Cellulase enzyme complex

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). 

Cellobiohydrolase I (CBH I, 1,4-jS-D-glucan-cellobiohydrolase, E.C. 3.2.1.91) is the main protein (ca. 60%) of the cellulase complex produced by T. reesei strains. CBH I hydrolyses crystalline cellulose, acid swollen cellulose and 4-methylumbelliferyl-cellodex-trins by cleaving off the terminal cellobiose unit from the non reducing end of the chain. It operates with retention of configuration in the reaction products 19,20. The abundance of this enzyme and its stability has facihtated its purification to homogeneity... 

The hypothesis put forth concerning steric effects in acid hydrolysis would have as its corollary the proposal that the role of the Ci component in cellulase enzyme system complexes is to disrupt the engagement of the C6 oxygen in the bifurcated intramolecular hydrogen bond and thus permit rotation of the C6 group into positions more favorable to hydrolytic attack. 

This study describes an absolute method for the evaluation of endo-/ -glucanase (Cx) or endocellulase activities in the cellulase complex without the need of a time-consuming isolation of the endocellulase fractions. In the method proposed by Almin et al. (3), an assumption was needed in the theoretical derivation of endocellulase activities, namely, that Mv/Mn is constant in the initial stages of the enzymic reactions. We found, however, a linear relationship between Mv and STn. They used CMC as substrate, and since the mode of action of endocellu-lases on CMC can be regarded as similar to that on HEC, it is possible that their assumption is not fulfilled. 

Hphe study of the synergism shown by enzymes of the cellulase complex in solubilizing highly ordered native cellulose has now progressed to a stage where it is possible to state with some certainty the principal enzyme components involved. Not everyone agrees, however. 

Leatherwood (49) interprets his observations made with roll tube cultures of the anaerobic bacterium Ruminococcus albus to indicate that a single cellulase complex is formed from enzymes diffusing from two different colony types. He discussed the mechanism in terms of a non-... 

Tt is a widely recognized fact that true cellulolytic microorganisms A produce three basic cellulase components IS), and that these enzyme components act in concert to hydrolyze crystalline cellulose to glucose (6). Many research laboratories have undertaken the task to purify cellulose components from various cellulolytic microorganisms and to study the mechanisms of cellulose hydrolysis. Much information has accumulated concerning the mode of action of cellulose hydrolysis since Reese et al. first proposed the Ci-C concept (7). In spite of this, however, conflicting reports still flourish concerning the composition of the "cellulase complex, the multiplicity of cellulase components, the biosynthesis of cellulose, and the mechanisms of cellulose hydrolysis. 

Crude Cellulases. The culture filtrate obtained from four-day-old cultures was concentrated by lyophilization. The protease inhibitors, phenyl methyl sulfonyl fluoride (PMSF, 2.3mM) and ethylenediamine-tetraacetic acid (EDTA, 0.2mM) were added to the culture solution and the salt concentration was adjusted to 0.5M with NaCl. After stirring at 4°C for at least 30 min to release cellulases from any enzyme-substrate complex which might have formed, the culture solution was centrifuged... 

A wide range of prokaryotic and eukaryotic microorganisms have the potential to produce cellulolytic enzymes when cellulose is present in the growth media (20,23,28,30). However, unlike some of the microorganisms that produce an incomplete cellulase system, T. reesei, a true cellulolytic fungus, produces an array of cellulase enzymes, i.e., the cellulase complex, which is able to hydrolyze cellulose to glucose (23). 

The mechanisms controlling the synthesis and activity of each of the cellulase complex of enzymes are summarized in Table I. 

All naturally occurring fungal strains of Trichoderma require an inducer for cellulase synthesis. In the absence of an inducer such as cellulose, cellobiose (21,22), or sophorose (12,13,14,23), Trichoderma does not make any detectable cellulase complex enzymes. The true physiological inducer of cellulase is currently unknown. Insoluble cellulose is presumably not such an inducer since there is no way for the internal cell machinery to sense the presence of this insoluble material. However, a small transglycosylation product such as sophorose, 2-0-/ -glucopyranosyl-D-glucose, may well be the natural inducer. 

Selection of Hyperproducing Mutants for Each of the Enzymes in the Cellulase Complex... 

NG14. This mutant is capable of elaborating 15 FP units/mL with a productivity of 45 units/L/hr. The final concentration of soluble extracellular protein is 21.2 mg/mL. Samples of this enzyme preparation were sent to G. Pettersson at the University of Uppsala, Sweden, for quantification of each of the enzymes in the cellulase complex using purified antibodies to the individual enzymes. The quantitative antigen-antibody reaction showed that 600 mg/g of this enzyme preparation was one enzyme, cellobiohydrolase. This represents a yield of 13 g/L of cellobio-hydrolase, which is a 100-fold increase over the amount of cellobiohydrolase obtained with strain QM 9414 (130 mg/L) the best previously existing cellulase mutant (G. Pettersson, personal communication). 

A number of selective-screening methodologies have been devised that have allowed isolation of a series of hyperproducing and catabolite repression-resistant mutants of T. reesei. Yields of cellulase of 15 units/ mL under controlled fermentor conditions have been achieved with both Rut-NG14 and Rut-C30. Quantitative reaction of Rut-NG14 enzyme preparation with purified antibodies to cellobiohydrolase shows that in this mutant, the cellobiohydrolase is specifically hyperproduced relative to the rest of the enzymes in the cellulase complex. Rut-C30, which was derived from Rut-NG14, shows resistance to catabolite repression for... 

The point to be emphasized in relation to reports of multiple cellulases in plants or microorganisms, is that not all of these are necessarily functional components of an extracellular "cellulase complex that are needed for optimal or complete cellulose breakdown. Though all of the forms may show a capacity for hydrolyzing 3-1,4-linkages in vitro, in vivo they could function in different intra- or extracellular loci on different substrates, and some could represent processed forms of inactive precursors. In general, not enough is known about the mechanisms whereby these enzymes are synthesized and excreted to enable an informed decision to be made on the roles that they perform. 

As understanding of the components of the cellulase complex has increased, we can better define the mode of action by which native cellulose is depolymerized. The Ci-Cx model has directed attention to the specific steps in the crystalline cellulose breakdown. The use of nomenclature describing the catalytic function of each specific enzyme should encourage higher standards of purity. The ubiquity of the substrate invites imaginative applications of cellulases. 

Enzyme s effect mechanism, i.e. enzyme catalysis, operates first of all to form an enzyme substrate complex [14]. Direct physical contact of enzyme and substrate is required to obtain the complex. The current proposed mechanism of cellulase action is illustrated in Fig. 15-1. However, the mechanism of enzymatic hy-... 

The detailed kinetics of the individual cellulase components is a subject of research and will not be discussed here. However, some characterizations have been made by taking the cellulase complex as a whole. In this case, the behavior can be expressed as a function of the enzyme dosage by Eq. (2) ... 

The Tricboderma viride Cellulase Complex. Many cellulolytic organisms secrete cellulase as a multiple component enzyme system. Separation of physically distinguishable components by electrophoretic or chromatographic techniques has been repeatedly demonstrated. It is more difficult to prove that these components are distinct enzymes with different functions in terms of substrate specificity and mode—or mecha-... 

As currently understood, the cellulase complex contains the following components (listed in the order in which their action on cellulose occurs) 1. Ci is an enzyme whose action is unspecified. It is required for the hydrolysis of highly oriented solid cellulose (Cotton, Avicel, etc.) by 0-1 — 4 glucanases. 

It should be emphasized that these phosphorolytic enzymes are intracellular. They are not found in the extracellular cellulase complex, and they are involved in the cellulolytic systems of relatively few microorganisms. 

The data further suggest that Chrysosporium pruinosum, and Pent-ctllium pusillum are good sources of exo-/ -l — 4 enzyme, if one does not object to the cellobiase present. Polyporus cinnabarinus produces less exo-enzyme, but as in Trichoderma viride, it is nearly free of cellobiase. Streptomyces sp. B814 is the only organism which appears to lack exo-glucanase in its cellulase complex. 

Failure to detect any function that Ci alone could perform, that was not equally a property of Cx or of mixtures of the enzymes led to the conclusion that both enzymes must be present on the substrate simultaneously when contributing to the solubilization of highly ordered forms of cellulose. Attempts were made, therefore, to interpret the action of Ci in terms of its contribution to the synergism displayed by the cellulase complex. 

An apparent protein-protein interaction has been observed in cultures of Ruminococcus that results in the formation of an enzyme complex that degrades cellulose. On the basis of these and other observations, we propose a new mechanism for cellulose degradation that involves a single cellulase complex. 

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