Heterologous expression and characterization of endoglucanase I (EGI) from Trichoderma viride HK-75

Endoglucanase I (EGI) secreted from Trichoderma viride HK-75 has a unique transglycosylation activity. The genomic and cDNA clones encoding EGI (egl1) of T. viride HK-75 were isolated and characterized. The coding region of egl1, composed of 1392 bp, was found to encode a polypeptide of 464 amino acids that has extensive similarity (93.8%) with EGI of T. reesei. Expression of the egl1 gene in E. coli as a fusion protein (with N-terminal thioredoxin and C-terminal histidine tag) led to a large production of a nonglycosylated protein of 62.5 kDa. However, it formed an insoluble inclusion body. Upon denaturation with 8 M urea followed by dialysis and successive purification, the enzymatically active recombinant EGI (rEGI) was obtained at a level as high as 18.3 mg/l of 1,000 ml of culture. The rEGI had 67.8% activity for carboxymethyl cellulose (CMC), compared to native EGI (nEGI). The optimum pH and optimum temperature of rEGI were lower than those of nEGI by 0.5 and 5 degrees C, respectively. The rEGI also had narrower CMCase ranges than nEGI in pH and temperature stabilities. However, the catalytic and transglycosylation abilities against cellotriose of rEGI were comparable to those of nEGI. These results suggest that the glycosylation is important for the stabilities of EGI but not critical for the essential enzymatic capacity.     

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.     

Affinity chromatography of endoglucanase of Trichoderma viride by concanavalin A-agarose.

Endoglucanase (C kappa cellulase) and cellobiase are often cross-contaminated in separation procedures by ion-exchange chromatography such as DEAE-cellulose. By using concanavalian A (Con A)-agarose chromatography, C kappa cellulase and cellobiase from Trichoderma virde can be separated. C kappa cellulase showed affinity toward Con A. indicating a glycoprotein containing alpha-D-mannopyransyl and alpha-D-glucopyranosyl end groups or internal 2-O-D-mannopyranosyl residues in sugar moieties. This method provides a way to estimate the quantities of C kappa enzyme produced by T. viride and possibly by other organisms.     

表面活性剂对绿色木霉产纤维素酶影响

利用绿色木霉,以稻草为唯一碳源,采用液态发酵的方法,分别加入生物表面活性剂鼠李糖脂和化学表面活性剂Tween 80,重点研究了生物表面活性剂对绿色木霉产纤维素酶的影响。实验分析了加入不同浓度的表面活性剂时滤纸酶活、羧甲基纤维素酶活、微晶纤维素酶活及酶液的表面张力随时间的变化情况。结果表明,添加鼠李糖脂能够促进绿色木霉产酶,分别使滤纸酶活、羧甲基纤维素酶活、微晶纤维素酶活最大提高了1.08倍,1.6倍和1.03倍。与Tween 80相比,鼠李糖脂促进产酶的效果明显优于Tween 80。     

绿色木霉葡聚糖内切酶EGIII基因在大肠肝菌中的表达、定点突变及其动力学特性的研究

将绿色木霉葡聚糖内切酶EGIII基因亚克隆到表达载体pET-22b(+),构建重组质粒pET-egl3,转化到大肠杆菌BL21(DE3).利用金属亲和层析对重组EGIII进行纯化,纯化后酶比活力达到6 U/mg蛋白,最适反应温度为60 ℃,最适pH为4.0.同时对EGIII催化区的氨基酸残基R130和E218进行定点饱和突变,各筛选到一株酶活有提高的突变子R130P和E218F,其比活力为野生型EGIII的2.8倍和3.45倍.突变酶E218F的Km提高了一倍,催化效率Kcat提高了5.4倍;而R130P的Km和Kcat没有明显变化.两个突变酶的最适酶解温度和pH分别都提高至65 ℃和4.4.     

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.     

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.     

Characterization of polyadenylate from the fungus Trichoderma viride.

Polyribonucleotide segments, about 60 nucleotides long and consisting of about 95% adenylic acid residues, were isolated from whole cell ribonucleic acid of the deuteromyceteous fungus Trichoderma viride. Similar findings in two other groups of the true fungi raise the possibility that short polyadenylate sequences may be a feature of these relatively simple organisms.     

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.     

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.     

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).     

Effects of different inoculum densities of Trichoderma harzianum and Trichoderma viride against Meloidogyne javanica on tomato

A greenhouse experiment was conducted to evaluate the effects of different inoculum densities of two Saudi isolates of Trichoderma harzianum and Trichoderma viride against Meloidogyne javanica on tomato. Four densities (10⁴, 10⁶, 10⁸ and 10¹⁰ spores/g of soil) of each fungus were used. The results indicate that all four inoculum densities of the two Trichoderma species suppressed the nematode reproduction and root galling; and increased the growth of tomato plants, compared to controls. Efficacy of both fungi increased as their inoculum densities increased. Generally, efficacy of T. harzianum was better than that of T. viride, especially at the highest used density (10¹⁰ spore/g soil) which resulted in the best control.     

Purification and characterization of a chitinase from Trichoderma viride

Usukizyme, a commercial enzyme preparation from Trichoderma viride, showed multiple chitin- degrading activities. One of these was purified to homogeneity by sequential DEAE Sepharose CL-6B, Q-Sepharose FF, and Sephacryl S-100 HR column chromatographies. The purified enzyme showed optimum activity at pH 3.5 and 50 degrees -55 degrees C and was stable in the pH range of 3.5-6.0 and up to 45 degrees C. It showed higher activity toward chitosan-7B, a 62% deacetylated chitosan, as opposed to highly deacetylated chitosan substrates. Products of degradation of a 1% (w/v) solution of partially deacetylated chitin (PC-100) were purified on CM-Sephadex C-25 and analyzed by HPLC, exo-glycosidase digestion, and nitrous acid deamination. The enzyme was unable to split the GlcN-GlcN linkages in the substrate. It produced mainly (GlcNAc)(2) and (GlcNAc)(3) along with mixed oligosaccharides. When subjected to nitrous acid degradation, some of the mixed oligosaccharides produced mainly 2-deoxyglucitol, implying the presence of GlcN at the reducing end of the oligosaccharides.     

Purification and characterization of laminaran hydrolases from Trichoderma viride

At least three extracellular laminaran hydrolases which hydrolyzed laminaran (beta-1,3:1,6-glucan) from Eisenia bicyclis were secreted in wheat bran solid medium by Trichoderma viride U-1. These three enzymes, lam AI, AII, and B, were purified to electrophoretic homogeneity. Their molecular masses were estimated to be 70.1, 70.4, and 45.0 kDa for lam AI, AII, and B, respectively, by SDS-PAGE. Whereas both lam AI and AII could hydrolyze laminarin from Laminaria digitata, lam AII showed higher activity against Laminaria laminarin rather than Eisenia laminaran. On the other hand, lam B preferentially hydrolyzed pustulan, a beta-1,6-glucan. Laminarioligosaccharide was hydrolyzed by lam AI and AII but not B, whereas gentiooligosaccharide was hydrolyzed by only lam B. It showed that lam AI and AII were specific for beta-1,3-linkages, but lam B was specific for beta-1,6-linkages. These results indicated that T. viride U-1 has a multiple glucanolytic enzyme system.     

Comparative study of the effect of L-lysine-L-oxidase from Trichoderma harzianum Rifai and Trichoderma viride on nucleic acid synthesis in human tumor cells in vitro

L-lysine-alpha-oxidase, a new fungal enzyme catalyzing oxidative deamination of L-lysine, exerts an inhibitory effect on DNA, RNA and protein synthesis in human cells of carcinoma ovarius (CaOv) in vitro.