Enzymes from Trichoderma

The core-enzymes, prepared in our laboratory, and containing the active centers, were successfully crystallized (Dr. Jones, Uppsala, communicated) and tertiary structures will be described in the near future. Chemical modification studies on these enzymes are currently being undertaken in our laboratory identification of important catalytic residues and location of the active centers will lead to more functional information on these enzymes. Other cellulases such as some endoglucanases from Clostridium thermocel-lum (EG A, EG B, EG D) (10) and EngA and Exg from Cellulomonas fimi (19) also contain sequences of conserved, terminally located and sometimes reiterated, amino acids. Some of these sequences are preceded by proline-serine rich domains. Thus, a bistructural-bifunctional organization seems to be a rather common feature among cellulases, at least for EngA and Exg from C. fimi and the enzymes from Trichoderma reesei. 

Kitomoto, Y, Moii, N., Yamamoto, M., Ohiwa, T, Ichiwaka, Y. A simple method of protoplast formation and protoplast regeneration fiom various fungi using an enzyme from Trichoderma harzianum. Appl Microbiol Biotechnol 1988,28,445—450. 

Enzymatic methods also yield a high conversion rate of L-lysine to AMV. Pukin et al. (2010) developed an enzyme (L-lysine a-oxidase enzyme from Trichoderma viride) immobilization system with an epoxy-activated solid support. They reported a 0.95 yield of 5-AMV acid. In this study, the enzyme showed an 8-fold lower activity as compared to the one determined in the standard assay under the experimental conditions, and the reason for the low enzymatic activity remained unclear. Liu et al. (2014) reported a simple composition of the two-enzyme coupled system and a 0.865 yield of 5-AMV. Considering all of the studies on AMV production, it seemed that L-lysine might be a suitable starting material for a higher production of AMV. 

Cellulase-gold was made and applied according to (Berg et al., 1988) with sections from material embedded in Quetol 651. Chromatographically purified cellulase complex from Trichoderma reesei was obtained from Worthington Enzymes (Cat. it CEL). 

This enzyme was shown to be specific for xylan oligomers and small acetylated synthetic substrates. Many characteristics have been published recently about this type of enzyme, purified from Trichoderma reesei, and A. oryzae [6], and a different A. n/ger preparation[7]... 

L-Pipecolic acid, a key component of many antibiotic and anticancer biomolecules, serves as an important chiral pharmaceutical intermediate. We have developed an enzyme-coupled system consisting of zl -piperidine-2-carboxylate reductase (Pip2C) from Pseudomonas putida, glucose dehydrogenase (GDH) from Bacillus subtilis, and L-lysine a-oxidase from Trichoderma viride, affording L-pipecolic acid from L-lysine in high yield with an excellent enantioselectivity (Figure 10.2). ... 

Matsumura and Bousch (1966) isolated carboxy lest erase (s) enzymes from the soil fungus Trichoderma viride und a bacterium Pseudomonas sp., obtained from Ohio soil samples, that were capable of degrading malathion. Compounds identified included diethyl maleate, desmethyl malathion, carboxylesterase products, other hydrolysis products, and unidentified metabolites. The authors found that these microbial populations did not have the capability to oxidize malathion due to the absence of malaoxon. However, the major degradative pathway appeared to be desmethylation and the formation of carboxylic acid derivatives. 

Chromophoric substrates were also used as tools in the study of the binding of several cellulase components to their natural substrates (such as Avicel). This is illustrated here in the investigation of the synergy in binding of CBH I and CBH II from Trichoderma reesei onto Avicel. The enzymes were differentiated with CNPL (see above), which was a substrate only for CBH I (core I). Thus, the amount of CBH II adsorbed when a mixture of both enzymes was added, either simultaneously or sequentionally, to Avicel was calculated from the amount of CBH I bound (activity measurements with CNPL) subtracted from the values for total protein binding (280 nm absorbance reading). The results obtained from these experiments are summarized as follows ... 

Table I. Xylanolytic Enzymes Isolated from Trichoderma reesei...

Cellulolytic enzy- Trichoderma reesei AOT/isooctane Recovery of enzymes from broth [90]... 

Enzymes. The mannanase was isolated from Aspergillus niger (11) fraction 4 b was used throughout the experiments (cf. Table 1 in (11)). The xylanase 2, the avicelase 1, and the avicelase 2 were isolated from Trichoderma viride (10). The properties of these enzymes have also been described in the papers cited above. The cellobiohydrolase C was kindly supplied by Dr. R. D. Brown, Jr., and Dr. E. K. Gum, Jr. (Virginia Polytechnic Institute, Blacksburg). The isolation (from Trichoderma viride) and the properties of the cellobiohydrolase C are described in their 1974 paper (12). 

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. 

Medve, J., Karlsson, J., Lee, D., and Tjerneld, F. 1998. Hydrolysis of microcrystalline cellulose by cellobiohydrolase I and endoglucanase II from Trichoderma reesei Adsorption, sugar production pattern and synergism of the enzymes. Biotech. Bioeng.,59, 621-634. 

Enzymatic Hydrolysis. Saccharification of wood polysaccharides to sugars can be accomplished by enzymatic techniques instead of acid hydrolysis. The U.S. Army Natick Laboratories developed a method for conversion of cellulose to glucose with a cellulose enzyme from an active strain of the fungus Trichoderma viride. However, extensive pretreatment of wood is necessary before sufficient enzymatic hydrolysis will take place. 

Cellulases are found in fungi and bacteria. Of commercial interest are fungal enzymes from Aspergillus or Trichoderma and a few bacterial enzymes. They are either used as a multicomponent, which contain all enzyme types and are found in Trichoderma reesei (Hypocrea jecorina), or as a monocomponent enzyme product, which consists of only one of the three types of enzymes. The multicomponent enzyme preparations can be produced from a selected cellulose overproducing strain of the wild-type organism, whereas the monocomponent cellulases are mainly produced in recombinant production systems. 

Lemos, M. A., Teixeira, J. A., Domingues, M. R. M., Mota, M., and Gama, F. M., The enhancement of the cellulolytic activity of cellobiohydrolase I and endoglucanase by the addition of cellulose binding domains derived from Trichoderma reesei. Enzyme Microbial Technol 2003, 32, (1), 35—40. 

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