytic enzymes, such as fructose-diphosphate-aldolase and 3-phosphoglycerolaldehydehydrogenase did not practically differ. Significant differences were found in the reactions of puryvic acid degradation: the activity of cytoplasmic pyruvatedecarboxylase in the mutant with high antibiotic production level was lower than that in the low productive strain, while oxidation of the pyruvate of the mitochondrial fraction was on the contrary more intensive than in the highly productive strain. Therefore, metabilism in the strains studied was characterized by ever-increasing biochemical changes with an increase in their antibiotic productivity. Lowering of the growth rate of the mutants as their capacity for antibiotic supersynthesis increased and subsequently the anabolic processes became more intensive was accompanied by increasing derepression of the key enzymes of carbohydrate metabolism and in particular NADR-deficient dehydrogenase of the pentose cycle and pyruvatedehydrogenase, significant for fusidin biosynthesis and providing production of the antibiotic of steroid nature by cofactor NADP-H and acetyl-KoA, the primary precursor.     

Ligninolytic characteristics of Pleurotus ostreatus strain F6 and its monokaryotic protoplast derivative P19

A stable isolate of Pleurotus ostreatus P19 differing in some morphological and physiological characteristics from its parental wild-type strain F6 was obtained via protoplast isolation during the preparation of strains with altered ligninolytic abilities. The isolate is monokaryotic, does not form clamp-connections, and produces much higher activities of enzymes involved in lignin modification (laccase, manganese peroxidase). Cellulase activity was comparable to that of wild-type strain F6, but the xylanase activity was slightly higher in isolate P19. However, this monokaryotic derivative degrades lignin at a slightly lower rate than its parental strain F6. Electron microscopy observations of wood degradation as a function of mycelium growth were performed on three zones of birch wafers delimited according to the distance from the point of inoculation. The different stages of fungal mycelium growth showed differences in the ultrastructural patterns of the decay not only between the strains P19 and F6, but also depending on the distance from the point of inoculation. This suggests a spatio-temporally controlled secretion of enzymes along the hyphae. The enhanced ability of P19 to degrade the condensed forms of lignin in middle lamellae is correlated to its higher laccase activity.     

中国弯颈霉的培养性状和药理作用研究

中国弯颈霉ZN923菌株分离自蔗褐蠹蛾PhragmatoeciacastaneaeHubner虫尸。其生长最适温度为25～28℃,最适pH6～7,在富氮培养基上菌丝生长茂盛、洁白,有香气。药理试验表明,该菌株培养物具有镇静、抗炎、增强耐缺氧能力、扩张气管和雄性激素样作用,急性毒性试验以80g／kg（最大允许容积）剂量对小鼠一次灌胃,未见不良作用。中国弯颈霉是与冬虫夏草Cordycepssinensis（Berk．）Sacc．无性型密切相关的真菌。具有潜在的药用价值。     

Expression of Intracellular Enzymes During Hyphal Aggregate Formation in a Fruiting-Impaired Variant of Agaricus bisporus

The specific activity and enzyme protein concentration of the developmentally regulated enzyme glucose 6-phosphate dehydrogenase (G6PD) were measured in the developing aggregates and supporting mycelium of a fruiting-impaired variant strain of Agaricus bisporus. The nonregulated enzymes mannitol dehydrogenase (MD) and hexokinase (HK) were assayed for comparison. G6PD activity was higher in aggregates than in the mycelium, whereas MD and HK activities varied little between mycelium and aggregates. Enzyme protein levels varied in a way different from enzyme activity, suggesting the presence of inactive enzyme at times during development. The raised level of G6PD in aggregates provides a possible mechanism for the increased mannitol concentration previously observed in aggregates. There was no parallel to the rapid increase in G6PD activity associated with primordium development of normally fruiting strains growing on compost.     

The role of fungi in the diet of the amphipod Gammarus pulex (L.): an enzymatic study

SUMMARY. 1. Sets of ten Gammarus pulex fed on controlled diets of sterile alder leaves, or fungal mycelium, or alder leaves incubated for 10 days with an aquatic hyphomycete, were assayed for cellulase, β-1,3-glucanase an d chiitinase activity and compared with (a) animals taken directly from the stream, (b) animals starved for 2 days, and (c) enzyme activity in fungal mycelium.
2. Gut enzyme activity was compared on natural substrates of sterile leaves, mycelium and inoculated leaves as well as on model substrates.
3. G. pulex secretes an endogenous coupled cellulase system capable of degrading native cellulose in plant cell walls. It also secretes β-1,3-glucanase and chitinase capable of degrading fungal cell walls thus affording access for gut enzymes to cell contents.
4. Secretion of enzymes active on native cellulose is enhanced on a diet of leaves already partially degraded by fungal enzymes. Gut enzymes extract more reducing sugar from this substrate than from sterile leaves. Specific enzyme secretion is enhanced by the presence in the diet of exposed, accessible substrates. Fungal enzymes do not appear to contribute to the digestive processes of G. pulex.     

环孢菌素8562的研究

中国弯颈霉Tolypocladium sinense C.L.Li 8562培养物用乙酸乙酯萃取,减压浓缩,获粗提品,经硅胶柱层析分离,葡聚糖凝胶LH-20纯化,活性炭脱色,得到2个成份,8562-成份1,8562-成分2,以成分1为主。经紫外光谱、红外光谱、高效液相色谱、质谱、核磁共振氢谱等分析表明8562-成份1、8562-成份2与环孢菌素A、环孢菌素C同质。8562-成分1的抗真菌试验显示了对半知菌和酵母菌具有明显拮抗活性,最低抑菌浓度(MIC)为2—150μg/ml。     

Extracellular hydrolases of strain Bacillus sp. 739 and their involvement in the lysis of micromycete cell walls

The mycolytic bacterial strain Bacillus sp. 739 produces extracellular enzymes which degrade in vitro the cell walls of a number of phytopathogenic and saprophytic fungi. When Bacillus sp. 739 was cultivated with Bipolaris sorokiniana, a cereal root-rot pathogen, the fungus degradation process correlated with the levels of the beta-1,3-glucanase and protease activity. The comparative characteristic of Bacillus sp. 739 enzymatic preparations showed that efficient hydrolysis of the fungus cell walls was the result of the action of the complex of enzymes produced by the strain when grown on chitin-containing media. Among the enzymes of this complex, chitinases and beta-1,3-glucanases hydrolyzed most actively the disintegrated cell walls of B. sorokiniana. However, only beta-1,3-glucanases were able to degrade the cell walls of native fungal mycelium in the absence of other hydrolases, which is indicative of their key role in the mycolytic activity of Bacillus sp. 739.     

ENZYME ACTIVITY AND CELL DIFFERENTIATION IN NEUROSPORA

Zalokar , Marko . (Yale U., New Haven, Conn.) Enzyme activity and cell differentiation in Neurospora. Amer. Jour. Bot, 46(7): 555–559. Illus. 1959.—Morphological differences were observed in vegetative cells of Neurospora of different ages and in different parts of the mycelium. The surface layer of mycelium grown in standing cultures could be separated from the deep layer. The first contained most of the growing hyphae rich in protoplasm, while the second contained heavily vacuolated hyphae laden with fat droplets. Specific activities of several enzymes were studied in conidia, young hyphae, and the surface and deep layers of mature mycelium. Succinic dehydrogenase was low in conidia and about 10 times more active in mature mycelium. The surface layer had twice the activity of the deep layer. Aldolase increased about 3 times after the germination of conidia; it was slightly lower in the surface than in the deep layer of mycelium. Tryptophan synthetase exhibited only small differences between conidia and mycelium and was slightly lower in the surface than in the deep layer. β-galactosidase was formed in appreciable amounts only after prolonged growth and had a much higher specific activity in the deep layer. The results were discussed in connection with cell differentiation and aging.     

Oxidative Enzymes and Pathways of Hexose and Triose Metabolism in Chlorella

Cell-free preparations of Chlorella pyrenoidosa Chick, van Niel's strain, were assayed for oxidative enzymes, utilizing isotopic and spectrophotometric techniques. The enzyme activity of heterotrophic and autotrophic cells was compared. The study was divided into categories, one concerned with the spectrophotometric detection of enzymes involved in the initial reactions of glycolysis and the hexose monophosphate shunt, and the other with the direct oxidation of glucose as compared with that oxidized via glycolysis. The reduction of pyridine nucleotides in crude extracts was studied with glucose, glucose-6-phosphate, 6-phosphogluconate, and fructose-1-6-diphosphate as substrates. Enzymes detected in both heterotrophic and autotrophic cells were hexokinase, fructose-diphosphate-aldolase, NAD-linked 3-phosphoglyceraldchyde dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and a NADP-linked 3-phosphoglyceraldchyde dehydrogenase. In addition to isotopic studies designed to make an appraisal of the hexose monophosphate shunt, a comparison of the rate of reduction of NADP by glucose-6-phosphate and 6-phosphogluconate in relation to the reduction of NAD by 3-phosphoglyceraldehyde was made in light- and dark-grown cells. The rate of reduction of NADP appeared to be lowered in the light-grown cells, suggesting, as did also the isotopic studies, that the hexose monophosphate shunt is less active in autotrophic metabolism than in heterotrophic metabolism.     

Morphological and genetic characterization of a cultivated Cordyceps sinensis fungus and its polysaccharide component possessing antioxidant property in H22 tumor-bearing mice

Cordyceps sinensis, one of the most precious traditional Chinese medicines, possesses the antitumor activity, antioxidant activity and the capability of modulating the immune system. In the present study, a fungus strain G1 isolated from wild C. sinensis was identified and initially characterized. A phylogenetic tree was generated based on the sequences of the internal transcribed spacer (ITS) region of related fungi. The analysis of ITS sequence showed that fungus G1 was clustered together with C. sinensis, Tolypocladium cylindrosporum and Tolypocladium inflatum in the phylogenetic tree. Both the morphological character and the ITS sequence analysis establish that fungus G1 is one of the anamorph strains of C. sinensis and belongs to Tolypocladium genus. Furthermore, the polysaccharide (PS) extracted from fungus G1 and its antioxidant activity on H22-bearing mice was investigated. H22 cells were hypodermically injected into the right oxter of each mouse after the ICR mice were treated with PS by means of gavage for 7 days. Then the same administration process continued for 9 days. At the end of the experiments, the tumor weight of each mouse was measured. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) level in mouse liver, brain and serum, as well as glutathione peroxidase (GSH-Px) activity in mouse liver and brain were assayed. The results showed that the H22 tumor growth was significantly inhibited by PS. Moreover, PS significantly enhanced SOD activity of liver, brain and serum as well as GSH-Px activity of liver and brain in tumor-bearing mice. PS also significantly reduced the level of MDA in liver and brain of tumor-bearing mice.     

Inducible trehalase enzyme activity of Morchella conica Persoon mycelium

Morchella conica Pers. strains of the study were isolated from fruit bodies collected in ash-mixed forests. At first, the strains were cultured on potato dextrose agar (PDA), then on modified Murashige and Skoog (MS) solid agar media. A normal-growing strain was chosen for the trehalase induction experiments. During the trehalase induction treatment, mycelia were grown in liquid culture containing different concentrations of trehalose. After the induction period of trehalase enzymes, physiological state of the mycelium and the oxidative stress were monitored in the vegetative mycelia by measuring the change of the malondialdehyde content, superoxide dismutase enzyme activity, the fresh and dry weight. The examined Morchella conica strain utilized the trehalose properly. The rising amount of the trehalose triggered the increase of the mycelial trehalase enzyme activity. Our results clearly proved that both neutral and acidic trehalase isoenzyme activity of the Morchella conica mycelium are inducible and are playing important role in the utilization of external trehalose.     

Study of the carbohydrate content in the mycelia of an active strain producing oxytetracycline and in an inactive mutant]

The content of carbohydrates in the mycelium of the active strain and inactive mutant of the oxytetracycline-producing organism under conditions favourable (starch medium) and unfavourable (glucose medium) for the antibiotic biosynthesis was studied. The mycelium of both organisms was fractionated and carbohydrate distribution according to the mycelium fractions and carbohydrate content in every fraction were investigated. No significant differences were observed between the active strain and inactive mutant with respect to the characteristics studied. The carbon source in the medium had the dominating effect on the chemical composition of the mycelium. The mycelium of both strains grown on the starch medium contained much more carbohydrates than that grown on the glucose medium. The carbohydrates of the mycelium grown on the starch medium were mainly found in fraction III and must be represented by polysaccharides.