Cellulase components, Trichoderma

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 ... 

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. 

The production of D-xylanase activity by fungi cultured on cellulose may also be due to the action of a cellulase component having multisubstrate activity. Cellulases have always been reported to be adaptive enzymes,226 and the multiplicity of such enzymes may imply that this pseudo-xylanase activity is produced constitutively when fungi are grown on cellulose as the sole source of carbon. Cellulases of this type have been shown203 also to attack D-xylan even when they are highly purified (for example, cellulase F-2 from Trichoderma viride203). 

The original cellulases used in denim washing were the crude enzymes of Trichoderma and Humicola, referred to as acid and neutral cellulase, respectively, based on the optimum pH range of use of the enzymes, which was pH 4 to 5 for the acid cellulase and 6 to 7 for the neutral cellulase. The Trichoderma cellulase, comprising the more complete set of EG and CBH components capable of the full hydrolysis of cellulose, works more quickly and is capable of a greater degree of abrasion and fading of the blue dye color than the Humicola cellulase. The Trichoderma cellulase also achieves certain desired finishes (appearances) that the Humicola cellulase does not. 

Table 6. Distribution of cellulase components from representative strains of Trichoderma and Humicola ...

It is often desirable to enhance or deplete the cellulase enzyme mixture of one or more individual cellulase components. This is done if one or two components are particularly desired, or if one or more are deleterious to the application. In the extreme, only a single cellulase component is produced. In one example of this, pure Trichoderma endoglucanase III is produced by extracting it with polyethylene glycol [16]. 

Discrete CBMs are usually produced by protein engineering methods however, with some enzymes, such as the cellulase components of Trichoderma reesei, the CBMs are connected to the catalytic by long, heavily glycosylated linker regions, which are susceptible to attack by proteases, so that protease hydrolysis of commercial cellulases can be practical method of obtaining CBMs. ... 

The conversion of cellulase component B into A may be a result of some enzymatic modification of the enzyme molecule. Similar type of in vitro conversion has also been reported, for example, for the extracellular cellulase of Trichoderma viride (38) and the cell-bound invertase of bakers yeast (15). The occurrence of another type of conversion where the reversible association and dissociation of active subunits are operative, has been proven on the intrawall and extracellular invertases of Neurospora crassa (25). 

A cellulase component has been purified from a crude cellulase preparation of Trichoderma viride until homogeneous on polyacrylamide disc gel electrophoresis. The enzyme (mol. wt. 4.5 x 10 by gel filtration, pH optimum 4.5—5.0, temperature optimum 50 °C) was thermostable but was inactivated by Hg +, Ag+, and Cu +. The ATm values for the enzyme decreased in parallel with increase of chain length of the substrates, and the enzyme produced cellobiose and D-glucose from various cellulosic substrates. The en me was also found to catalyse the rapid synthesis of cellotetraose from cellobiose. 

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). 

Component enzymes of the cellulase system have been purified from several microbial species (1-13), among which mutants of the imperfect fungus Trichoderma provide the highest levels of extracellular enzyme activity (14). From this organism have been purified / -glucosi-dases (EC 3.2.1.21), endo-l,4-/ -D-glucanases (EC 3.2.1.4) and 1,4-/ -d-... 

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. 

Not all cellulase enzymes give identical results, even with similar fabrics in similar equipment. Cellulases derived from Trichoderma typically are the most aggressive in their action, whereas mono-component endo-glucanases often require the most mechanical action to achieve the desired effects. Slow deactivation of the cellulases during transport and storage can adversely affect the reproducibility of the resulting effects. If cotton is not washed carefully before bio-finishing,... 

With the example provided by the characterization of these chemically and enzymologically pure cellulases, we decided to purify and describe the enzyme components of a cellulolytic organism, Trichoderma viride. Brief descriptions of the properties of partially purified exo-fi (1 4)glucanase (20), Ci or hydrocellulase (48), and endo-fi-(l 4)-... 

By the mid-1990s, several new cellulase enzyme products had evolved for denim washing. Fower degrees of fabric strength loss were achieved with individual cellulase enzyme components, most prominently Trichoderma EG3 [16] and Humicola EGS [17]. Trichoderma cellulase used in combination with protease improved its performance to close to that of the Humicola cellulase [18]. 

The distribution of individual cellulase enzymes in two prominent crude cellulases, those produced by Trichoderma longibrachiatum Rut C30 and Humicola insolens DSM 1800, are listed in Table 6. Note that the component designations... 

The Trichoderma viride complex is a true cellulase in the most rigid sense, being able to convert crystalline, amorphous, and chemically derived celluloses quantitatively to glucose. It has been established that (a) the system is multi-enzymatic, (b) that at least three enzyme components are both physically and enzymatically distinct, and (c) that all three components play essential roles in the overall process of converting cellulose to glucose. 

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