Chemical and structural differences in mycelial and regeneration walls of Trichoderma viride

When incubated in Winge medium, protoplasts from Trichoderma viride obtained by treatment with Micromonospora chalcea or Streptomyces venezuelae RA lytic systems first synthesized an aberrant wall, different from the normal one; it was aseptate, larger and irregular in size and length. They then regenerated a new wall, similar to the original one from which they were liberated. Analysis showed that the wall polymers were mainly β-(1–3) glucan, β-(1–6) glucan and chitin in the normal walls, whereas chitin was absent in aberrant tubes. These results are discussed below, together with electron micrographs of aberrant and normal walls.     

Layered structure of trichoderma cell walls

Trichoderma pseudokoningii mycelium was treated sequentially with the following enzymes; (1) lysing enzyme and laminarinase, (2) pronase, and (3) chitinase. (1) Removed the outermost amorphous glucan layer and revealed a striated layer which was removed by (2). Microfibrils were exposed and removed by (3). The wall thus has a layered structure comprising glucan, protein and/or glycoprotein, and chitin.Glucan was prepared and subjected to methylation and hydrolysis. 1,3,5,6-Tetra-O-acetyl-2,4-di-O-methyl-D-glucose was identified suggesting that the 1–3 and 1–6 glucans are cross-linked at intervals.     

Energetic efficiency of complex substrate utilization by trichoderma viride

The efficiency of carbon substrate utilization is evaluated as the thermodynamic efficiency (η_x) of microbial growth. Three methods based on mass-energy balance are used for the efficiency studies of complex substrates (straw, plant juices, lye) utilization by microfungi Trichoderma viride.

• 1 According to substrate and biomass balance η_x = 0.55, 0.37 and 0.36 for Trichoderma viride growth on alkali pretreated wheat straw during 23, 34 and 50 hours. Cellulose biodegradation increases with cultivation time. However, the efficiency of cellulose utilization for cell mass growth decreases at the same time.
• 2 In accordance with oxygen-balance calculations η_x = 0.75 and 0.71 for the same processes. The discrepancy in results from the above two methods probably can be explained by the following:
  • A Substrate and biomass balance gives underestimated results.
  • B Oxygen balance method includes the part of energy for extracellular product formation and therefore η_x can be overestimated.
  • C The efficiency of complex soluble substrate utilization (lye, green juice, deproteinized brown plant juice) tested by means of pulse method gives the values of η_x = 0.72 – 0.88. Similar high estimates of η_x in C-limited batch culture are observed for soluble carbohydrates (glucose, galactose, lactose, xylose) but not for acetate. The pulse method is advantageous for testing the “true” efficiency of carbon substrate utilization in a definite physiological environment.

     

Kinetics of solka floc cellulose hydrolysis by trichoderma viride cellulase

A product inhibition model is developed to describe the hydrolysis of cellulose by the Trichoderma viride enzyme system. It is assumed that noncompetitive inhibition by cellobiose dominates the reaction kinetics. Experiments show that this is indeed a reasonable assumption for initial cellulose concentrations of up to 15 g/liter and at hydrolysis extents up to 65′. Kinetic parameters were determined for the noncompetitive inhibitionmodel in batch experiments with durations of up to 1.5 hr. These parameterswere then used in predicting reaction progress for up to 10 hr. Cellobiose was added to the reaction mixture at the onset of some runs and againreliable predictions were obtained for up to 8 hr of hydrolysis. Finally reaction was carried out in a membrane reactor whereby the product cellobiose was being continuously removed and again reasonable predictability was obtained with a higher net reaction rate.     

Protoplasts of Trichoderma viride: formation and regeneration.

High yields of protoplasts from the 18-hr old mycelium of Trichoderma viride were obtained by using the lytic system, produced by Streptomyces venezuelae RA and Micromonospora chalcea grown on a synthetic medium containing laminarin and chitin, when 0.7 M MgSO4 or (NH4)2SO4 were used as osmotic stabilizers. Regeneration of these protoplasts occurred through the production of an abortive tube and direct germination of the protoplasts. Regeneration could also take place in the medium used to produce protoplasts, but the process was different in many details.     

Continuous enzymatic saccharification of cellulose with culture filtrates of trichoderma viride QM 6a.

Continuous saccharification of Solka Floc (cellulose pulp) in single and four-vessel stirred-tank reactor systems has been possible employing enzymes obtained directly from submerged fermentation of Trichoderma viride QM 6a. Studies on the effect of modification of the solid substrate, enzyme stability, substrate concentration, and the influence of reducing sugar concentration on the rate of hydrolysis are reported. While susceptibility of substrate to digestion is not affected by heating alone, it is strikingly increased by heating plus grinding, or by grinding following heating. Batch and steady state continuous saccharification experiments have yielded more than 5% reducing sugar in the effluent with a dilution rate of 0.025 hr^?1 at 50°C, at a substrate level of 10%. An average glucose concentration of 3.4% has been obtained in the effluent of a continuous saccharification using 5% substrate at the same dilution rate and temperature.     

Production of extracellular enzymes in mutants isolated from trichoderma viride unable to hydrolyze cellulose

Mutant strains not producing cellulases were induced and isolated from the cellulolytic fungus Trichoderma viride. Enrichment of mutants was carried out with the aid of nystatin selection. Mutants were shown to lack the ability to hydrolyze both soluble and crystalline cellulose. Mannanase and xylanase activities were also absent, indicating a common regulation for all these enzymes in T. viride. In some strains aryl-beta-glucosidase activity was also missing. Mutants grew normally, but the amount of proteins secreted into the medium was very low, and in most cases these proteins were qualitatively different from the proteins of the parent strain.     

Chemical and ultrastructural studies on the cell walls of the yeastlike and mycelial forms of Histoplasma farciminosum

The cell wall of the yeast form of Histoplasma farciminosum contains 13.2% beta-1,3-glucan, 1.0% galactomannan, and 25.8% chitin, whereas the cell wall of mycelial form has 21.8, 4.5, and 40%, respectively, for the same polymers. Also, the cell wall of the yeast form contains alpha-1,3-glucan (13.5%) and an unidentified polymer (21.5%). Chitin, one of the structural polymers of both yeast and mycelial cell walls, is identified as thin isolated fibers (4 nm wide) or in thick bundles (50 nm wide) of fibers. beta-(1-3)-Glucan is also found as thin isolated fibers indistinguishable from isolated fibers of chitin. Fibers 14 nm wide and resembling alpha-(1-3)-glucan fibers of other fungi are found in the yeast form. The results reported here do not give support to the proposal for a different taxonomic classification.     

β-1, 3-Glucanase from trichoderma viride. Purification and characterization

The extracellular complex of β-glucanases produced by the mould Trichoderma viride hydrolyzes β-1,3-; β-1,4- and β-1, 6-bonds of β-glucans, as well as mixed β-1,3- and β-1,4-glycosidic linkages. This complex contains also xylanase. The enzymes were isolated from liquid culture medium by centrifuge techniques, concentration and precipitation with acetone. Isolation and purification of β-1,3-glucanase was carried out according to a procedure involving filtration on Bio-Gel P-100, DEAE-Sephadex A 50 and CM-cellulose C-11 chromatography, ultrafiltration and selective adsorption on xylan. The homogeneity of the enzyme was determined by polyacrylamidegel electrophoresis. The purified homogeneous preparation of the isolated β-1,3-glucanase from Tr. riride was subjected to detailed characterization. Amino acid composition, molecular weight and optimum conditions for the enzymatic activity of the protein were determined. The isolated enzyme was shown to be highly specific to substrates with β-1,3-glycosidic linkages; the rate of degradation was found to be proportional to the degree of polymerization of the substrate.     

Comparative mycelial and spore yield by Trichoderma viride in batch and fed-batch cultures

The effects of cultural parameters such as carbon and nitrogen source and environmental factors including temperature and pH were investigated on spore and mycelial yield of Trichoderma viride, which has potential as a biocontrol agent against species of Fusarium in batch culture and fed-batch culture where there was limiting nutrient. The results obtained indicated that growth and sporulation of T. viride were greatly influenced by various carbon and nitrogen sources, and by environmental factors such as pH and temperature. Mannitol, wheat bran and rice bran as sole carbon sources appear to stimulate high mycelial growth and spore yield in fed-batch culture. Growth and sporulation were also favoured by NaNO₃, peptone and NH₄SO₄ as the nitrogen sources in fed-batch and batch cultures_. Maximum growth and sporulation was between pH 4.5 and 6.0. Temperatures between 30 and 37 °C were good for mycelium growth of T. viride while temperatures between 30 to 45 °C were good for sporulation. The amount of spore and mycelium produced and the time required for attainment of maximum spore yield increased with increasing carbon and nitrogen source in batch culture. The final spore yield obtained in fed-batch culture was two times higher than the apparent spore-carrying capacity of batch culture. These results show that T. viride is capable of growing and sporulating with varied nutritional and environmental conditions, and, therefore, this strain of T. viride may be useful as a biocontrol agent under diverse physiological and environmental conditions.     

Development,persistence and regeneration of foraging ectomycorrhizal mycelial systems in soil microcosms

Development of extraradical mycelia of two strains each of Paxillus involutus and Suillus bovinus in ectomycorrhizal association with Pinus sylvestris seedlings was studied in two dimensions in non-sterile soil microcosms. There were significant inter- and intra-specific differences in extraradical mycelial growth and morphology. The mycelial systems of both strains of P. involutus were diffuse and extended more rapidly than those of S. bovinus. Depending on the strain, P. involutus mycelia were either highly plane filled, with high mass fractal dimension (a measure of space filling) or sparse, low mass fractal dimension systems. Older mycelial systems persisted as linear cords interlinking ectomycorrhizal tips. S. bovinus produced either a mycelium with a mixture of mycelial cords and diffuse fans that rapidly filled explorable area, or a predominately corded mycelium of minimal area cover. In the soil microcosms, mass fractal dimension and mycelial cover tended to increase with time, mycelia encountering litter having significantly greater values. Results are discussed in terms of the ecology of these fungi, their foraging activities and functional importance in forest ecosystems.     

Incorporation of specific wall proteins during yeast and mycelial protoplast regeneration in Candida albicans

The kinectics of incorporation of two precursor mannoproteins into the regenerating cell wall of Candida albicans protoplasts have been followed at 28°C and 37°C using two monoclonal antibodies specific for protein epitopes (MAb 1B12 and 4C12) as probes. Both molecules were secreted from the beginning of the regeneration process, and their incorporation was retarded significantly. Analysis of the secreted materials by Western immunoblotting with MAb 1B12 allowed the identification of two closely migrating bands at apparent Mr higher than 170 kDa and significant amounts of a highly polydisperse material of even greater molecular mass. Some of these mannoproteinaceous species carried both N- and O-glycosidically linked mannose residues, as deduced from their drop in apparent Mr when synthesized in the presence of tunicamycin and by their reactivity with Concanavalin A. Following secretion, the molecules reacting with MAb 1B12 were incorporated into the regenerating walls by covalent binding. Then, when the regenerating walls by covalent binding. Then, when the antigen molecules were solubilized from partially regenerated walls, their mobility differed when regeneration took place at 28°C (blastoconidia) or 37°C (mycelial cells).Abbreviations used IIF indirect immunofluorescence - MAb monoclonal antibody - PBS phosphate-buffed saline - PMSF phenyl methyl sulfonyl fluoride - SDS sodium dodecyl sulfate     

Chemical differences between thyrotropin isohormones.

Amide determinations have been carried out, with a newly developed g.l.c. method, on thyrotropin hormones prepared by isoelectric focusing. The observed differences in amide content fully account for the differences in the electrochemical properties of the isohormones.     

Chemical modification of the surfaces of bacterial cell walls.

The surfaces of the isolated cell walls of four bacterial species were studied by microelectrophoresis following chemical treatments intended to remove specific charged groups. Acid-base titrations of the walls were used to assess specificity and extent of the modifications. Carboxyl groups were specifically and completely modified by activation with a water-soluble carbodiimide and subsequent reaction with a nucleophile, such as glycinamide, to give an uncharged pH-stable product. Aqueous media and mild reaction conditions make the method suitable for modifying carboxyl groups on cell surfaces too labile to withstand the harsh conditions required for conventional esterification reactions. Use of the carbodiimide-mediated reaction for discharging carboxyl groups, along with fluorodinitrobenzene for discharging amino groups and extraction procedures for removing constituents carrying phosphoester groups (teichoic acids), made it possible to obtain information about the spatial arrangement of charged groups on the wall surfaces. Removal of the exterior negative charge dominating wall surfaces allowed underlying amino groups to become electrokinetically effective and, in the case of E. coli, also revealed a lipophilic region with an affinity for a cationic surfactant.     

Detection and characterization of two antigens specific for cell walls ofCandida albicans mycelial growth phase

Antiserum againstCandida albicans ATCC 10231 mycelium growth phase absorbed with yeast cells and intracellular material of mycelium cells showed a positive reaction with mycelium cells in immunofluorescence assay, whereas with yeast cells the reaction was negative. Mycelium and blastospore cell wall were extracted with dithiothreitol (DTT_My- and DTT_B-extract). When DTT_My was separated in gel-electrophoresis, two glycoprotein bands of 87 and 67 Kd could be detected. In immunoblot these bands showed a strong reaction with mycelium cell wall-specific antiserum, but also a weak reaction with the blastospore antiserum. Whereas pronase treatment destroyed antigenicity, mannanase treatment did not. After enzymatic digestion with endoglycosidase H, four major enzymatic digestion products were found at 37, 35, 30, and 27 Kd when protein staining was performed. The digestion products at 37 and 35 Kd could be made visible through glycoprotein staining. Antibodies of yeast-phase-immunized animals reacted only with the 37 and 30 Kd bands, whereas the digestion products at 35 and 27 Kd were also detected by mycelium cell wall-specific antiserum. This proves the existence of two mycelium cell wall-specific antigens (35 Kd and 27 Kd) that can be isolated from the DTT-mycelium cell wall extract by endoglycosidase H treatment.