Protoplasts of Trichoderma viride

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.     

The effect of acid pH on the growth kinetics of Trichoderma viride.

Batch cultures of Trichoderma viride have been carried out in a 10 liter stirred fermenter a controlled pH values of 2.5, 2.7, 3.0, and 4.0 and without pH control at a temperature of 28 degrees C. Cell and glucose concentrations and dissolved oxygen values are reported. The yield coefficient was found to be constant at 0.40 kg cells/kg glucose and the maximum specific growth rate was linearly correlated with the hydrogen ion concentration.     

The effects of deuterium oxide on bacteria

Summary The growth of several strains of bacteria was inhibited by D₂O-media. The degree of inhibition was strain specific. The incorporation of 0.5% NaCl (w/v) to the D₂O-medium decreased the inhibition of growth. No adaptation to better growth in deuterium was obtained in any of the strains tested; however a deuterium resistant mutant was obtained from one strain. Two strains tested formed enlarged, distorted cells when grown for more than eight generations in D₂O-media. Cells grown in D₂O-media and washed and resuspended in H₂O-0.85% NaCl (w/v), were up to sixty times more sensitive to ultraviolet irradiation than control cells. Deuterium induces the occurrence of forward mutants in some strains. Many loci, but not all, showed an increase in the back-mutation rate over the spontaneous level, indicating some specificity of action by deuterium. Deuterated bases, obtained from a strain grown in D₂O-medium, did not induce any phenotypic or genotypic effects when supplied to specific baserequiring strains. The amout of deuterium incorporated into the bacteria may be related to the mutagenic effect induced. The genotypic and phenotypic effects induced by deuterium are probably the result of an overall isotope effect.With 2 Figures in the TextThe work reported here was supported by the Atomic Energy Commission research grant AT(30-1) 2184 to Dr.Stephen Zamenhof, Columbia University. This work was submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Faculty of Pure Science, Columbia University.I dedicate this paper to Prof.L. C. Dunn with thanks for his enthusiastic and inspiring instruction.     

The infection of oranges by Trichoderma viride and mixed infection by Trichoderma viride and Penicillium digitatum

Trichoderma viride spores applied in water to apparently uninjured skin of oranges do not cause lesions. Adding orange juice, rind extract, citric acid or orange essential oil did not influence infection. Oranges became infected only when the stem-end cuts or wounds deeper than 6 mm into oil vesicles were inoculated. Sound oranges in contact with decayed oranges did not become infected. Diphenyl-impregnated wrappers reduced infection. A mixed inoculum of T. viride and Penicillium digitatum caused as fast rotting as P. digitatum, which caused faster rotting than T. viride alone. Lesions infected with P. digitatum could become infected by T. viride but those caused by T. viride did not become infected by P. digitatum. T. viride was antagonistic to P. digitatum in vivo and in vitro, possibly because it produces a heat-labile diffusible substance toxic to P. digitatum.     

The glycosulphatase of Trichoderma viride

The growth of the mould Trichoderma viride on a defined medium containing either potassium d-glucose 6-O-sulphate or potassium d-galactose 6-O-sulphate as sole sources of both carbon and sulphur is marked by the production of an enzyme system capable of liberating inorganic SO(4) (2-) ions from either of the sulphate esters. The enzyme is not produced when the organism is grown with glucose (or galactose) and potassium sulphate or with glucose and methionine as sole sources of carbon and sulphur. Experimental conditions are described whereby inorganic SO(4) (2-) ions liberated from potassium glucose 6-O-sulphate by the growing mould appear in the culture medium after a constant lag period of 21-24hr. The enzyme has been shown to be a simple glycosulphatase that is active towards the 6-O-sulphate esters of d-glucose and d-galactose but not towards potassium glucose 3-O-sulphate. The properties of the crude glycosulphatase show the enzyme to be appreciably different from analogous molluscan enzymes that can degrade monosaccharide sulphate esters.     

Continuous cultivation of Trichoderma viride on cellulose

Using ball milled cellulose as the only carbon source Trichoderma viride was grown in a continuous flow culture at pH = 5.0 and T = 30°C. Steady-state values for cell protein, cellulose, and cellulase for different substrate concentrations (4–11 g/liter) and dilution rates (0.033–0.080 hr^?1) were obtained. Under steady-state conditions, 50–75% of the cellulose was consumed indicating a critical dilution rate on 0.17 hr^?1. Cellulase activity (U/ml) in the fermentation broth increased slightly with increasing substrate concentration and decreased with increasing dilution rate, while the specific cellulase productivity (U/mg cell protein·hr) was fairly independent of the dilution rate, with a maximum around D = 0.05 hr^?1. Following step changes in substrate concentration and dilution rate, new steady-state values were reached after three to five residence times (cell protein and cellulose) and four to six residence times (celullase activity).     

绿色木霉木聚糖酶的纯化和性质

绿色木霉木聚糖酶经分离纯化后,获得三个组分木聚糖酶,称为XⅠ,XⅡ和XⅢ,它们最反应温度分别为60℃、60℃、50℃,pH分别为5．5、5．0、0、4．5,pⅠ分别为XⅠ3．8,XⅡ3．4,XⅢ3．6。半失活温度分别为XⅠ37℃,XⅡ44℃,XⅢ40℃。     

Bioaccumulation of copper by Trichoderma viride

Studies were carried out on interaction of Trichoderma viride with copper and reports bioaccumulation as a mechanism of copper tolerance during growth. There was a marked increase in the lag phase of the growth, which was concentration dependent. At a concentration of 100 mg/L of CuCl2.2H2O, 81% of Cu(II) were removed by 3.4 g/L of the biomass in 72 h. The process was temperature and pH dependent. The maximum copper bioaccumulation occurred at 30 degrees C, pH 5.0. Metabolic inhibitors such as sodium azide (NaN3) and 2,4-dinitrophenol (2,4-DNP) drastically reduced the extent of Cu(II) bioaccumulation. Electron microscopy and cell fractionation studies revealed that 70-80% of copper was present as a layer on the cell wall surface.     

Adsorption of cellulose by Trichoderma viride

Adsorption of cellulase by Trichoderma viride QM 9414 has been studied with resting and growing cells and equations have been derived to describe the process quantitatively. It has been observed that the adsorption is a purely physical process being dependent only on cell and cellulose concentrations. It has also been demonstrated that adsorption isrequired for the induction of cellulases; some discussions are devoted to this point.     

绿色木霉代谢产物的植物毒性研究

通过人工固体培养和液体发酵研究发现绿色木霉的代谢产物对植物的幼苗生长有抑制作用,其代谢产物大量分泌到它所生长的环境中,在不同的营养基质中其代谢产物的抑制作用有差异．     

Cytostatic effect of L-lysine-alpha-oxidase from Trichoderma harzianum Rifai and Trichoderma viride

L-lysine-alpha-oxidase, a new fungal enzyme catalyzing oxidative L-lysine deamination, was shown to have an inhibitory effect on the in vitro synthesis of DNA, RNA and proteins in human carcinoma ovarian (CaOv) cells.     

Adsorption of cellulase from Trichoderma viride on cellulose

The adsorption of cellulase from Trichoderma viride (Meicelase CEP) on the surface of pure cellulose was studied. The adsorption was found to obey apparently the Langmuir isotherm. From the data concering the effects of temperature and the crystallinity of cellulose on the Langmuir adsorption parameters, the characteristics of the adsorption of the individual cellulase components, namely CMCase (endoglucanase) and Avicelase (exoglucanase), were discussed. While beta-glucosidase also adsorbed on the surface of cellulose at 5 degrees C, it did not at 50 degrees C.     

Differential responses of wood-rot fungi cellulases towards polyclonal antibodies against Trichoderma viride cellobiohydrolase I

Cellobiohydrolase (CBH) I, a main component of Trichoderma extracellular protein, was purified to an electrophoretically homogeneous state from a commercial cellulase preparation (Meicelase from T. viride) by column chromatography on anion and cation exchangers. The difference in the cross-reactivity of cellulolytic enzyme systems of brown-rot and white-rot fungi with the polyclonal antibodies to the CBH I was studied by enzyme-linked immunosorbent assay (ELISA). The antibodies were observed to react quantitatively and with great sensitivity with the antigen (CBH I), and at the same time to cross-react to some extent with T. viride cellulase components other than the CBH I. Nevertheless, the intensity of cross-reactivity of wood-rot fungi cellulases with the antibodies was parallel to the activity of exo-1,4-ß-glucanase. The cellulase system from brown-rot fungi, believed to lack exo-1,4-ß-glucanases, gave a negative response towards the antibodies. These results suggested the presence of some homologous sequences and structures with the T. viride CBH I in the enzymes of white-rot fungi and their absence in those of brown-rot fungi. Correspondence to: M. Ishihara     

Specific and nonspecific glucanases from Trichoderma viride

Six endoglucanases (Endo I, II, III, IV, V, and VI), three exoglucanases (Exo I, II, and III), and a beta-glucosidase (beta-gluc I) isolated from a commercial cellulase preparation of Trichoderma viride origin were examined as to their activities on xylan ex oat spelts. Endo I, II, and III as well as Exo II and III showed no activity toward xylan and were classified as specific glucanases. Less specificity was found for the endoglucanases Endo IV, V, and VI, Exo I, and beta-gluc I, whose enzymes were able to hydrolyze xylan. With respect to product formation these xylanolytic cellulases fit the classification of xylanases generally accepted in the literature. Kinetic experiment with xylan, CM-cellulose, and p-nitrophenyl-beta-D-glucoside revealed that Endo IV, V, an VI and Exo I prefer to hydrolyze beta-1, 4-D-glucosidic linkages. beta-Gluc I showed no clear substrate preference.     

Cellulase Production by Trichoderma viride on Feedlot Waste

Feedlot waste contains essentially all the necessary nutrients for batch fermentation with the fungus Trichoderma viride. The organism utilizes two-thirds of the carbohydrate in feedlot waste while elaborating cellulase in quantities comparable to commercial preparations. Essentially odor-free, the fermented waste contains all of the original nitrogen but has 24% less organic matter.     

Location and Formation of Cellulases in Trichoderma viride

The location and formation of cellulases and β-glucosidase in the fungus Trichoderma viride were studied on different substrates. The cellulases were found to be cell-bound during active growth on cellulose, CMC, and cellobiose. On CMC, much CM-cellulase was found cell-free but sonication released cellulase from the washed mycelium. Analysis of the carbohydrates of the mycelial cell wall after hydrolysis revealed glucose, mannose, and galactose—the same carbohydrates as reported to be present in purified cellulase from the same organism. Glucose repressed the formation of both CM-cellulase and Avicelase and cellobiose apparently the formation of Avicelase. Relatively little CM-cellulase was formed on cellobiose but a straight line was obtained when a differential plot of CM-cellulase versus protein was made.