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PubMed1975

Fungal cellulase and hemicellulase prediction of forage digestibility.

An enzymatic procedure using Trichoderma viride carbohydrases, a fungal hemicellulase, and pepsin was developed to provide a laboratory method for predicting forage digestibility. The amount of forage dry matter solubilized by enzymes and incubation buffer was less than that in vivo or by in vitro fermentation by rumen microorganisms. Total forage dry matter solubilized by the enzymatic procedure was correlated (.92) with in vitro digestibility. Simple correlation coefficients between in vivo true digestibility of 18 forages and total dry matter solubilized by enzymes and buffer was .87; that soluble in the buffer only, .65; and that solubilized by the enzymes, .82. Correlation coefficients with in vivo true digestible amount of cell-wall and protein were greater when the forage species were considered separately than when pooled together. With this restriction, the enzymatic procedure can be a useful method for predicting forage digestibility. Fermentations with rumen inoculum however, provided more accurate predictions of in vivo digestibility in a variety of forage species.

Animal Feed↗

PubMed1996

Kinetics of cell-wall digestion of orchardgrass and alfalfa silages treated with cellulase and formic acid.

The objectives of this study were to determine the effects of cellulase (from Trichoderma longibrachiatum) combined with formic acid, applied before ensiling, on the subsequent concentration and composition of the cell wall and on the extent and rate of in situ cell-wall digestion of orchardgrass (Dactylis glomerata L.) and alfalfa (Medicago sativa L.). Treated and control forages of both plant species were ensiled for at least 60 d before being ruminally digested by two fistulated cows. Analyses of NDF, ADF, and acid detergent lignin were conducted sequentially on original and digested samples. Data were fitted with a first-order, nonlinear model to estimate extents and rates of digestion of potentially digestible NDF, cellulose, and hemicellulose. The concentration of indigestible residue and the discrete lag time before digestion were also determined for the cell-wall components. After ensiling, the mean NDF concentration of treated silages was 19% lower than that of control silages; the effect was greater for orchardgrass than for alfalfa. The extent of digestion of NDF, cellulose, and hemicellulose, respectively, was 33, 37, and 27% lower for treated silages than for control silages. Treatment effects on the extent of digestion varied between plant species. Cellulose from treated orchardgrass was digested 19% more slowly than cellulose from the control silage. Indigestible residue concentrations of NDF, cellulose, and hemicellulose, respectively, were 7, 8, and 7% lower in treated silages than in control silages. Thus, extensive cell-wall degradation by cellulase during ensiling resulted in less digestible cell-wall material for ruminal digestion but greater total cell-wall degradation, including that during ensiling and ruminal incubation, especially during early digestion in the rumen.

Cell Wall↗

PubMed1991

The effects of enzyme supplementation on the nutritive value of rye grain (Secale cereale) for the young broiler chick.

Two dose-response experiments utilizing differing enzyme concentrations were carried out to determine the level of a crude enzyme preparation (Trichoderma viride cellulase) required to obtain maximum growth performance in young broiler chicks fed a 60% rye diet. Enzyme concentrations utilized in these studies ranged from .05 to 12.8 g enzyme/kg diet. Enzyme supplementation at the 3.2 g/kg diet level yielded increases in feed consumption and weight gain of 71 and 193%, respectively; and the feed to gain ratio was improved by 43% compared with that for the unsupplemented rye control diet. As no further growth response was obtained by increasing the enzyme concentration, the enzyme level utilized in a subsequent study was 3.2 g/kg diet. A balance study using 2-wk-old broiler chicks was carried out to determine the effect of enzyme supplementation on the bioavailable energy (AMEn) and apparent protein digestibility (APD) of a 60% wheat diet replaced by rye in 10% increments. In the 60% rye diets, the enzyme addition increased the dietary AMEn and APD by 23 and 12%, respectively, compared with the unsupplemented diet. The AMEn and APD of the 60% wheat control diet were increased 9.7 and 4.6%, respectively, by enzyme addition. The data suggest that the AMEn of rye grain is increased by enzyme supplementation; however, alternate methodology will have to be employed to quantitatively determine the extent of these improvements.

Animal Feed↗

PubMed2006

Biological control of peach fungal pathogens by commercial products and indigenous yeasts.

The potential use of the commercial biocontrol products Serenade (Bacillus subtilis QST-713) and Trichodex (Trichoderma harzianum Rifai strain T39) to inhibit the postharvest pathogenic molds Penicillium crustosum and Mucor circinelloides was investigated. Both products exhibited antagonistic activity in vitro against the pathogens, reducing their growth at different levels. In addition, epiphytic yeasts isolated from peaches were identified as Candida maltosa, Pichia fermentans, and Pichia kluyveri by PCR-restriction fragment length polymorphism of internal transcribed spacer regions and screened for antagonistic activity against the same molds. The efficacy of biocontrol in vitro was dependent on the concentration of the yeast cells. Optimal yeast concentrations were above 10(7) CFU ml(-1). However, C. maltosa and P. fermentans were more effective than P. kluyveri in inhibiting molds. The exclusion of antifungal metabolite production and direct competition for nutrients or space with the pathogens was proposed as the mechanism of biocontrol. Application of biocontrol agents directly on artificially wounded peach fruits significantly reduced the incidence of mold rot during storage at 20 degrees C.

Antibiosis↗

PubMed1980

A new sesquiterpene antibiotic, heptelidic acid producing organisms, fermentation, isolation and characterization.

A new sesquiterpene antibiotic, heptelidic acid, was found in the culture filtrate of three different strains of fungi isolated from soil samples. These strains were identified as Gliocladium virens, Chaetomium globosum and Trichoderma viride. Heptelidic acid was produced by conventional submerged culture and purified by successive column chromatography on silica gel and Sephadex LH-20 and finally by preparative TLC on silica gel. The molecular formula of heptelidic acid was determined as C15H20O5 on the basis of elementary analysis and high resolution mass spectrometry of its monomethyl ester. The antimicrobial spectrum of the antibiotic revealed its specific activity against anaerobic bacteria, especially against Bacteroides fragilis.

Animals↗

PubMed1985

Specific inhibition of glyceraldehyde-3-phosphate dehydrogenase by koningic acid (heptelidic acid).

A sesquiterpene named koningic acid has been isolated from a strain of Trichoderma koningii as a potent inhibitor of ATP generation in the glycolytic pathway. From experiments with both cultured mouse carcinoma FM3A cells and isolated enzymes, it was shown that koningic acid is a specific inhibitor of glyceraldehyde 3-phosphate dehydrogenase that catalyzes the conversion of glyceraldehyde 3-phosphate to 3-phosphoglycerate.

Adenosine Triphosphate↗

PubMed1996

Melanoxazal, new melanin biosynthesis inhibitor discovered by using the larval haemolymph of the silkworm, Bombyx mori. Production, isolation, structural elucidation, and biological properties.

A new melanin biosynthesis inhibitor, melanoxazal, was isolated from the fermentation broth of Trichoderma sp. ATF-451 by successive purification procedures of carbon adsorption, ethyl acetate extraction and silica gel column chromatography. The inhibitor possesses a novel oxazole-containing structure with molecular formula, C8H9NO3. The structure was determined by means of NMR analyses to be (E)-4-(2'-formyl-3'-hydroxybuten-1'-yl) oxazole, which is related to melanoxadin. Melanoxazal inhibited melanin formation in the larval haemolymph of the silkworm, Bombyx mori; IC50 value = 30.1 micrograms/ml. Melanoxazal also showed a strong inhibitory activity against mushroom tyrosinase with IC50 value = 4.2 micrograms/ml.

Animals↗

PubMed1998

Cytotoxic substances produced by a fungal strain from a sponge: physico-chemical properties and structures.

Five novel metabolites, trichodenones A-C (1-3), harzialactone A (4) and B (5), have been isolated together with known R-mevalonolactone (6) from the culture broth of a strain of Trichoderma harzianum OUPS-N115 originally separated from the sponge Halichondria okadai. Their structures have been elucidated by spectral evidence. Among them, 1-3 exhibited significant cytotoxicity against cultured P388 cells.

Animals↗

PubMedDate not supplied

Allocation of ATP to synthesis of cells and hydrolytic enzymes in cellulolytic fermentative microorganisms: bioenergetics, kinetics, and bioprocessing.

Under anaerobic, carbon limited conditions, celluloytic fermentative microorganisms face a metabolic choice with respect to the allocation of relatively scarce ATP: to invest it in cells or in hydrolytic enzymes. A model is proposed that defines an allocation parameter reflecting the fractional expenditure of ATP on cell synthesis relative to the total ATP available (gross ATP synthesized less maintenance). This parameter is then incorporated into an ATP-centered model of anaerobic cellulose fermentation based on the ethanol fermentation of yeast and the cellulase system of Trichoderma reesei. Results indicate that high processing rates are possible via a consolidated bioprocessing strategy, especially at high cellulase specific activities, and that cell/cellulase allocation represents an interesting system in which to study, and perhaps exploit, microbial evolution and metabolic control.

Adenosine Triphosphate↗

PubMed1999

Gallotannin hydrolysis by immobilized fungal mycelia in a packed bed bioreactor.

Hydrolysis of gallotannin to gallic acid by immobilized mycelia of Aspergillus niger MTCC 282, Aspergillus fischerii MTCC 150, Fusarium solani MTCC 350 and Trichoderma viride MTCC 167 in a packed bed bioreactor was studied. Fungal mycelia preinduced with 5 g L-1 gallotannin were immobilized in calcium alginate gel (1.5%) and the resultant beads were packed in a column to a bed volume of 175 mm3. Gallotannin dissolved in distilled water was passed through the column and the eluate was recycled after adjusting pH to 6 with ammonium hydroxide (10%). Maximum hydrolysis of gallotannin was recorded by immobilized mycelia of F. solani and T. viride at 35 degrees and 45 degrees C after 175 and 60 min of residency period respectively. Optimum substrate concentration required for maximum hydrolysis was 10 g L-1 at pH 5 for both the fungi. Immobilized mycelia of A. niger and A. fischerii revealed maximum operational stability. Loss of activity after eighth run was in the order of-A. niger (no loss), A. fischerii (7.5%), F. solani (18%) and T. viride (18%). Stability in terms of retention of enzyme activity after 150 days of storage at 4 degrees C was A. niger (58%), A. fischerii (26.8%), F. solani (83%) and T. viride (85.1%).

Aspergillus↗

PubMed1998

Construction and application of MCBL plate for facilitation of chromosome recombination in fungi.

A medium with camphor and benomyl MCBL plate was designed and constructed based on the proposed mechanism that d-camphor could induce the fusion of nuclear membrane while benomyl could induce the nondisjunctional recombination of chromosome in fungi. This so-called co-induction plate consisted of 0.1% d-camphor (W/V) and 0.5 microgram/L benomyl contained in Czapek's minimal medium. The precautions to be taken in the construction procedure of this plate was described in detail. One typical example of intergeneric fusion-cross, Aspergillus niger x Trichoderma reesei, was investigated, comparing the ratios of genotypes and phenotypes of fusant progenies produced by the co-induction of MCBL plate and by the step-by-step induction with camphor and benomyl separately. The results showed that the heterodiploid state was extremely transient and the recombinant haploid was hardly obtained when induced by the routine step-by-step method, whereas the ratios of apparent diplodization and nondisjunctional recombinant haplodization among all types of varied segregates on MCBL plate were greatly improved, compared with those on the routine plates containing single reagents, which indicated that the transient heterodiploid could be grasped and transformed further into nondisjunctional recombinant haploid when co-induced on the MCBL plate. The age of regenerated mycelium to be induced was found to have a dominant influence on the co-induction effects of MCBL plate. The mechanism of co-induction by MCBL plate and its promising applications were discussed.

Aspergillus niger↗

PubMed1999

Enhancement of the affinity of cellobiohydrolase I and its catalytic domain to cellulose in the presence of the reaction product--cellobiose.

The catalytic domain of cellobiohydrolase I from Trichoderma reesei has been obtained by papain treatment of the native enzyme adsorbed onto the surface of microcrystalline cellulose. Both the intact and the truncated enzyme are almost equally active toward soluble fluorogenic derivatives of cellobi-, -tri-, -tetra-, and -pentaose, the fastest and the slowest fluorophore liberation being observed for MUF-cellopenta- and -tetraose, respectively. Titration of the active centers of the intact enzyme and its catalytic domain with MUF-cellotetraose showed their molecular masses to be 49 and 39 kD, respectively, the dissociation constants of the enzyme-soluble ligand complexes being almost equal (65 and 70 nM at 20 degrees C, respectively). In contrast, the intact enzyme and its catalytic core have been shown to significantly (50-60 times) differ in their affinity to insoluble microcrystalline cellulose at low enzyme loading (up to 10 mg per g of the substrate). At 20 degrees C the dissociation constants for the two forms of the enzyme are estimated to be 10 and 500 nM, respectively. Surprisingly, under these conditions the reaction product and inhibitor, cellobiose (Ki = 10 microM), at the concentration 10 mM, increased 3-4-fold the affinity of both the intact cellobiohydrolase and its catalytic domain to cellulose.

Cellobiose↗

PubMed2000

Epoxyalkyl glycosides of D-xylose and xylo-oligosaccharides are active-site markers of xylanases from glycoside hydrolase family 11, not from family 10.

A series of omega-epoxyalkyl glycosides of D-xylopyranose, xylobiose and xylotriose were tested as potential active-site-directed inhibitors of xylanases from glycoside hydrolase families10 and 11. Whereas family-10 enzymes (Thermoascus aurantiacus Xyn and Clostridium thermocellum Xyn Z) are resistant toelectrophilic attack of active-site carboxyl residues, glycosidehydrolases of family 11 (Thermomyces lanuginosus Xyn and Trichoderma reesei Xyn II) are irreversibly inhibited. Theapparent inactivation and association constants (k(i), 1/K(i)) are one order of magnitude higher for thexylobiose and xylotriose derivatives. The effects of the aglycone chainlength can clearly be described. Xylobiose and n-alkyl beta-D-xylopyranosides are competitive ligands and provide protectionagainst inactivation. MS measurements showed 1:1 stoichiometries inmost labelling experiments. Electrospray ionization MS/MS analysisrevealed the nucleophile Glu(86) as the modified residue inthe T. lanuginosus xylanase when 2,3-epoxypropyl beta-D-xylopyranoside was used, whereas the acid/base catalyst Glu(178) was modified by the 3,4-epoxybutyl derivative. The active-site residues Glu(86) and Glu(177) in T. reesei Xyn II are similarly modified, confirming earlier X-raycrystallographic data [Havukainen, Törrönen, Laitinen and Rouvinen (1996)Biochemistry 35, 9617-9624]. The inability of the omega-epoxyalkyl xylo(oligo)saccharide derivatives to inactivate family-10enzymes is discussed in terms of different ligand-subsiteinteractions.

Alkylation↗

PubMedDate not supplied

[Enzymatic hydrolysis of willow treated with a steam burst without preliminary water extraction with a high concentration of substrate].

A laboratory reactor equipped with a screw press was used for hydrolysis of steam-SO2 exploded willow Salix caprea by a composition of Trichoderma reesei and Aspergillus foetidus enzyme preparations at high substrate concentrations. Optimal conditions providing the maximal volume of hydrolysis syrup with maximal sugar concentrations were determined. Two different hydrolysis procedures were developed in order to exclude initial washing of steam-pretreated plant raw material by large volumes of water, which is necessary to eliminate the inhibitory effect of explosion by-products on enzymatic hydrolysis. The first procedure included a one-hour-long enzymatic prehydrolysis of the substrate, then separation of sugar syrup containing 40-60 g/l of glucose, 20-25 g/l of xylose, and up to 10% of disaccharides, as well as up to 35% of the initial enzymatic activity, then addition of a diluted acetate buffer (pH 4.5), and subsequent hydrolysis of the substrate by the adsorbed enzymes leading to the final accumulation of up to 140 g/l glucose and up to 15 g/l xylose. In the second scenario, the exploded willow was initially adjusted by alkali to pH 4.5 and then hydrolyzed directly by added enzymes for 24 hours. This procedure resulted in a nearly total polysaccharide hydrolysis and accumulation of up to 170 g/l glucose and 20 g/l xylose. The reasons of inhibition of enzymatic hydrolysis are discussed.

Aspergillus↗

PubMedDate not supplied

[Enzyme regeneration during hydrolysis of steam-pretreated willow and requirement for cellulase complex composition].

In order to reduce the total enzyme consumption in high-solids static hydrolysis of nonwashed steam-exploded willow Salix caprea by mixed cellulase of Trichoderma reesei + Aspergillus foetidus, two different approaches were proposed. In the first case, the enzyme activity adsorbed on residual solids after extended hydrolysis was used for hydrolysis of the newly added substrate. The initial mixing of fresh and hydrolyzed substrates was sufficient for the adsorbed enzyme redistribution and conversion of the new substrate portion, and permanent mechanical stirring was not required. Feeding of two additional portions of the exploded hardwood adjusted to pH 4 with dry caustic into the reactor with simultaneous replacement of accumulated sugars with fresh buffer (pH 4.5) resulted, on average, in a 90% conversion of cellulose at the final enzyme loading 8 IFPU per g ODM substrate, an average sugar concentration of 12%, and a glucose/xylose ratio of 5:1. In the second approach, weakly adsorbed cellulase fractions were used for static high-solids hydrolysis followed by their ultrafiltration recovery from the resultant sugar syrup. In contrast to the initial cellulase mixture whose residual activity in a syrup did not exceed 5-10% at the end of hydrolysis (48 h), up to 60% of weakly adsorbed enzyme fraction could be separated from sugar syrups by ultrafiltration and then reused. Weakly adsorbed enzymes displayed a hydrolysis efficiency of not less than 80% per IFPU enzyme consumed in extended hydrolysis of pretreated willow as compared to the original enzyme mixture. An electrophoretic study of the weakly adsorbed enzyme fraction identified T. reesei cellobiohydrolase II as the predominant component, whereas clear domination of T. reesei cellobiohydrolase I was found by electrophoresis of proteins tightly bound to hydrolysis residual solids.

Aspergillus↗

PubMed2002

Verticillium wilt of cauliflower in Belgium.

Since several years commercial cauliflower in Belgium is severely affected by a vascular wilt disease. Plants wilt and a vascular discoloration of the cauliflower stems can be observed. The pathogen causes significant yield and quality losses. The first objective of this study was to isolate and characterise the causal agent of this disease. Verticillium species could be isolated out of the vascular tissue of symptomatic cauliflower plants. Morphological and physiological characteristics indicated that the isolates from cauliflower could be identified as the new and brassica-related species V. longisporum. Secondly, the susceptibility of several cauliflower cultivars to V. longisporum was evaluated by way of field experiments in naturally infested soil. At harvest, vascular discoloration was evaluated by a visual score. These experiments pointed to considerable differences between cultivars in their susceptibility to V. longisporum. The final goal of this study was to find a way to control the wilt disease on cauliflower. A field experiment showed that vascular discoloration of cauliflower was significantly reduced when soil was incorporated with broccoli residues, two Brassica green manures (B. juncea or B. nigra) or two commercial biological products based on Talaromyces flavus or Trichoderma sp..

Belgium↗

PubMedDate not supplied

[Catalytic properties of induced L-lysine-alpha-oxidase].

Regulation of biosynthesis of L-lysine-alpha-oxidase from Trichoderma sp using biostimulators was studied. Specific activity of L-lysine-alpha-oxidase was increased 2.7-fold in presence of biostimulators; this occurred due to induction of the enzyme, properties of which were described previously. Catalytic and physico-chemical properties of the enzyme studied were not altered after addition of stimulators into the cultivation medium of the producing strain. Small amounts of L-lysine-alpha-oxidase isozyme were detected in the medium during cultivation; the isozyme was less active and had lower molecular mass as compared with the enzyme studied.

Amino Acid Oxidoreductases↗

PubMed2003

[Applied studies of biological control techniques on healthful Chinese crude drugs].

Medicinal plants always suffer from diseases and pests in their cultivation and growing, and the yield and quality are reduced. At present, chemical pesticide is mainly used to control the diseases and pests. The long-term use and over-use of pesticide result in pesticide residue, pests resistance to pesticide, water and soil pollution and other environmental contamination. It is necrssary to provide the integrated pests management, including new technology of biological control for developing green drug. This project makes research on the following aspects: 1. Applied studies on bethyloid wasp (Scleroderma guani) control for stem borer; 2. To develop Trichoderma spp. and use it to control the diseases of medicinal plants; 3. Applying Agro-antibiotics 120 to control phytophthora root rot of ginseng; 4. Researches on application of entomopathogenic nematodes against ten-spotted lema (Lema decempuntata) et al.

Animals↗