Cellulases from Trichoderma viride

Cellulase solution - dissolve 6.25 g cellulase from Trichoderma viride (Merck Ltd) in 1 I citrate-phosphate buffer, pH 4.6, immediately before use. 

In 1968 Nisizawas laboratory (55) purified three cellulase components from Meicelase, which is a commercial cellulase from Trichoderma viride. These three components—Cellulases II, III, and IV—contained 16.8, 15.6, and 10.4% carbohydrate, respectively, and were active in hydrolyzing cellooligosaccharides, CM-cellulose, and cotton. They were inactive toward cellobiose and p-nitrophenyl-yS-D-glucoside. 

Enzymatic synthesis of cellulose has been achieved by cel-lulase. For example, incubation of fS-cellobiosyl fluoride with a cellulase from Trichoderma viride can produce cellulose in 54% yield with DP around 22 after 12h. In addition, change of the reaction conditions (substrate concentration or organic solvent concentration) enabled the selective synthesis of the water-soluble celloohgosaccharides (118). 

Kobayashi et have also investigated other modes of enzymatic polymerization to produce a novel cellulose-chitin hybrid polysaccharide. A sugar fluoride monomer of GlcNAc/3(l 4)Glc (17) was designed as a TSAS monomer for polymerization catalyzed by cellulase from Trichoderma viride. In the polymerization, monomer 17 was recognized by cellulase from T. viride, leading to a cellulose-chitin hybrid polysaccharide 18 with perfect regioselectivity and stereochemistry (Scheme 17). 

Enzymatic Polymerization of 1 Catalyzed by Cellulase from Trichoderma Viride... 

Cellulase from Trichoderma viride was the most effective enzyme for the synthesis of cellulose when compared with cellulases from ANL or Polyporus tulpiferae. 

Hong, S. P. Kim, D. S. (1998).Chitosanolytic characteristics of cellulases from Trichoderma viride and Trichoderma reesei. Korean J Food Sci. Technol, 30, 245-252. 

Liu, B. D. (2004). Clone and expression of cellulases from Trichoderma viride.PhD Dissertation. Harbin Institute of Technology, Biological Engineering Department. Harbin,China. 

Sprucewood holocellulose was treated with an endo-p-1,4-mannanase isolated from Aspergillus niger and an endo-/3-1,4-xylanase, two avicelases, and a cellobiohydrolase C isolated from Trichoderma viride. The mannanase hydrolyzed about a quarter of the mannan in 2-3 days without xylan or cellulose degradation. The xylanase hydrolyzed about half the xylan with 10% mannan solubilization. The three cellulases hydrolyzed up to 45% of the cellulose and 20% of the xylan, accompanied by 40-70% solubilization of the mannan. Combined xylanase-mannanase treatment hydrolyzed about half the xylan and mannan. Addition of mannanase to to cellulose-treated samples increased the degradation of the cellulose and mannan. Micromorphological studies of the variously treated specimens revealed a loss of substances in P/Slf T, and adjacent zones of S2 of the tracheid wall. 

The three cellulases decomposed about 25-45% of the cellulose accompanied by solubilization of about 40-70% of the mannan and, by partial hydrolysis, of about 20% of the xylan present in the untreated sprucewood holocellulose. Based on the degradation products (cf. Table III, Columns 13-15, and Table II), the catalytic actions of the three cellulases—all isolated from Trichoderma viride—are similar or identical. The lower absolute degradation values obtained with cellobiohydrolase C might merely be a result of enzyme concentration. 

Capitalizing on this metabolic difference between higher forms of life and micro-organisms is the basis for this research approach to wood protection. Compounds are available which inhibit the cellulase enzyme systems however, their specificity has not been determined. Mandels and Reese (21,22) found that the extracts from the immature fruit of persimmon or the extract from leaves of bayberry were very effective inhibitors of the cellulase system. At concentration levels of. 00005 and. 00018%, respectively, these two extracts inhibited the cellulase enzymes isolated from Trichoderma viride. It is not known what the active component(s) are in these two extracts. 

The D-xylanase system of Stereum sanguinolentum,199 the only D-xylanase for which an amino acid composition has as yet been published, was found to contain a high proportion of acidic and aromatic amino acid residues. The M.W., as determined from the amino acid composition, is 23,900, compared with 21,600 as calculated from ultracentrifugation data. Other physical parameters that have been determined199 for this D-xylanase include the sedimentation coefficient [2.8S, which is similar to that reported for a D-xylanase isolated from Trichoderma viride,203 namely, 2.IS], the partial specific volume (0.71 cm3.g 1), and the molar extinction coefficient (6.25 X 104). Activation energies have been reported for D-xylanases from Schizophyllum commune233 (EA 28.6 kj.mol-1) and from a commercial cellulase preparation229 (EA 34.0 kj.mol-1). 

Gong C-S, Ladisch MR, Tsao GT (1977) Cellobiase from Trichoderma viride-. purification, properties,kinetics, and mechanism. Biotechnology and Bioengineering 19 959-981 Gong C-S, Ladisch MR, Tsao GT (1979) Biosynthesis, purification and mode of action of cellulase of Trichoderma reesei-. hydrolysis of cellulose mechanisms of enzymatic and acid catalysis. Advances in Chemistry Series vol. 181, American Chemical Society, Washington, DC, pp 261-287... 

A number of commercial cellulases, samples from other workers, and acetone precipitated powders were compared (Table II). All of these demonstrated good activity on carboxymethylcellulose. In fact a number of these, notably the enzyme from Poria, were more active on CMC than the T. viride enzyme. About half of the enzymes showed good activity on filter paper. But T. viride enzymes show the highest activity on cotton, the most resistant substrate. In case inactivation at 50 °C. affected the activity on cotton and filter paper, these enzymes were also tested for hydrolysis at 25°C. (data not shown). The relative activities were the same as those found at 50°C. Selby (31) made a similar study of a number of cellulase preparations. He also found that the cellulase of Trichoderma viride had the highest activity on cotton. 

In order to polymerize a fluoride-type monomer 10, cellulase and xylanase were chosen for the catalyst. In an acetonitrile-acetate buffer mixed solution, the consumption of 10 was accelerated by the addition of xylanase from Trichoderma viride, giving rise to a white precipitate as the progress of the reaction in a 58% yield. From the analysis of the resulting products, it is a cellulose-xylan hybrid polysaccharide 11 and polymers with the degree of polymerization up to 12 (24 saccharide units) were detected from the MALDI-TOF/MS. 

Separation of cellulase and cellobiase from Trichoderma viride... 

Continuous culture studies for the production of cellulase have been carried out growing Trichoderma viride on a medium containing commercial D-glucose as the carbon source. Some data on cellulase biosynthesis were examined and correlated in terms of a maturation time model. Specific oxygen uptake rates were correlated with specific production rates. Mutant strains not producing cellulases have been induced and isolated from Trichoderma viride. Enrichment of the mutants was carried out by nystatin selection. 

Xylan is a polysaccharide of xilose with a P(1 4)-glycosidic linkage and it is an important component of hemicellulose in plant cell walls. Xylan was synthesised by a transglycosylation reaction catalysed by cellulase with the use of P-xylobiosyl fluoride as the substrate [207]. Cellulose-xylan hybrid polymers were synthesised by the polycondensation of P-xylopyranosyl-glucipyranosyl fluoride catalysed by xylanase (EC 3.2.1.32) from Trichoderma viride, in a mixed solvent acetonitrile/ acetate buffer (Scheme 12.23) [208]. 

Liu, P. (2009). Study on the structure and function of bifuntional cellulase-chitosanase from Trichoderma.viride. PhD Dissertation. Jiangnan University,School of food science and technology, Wuxi,China. 

Evidence for Ex-1 to be an Exo-type Component. The time course of CMC hydrolysis by Ex-1 is shown in Figure 10. The hydrolysis proceeded rapidly at first, but it reached a plateau and seemed to stop after 3 hr. This is characteristic of the hydrolysis by exo-type cellulase, as has been reported for exocellulase of glucosidase type from T. viride (7) and for another Trichoderma exocellulase of Avicelase type (10). 

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