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.     

Biodegradation of wastepaper by cellulase from Trichoderma viride

Environmental issues such as the depletion of non-renewable energy resources and pollution are topical. The extent of solid waste production is of global concern and development of its bioenergy potential can combine issues such as pollution control and bioproduct development, simultaneously. Various wastepaper materials, a major component of solid waste, were treated with the cellulase enzyme from Trichoderma viride, thus bioconverting their cellulose component into fermentable sugars. All wastepaper materials exhibited different susceptibilities towards the cellulase as well as the production of non-similar sugar releasing patterns when increasing amounts of paper were treated with a fixed enzyme concentration. The hydrolysis of wastepaper with changing enzyme concentrations and incubation periods also resulted in dissimilar sugar-producing tendencies. A general decline in hydrolytic efficiency was observed when increasing sugar concentrations were produced during biodegradation of all wastepaper materials.     

Production of cellulase and protein from barley straw by Trichoderma viride

The cellulase production by two strains of the cellulolytic fungus Trichoderma viride was examined. The fungi were grown on different preparations of barley straw pretreated with NaOH under high pressure. The production of cellulases and microbial protein by the better strain (QM 9123) was investigated in an aerated 5-liter fermenter under varying stirring rates (200-350 rpm) and straw concentrations (1–2%). The pH was kept between 3.5 and 4.5. The growth of the fungus was followed by measuring the quantity of CO₂ produced and the cell protein. After 2–6 days growth ceased, the lag phases lasting 0–2 days, increasing with increasing straw concentrations. The maximum enzyme yields were reached after 4–10 days. The protein content of the product was 21–26% and up to 70% of the straw was utilized. The yield constants were calculated to be 0.40–0.56; of the same order as those which can be obtained by growing the fungus on glucose.     

Transglycosylation of cellobiose by partially purified Trichoderma viride cellulase.

A commercial cellulase from Trichoderma viride was fractionated into three fractions, F1, F2, and F3, in order to investigate transglycosylation activities. Among these fractions, F3, which demonstrated highly hydrolytic activity toward p-nitrophenyl beta-D-glucopyranoside and Avicel, most effectively catalyzed the transglycosylation of cellobiose and converted cellobiose into beta-Glc-(1-->6)-beta-glc-(1-->4)-Glc and beta-Glc-(1-->6)-beta-Glc-(1-->6)-beta-Glc(1-->4)-Glc. The F3 fraction contained the enzyme to catalyze beta-glucosyl transfer toward only the C-6 position of the sugar acceptor, and thus it is expected to be of use for syntheses of functional oligosaccharides.     

Enzymatic studies on a cellulase system of Trichoderma viride. III. Transglycosylation properties of two cellulase components of random type.

Two highly purified cellulases [EC 3.2.1.4], II-A, and II-B, were obtained from the cellulase system of Trichoderma viride. Both cellulases split cellopentaose retaining the beta-configuration of the anomeric carbon atoms in the hydrolysis products at both pH 3.5 and 5.0. The Km values of cellulases II-A and II-B for cellotetraose were different, but their Vmax values were similar and those for cellooligosaccharides increased in parallel with chain length. Both cellulases produced predominantly cellobiose and glucose from various cellulosic substrates as well as from higher cellooligosaccharides. Cellulase II-A preferentially attacked the holoside linkage of rho-nitrophenyl beta-D-cellobioside, whereas cellulase II-B attacked mainly the aglycone linkage of this cellobioside. Both cellulases were found to catalyze the synthesis of cellotriose from rho-nitrophenyl beta-D-cellobioside by transfer of a glucosyl residue, possibly to cellobiose produced in the reaction mixture. They were also found to catalyze the rapid synthesis of cellotetraose from cellobiose, with accompanying formation of cellotriose and glucose, which seemed to be produced by secondary random hydrolysis of the cellotetraose produced. The capacity to synthesize cellotetraose from cellobiose appeared to be greater with cellulase II-B than with cellulase II-A.     

Sugar production from agricultural woody wastes by saccharification with Trichoderma viride cellulase.

The saccharification of agricultural woody wastes was studied using a commercial enzyme preparation, Cellulase onozuka, derived from Trichoderma viride or the solid culture extracts of the fungus. With the intention of producing sugar at low cost, a simple procedure of enzymatic saccharification of rice straw, bagasse, and sawdust was studied. Delignifying methods of these wastes were investigated using dilute sodium hydroxide solution and dilute peracetic acid. Rice straw and bagasse were effectively delignified by boiling in a 1% sodium hydroxide solution for 3 hr or by autoclaving at 120 degrees C in a 1% sodium hydroxide solution for 1 hr. The sawdust from a broad leaved tree (Machilus thunbergii) was delignified by autoclaving at 120 degrees C in a 1% sodium hydroxide solution for 1 hr and by subsequent boiling in diluted 1/5 peracetic acid for 1 hr. This type of sawdust was also delignified by boiling in 1/5 peracetic acid for 1 hr and by subsequent autoclaving at 120 degrees C in a 1% sodium hydroxide solution for 1 hr. The sawdust from a coniferous tree (Cryptomeria japonica) was delignified by boiling in 1/5 peracetic acid for 1 hr and by subsequent autoclaving at 120 degrees C in a 1% sodium hydroxide solution for 1 hr; however, the successive treatment by autoclaving with alkali solution and subsequent boiling with diluted peracetic acid failed to bring about the desired effect. The saccharification of delignified rice straw, bagasse, and sawdust was examined using Cellulase onozuka, wheat bran or rice straw solid culture at various substrate concentrations, resulting in the formation of 5 to 10% sugar solutions after incubation at pH 5.0, 45 degrees C for 48 hr. The optimum substrate concentration existed at around 10%. Reuse of cellulase solution and resaccharification of residual sawdust were considered to be inadequate.     

The cellulase of Trichoderma viride: Separation of the components involved in the solubilization of cotton

1. Culture filtrates from Trichoderma viride have been fractionated by gel filtration on Sephadex G-75 followed by ion-exchange chromatography on DEAE- and SE-Sephadex. 2. The components essential for attack on cotton are a carboxymethylcellulase, a cellobiase and a third (C₁) component which has no action on CM-cellulose, cellobiose or cotton. 3. These components, which together can completely convert cotton into water-soluble products, lose this ability when separated and regain it quantitatively when recombined in their original proportions.     

Growth kinetics and cellulase biosynthesis in the continuous culture of Trichoderma viride.

Continuous culture studies have been carried out growing Trichoderma viride QM 9123 in a 10 liter stirred fermentor on a medium containing commercial glucose as the carbon source. Experiments were carried out at 30 degrees C and at three controlled pH values of 2.5, 3.0, and 4.0 over a range of dilution rates from 0.01 to 0.11 hr-1. Steady-state values of cell, glucose, and cellulase concentration oxygen tension, and outlet gas oxygen partial pressure were recorded. Values of maximum specific growth rate, endogenous metabolism coefficient, Michaelis-Menten coefficient, yield and maintenance coefficient for glucose were derived and correlated the effect of the hydrogen ion concentration. Specific oxygen uptake rates were correlated with specific growth rates and absorption coefficients were shown to be a function of dilution rate independent of pH. Some data on cellulase biosynthesis were examined and correlated in terms of a maturation time model.     

Purification and characterization of endoglucanase and exoglucanase components from Trichoderma viride

Major cellulase components—four endoglucanases (Endo I, II, III and IV) and one exoglucanase (Exo II)—were isolated from a commercial cellulase preparation derived from Trichoderma viride by a series of chromatographic procedures. The average molecular weights were determined by SDS-polyacrylamide gel electrophoresis. Endos I, III and IV, with M_rs of 52,000, 42,000 and 38,000, respectively, exhibited a more random hydrolytic mode on carboxymethylcellulose (CMC) than Endo II, which has an M_r of 60,000. Endo II showed low activity towards CMC, but out of the four purified endoglucanases this enzyme had the highest specific activity against Avicel. In the hydrolysis of H₃PO₄-swollen cellulose by Endos I, III and IV, cellobiose was the major product, but equimolar amounts of glucose and cellobiose were formed by Endo II. Exo II, with an M_r of 62,000, released cellobiose as the main product in the hydrolysis of H₃PO₄-swollen cellulose, but glucose was negligible. The combination of Endo I, II, III or IV with Exo II resulted in a synergistic effect in the degradation of Avicel at various combination ratios of these enzymes; the specific optimum ratio of endoglucanase to exoglucanase was largely dependent upon the random hydrolytic mode of the endoglucanase. On the other hand, adsorption of cellulase components was found apparently to obey the Langmuir isotherm, and the thermodynamic parameter (ΔH) was calculated from the adsorption equilibrium constant (K). The enthalpies of adsorption of the endoglucanases were in the range of −2.6–−7.2 KJmol⁻¹, much smaller than that of Exo II (−19.4 KJmol⁻¹). This suggest that Exo II shows stronger preferential adsorption than endoglucanases, and that the enthalpy of adsorption will be effective in distinguishing endoglucanase from exoglucanase.     

Structural characterization of a glycoprotein cellulase, 1,4-beta-D-glucan cellobiohydrolase C from Trichoderma viride.

A glycoprotein enzyme, 1,4-beta-D-glucan cellobiohycrolase (EC 3.2.1.91) form C, was purified to electrophoretic homogeneity by a procedure which permitted isolation of gram quantities from a commercial Trichoderma viride culture filtrate preparation. Purified cellobiohydrolase C has an E1%/280 nm = 14.2 and degrades both microcrystalline and phosphoric acid-swollen cellulose to cellobiose. The cellobiohydrolase C contains 26.4, 4.8, 2.4 and 3.4 mol of mannose, glucose, galactose and glucosamine, respectively, per mol of enzyme (molecular weight, 48 400). Methylation analysis of cellobiohydrolase glycopeptides indicates an average carbohydrate chain length of two residues. Alkaline borohydride treatment of cellobiohydrolase C released neutral carbohydrate which is bound through an average of 16.7 O-glycosidic linkages to serine and threonine per molecule of enzyme. Glucosamine was not released from the protein by alkaline treatment. Analysis of alkaline borohydride-released carbohydrate by high pressure liquid chromatography demonstrated that an average enzyme molecule contains 8.8 mono-, 1.8 di-, 4.6 tri-, 1.2 tetra-, and 0.4 pentasaccharide chains. The linkages between the neutral monosaccharides are (1 leads to 6) as shown by gas chromatography - mass spectrometry of partially methylated residues. The (1 leads to 6) linkage is consistent with the stability of the linkages to alkaline conditions and the destruction of all neutral carbohydrate by periodate. Action of alpha-mannosidase indicates that some oligosaccharide chains contain alpha-mannose as the terminal residue.     

Low-molecular-weight xylanase from Trichoderma viride.

An endo-1,4-beta-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) has been isolated from a commercial preparation of Trichoderma viride. The molecular weight was 22,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the pI value was 9.3. The xylanase was a true xylanase without cellulase activity. When the N-terminal amino acid sequence of the first 50 residues was compared with that of a xylanase from Schizophyllum commune, strong evidence for homology was found, with more than 50% amino acid identity. T. viride xylanase also possessed extensive identity with a proposed amino-terminal consensus sequence of xylanases from bacteria.     

Enhanced cellulase production of the Trichoderma viride mutated by microwave and ultraviolet

Cellulase-producing fungi Trichoderma viride were cultured and fermented on the solid-state wheat bran fermentation medium. The characteristics of its carboxymethyl cellulase (CMCase) in the condition of this solid-state fermentation were evaluated, and the optimum culture time, optimum pH and optimum temperature for CMCase activity of T. viride fermented in this solid state were 60 h, 5.0 and 50 °C, respectively. Carboxymethyl cellulose sodium (CMC-Na) and Congo red were used to screen the strains that had stronger ability to produce enzymes. After the compound mutagenesis by microwave and ultraviolet, seven mutant strains (M-B1–M-B7) were selected and their CMCase activities were assayed. Five of them (M-B1, M-B2, M-B3, M-B5 and M-B7) had significantly stronger ability to produce enzymes than the normal wild type, and they were also very stable for a long period up to 9 generations to produce cellulase. Molecular studies showed that there were some base mutations in endoglucanase I (EG I) genes of mutants M-B1, M-B2, M-B3 and M-B5, but no change in M-B7, suggesting that some amino mutations in EG I proteins caused by base mutations could lead to enhanced cellulase production.     

Strain improvement for enhanced production of cellulase in Trichoderma viride

The filamentous fungi Trichoderma species produce extracellular cellulase. The current study was carried out to obtain an industrial strain with hyperproduction of cellulase. The wild-type strain, Trichoderma viride TL-124, was subjected to successive mutagenic treatments with UV irradiation, low-energy ion beam implantation, atmospheric pressure non-equilibrium discharge plasma (APNEDP), and N-methyl-N'-nitro-N-nitrosoguanidine to generate about 3000 mutants. Among these mutants, T. viride N879 strain exhibited the greatest relevant activity: 2.38-fold filter paper activity and 2.61-fold carboxymethyl cellulase, 2.18-fold beta-glucosidase, and 2.27-fold cellobiohydrolase activities, compared with the respective wild-type activities, under solid-state fermentation using the inexpensive raw material wheat straw as a substrate. This work represents the first application of APNEDP in eukaryotic microorganisms.     

Enzymatic treatment of primary municipal sludge with Trichoderma viride cellulase

Enzymatic hydrolysis of the cellulose in raw primary settled municipal sludge by Trichoderma viride cellulase achieved conversions of up to 75% of the cellulose, primarily to cellobiose, in 24 hr. Simultaneously the gel-like characteristic of raw primary sludge was changed to that of a slurry of fine particles in less than 2 hr, causing a radical change in the ability to ultrafilter the sludge. The use of raw primary sludge as a growth medium for T. viride cellulase production was also investigated. It was possible, with nitrogen supplements, to obtain an enzyme with a filter paper activity (FPA) of two compared to over four which is attainable on defined medium of similar strength. The potential use of cellulase treatment as a pretreatment or integral part of waste treatment processes is discussed and the alternatives evaluated.     

Mutarotation of hydrolysis products by different types of exo-cellulases from Trichoderma viride.

Mutarotation of products from p-nitrophenyl beta-D-cellobioside and cellopentaitol by two different types of exo-cellulases from Trichoderma viride was investigated. It was found that an exo-cellulase of glucosidase type produced from the former substrate D-glucose which was mutarotated in a downward direction, while another exo-cellulase of Avicelase type produced from the latter substrate cellobiose which was mutarotated in an upward direction.