Complex cellulase

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... 

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-... 

Table X. Solubilization of Dewaxed Cotton by Reconstituted Mixtures of the Components of the Cellulase Complex of T. koningiia...

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. 

While cellobiose is a poor inducer, lactose, the milk sugar, is a good inducer of cellulase biosynthesis. Lactose-induced fungus produces a cellulase complex which is identical to those cellulases induced by cellulose (for SDS gels see Figure 9b, e, h, i). 

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). 

To hydrolyze crystalline cellulose efficiently by enzymatic means, the inaccessibility of crystalline structures must be overcome. T. reesei and some other true cellulolytic microorganisms produce a cellulase complex that is capable of efficiently hydrolyzing crystalline cellulose. One explanation of this capability was first proposed by Mandels and Reese (7). In this model, two factors, Ci and C worked together to disrupt and hydrolyze cellulose. Ci first disrupted the crystalline structure of the cellulose while Cx attacked the available sites formed by Ci. In other words, Ci and C exhibit synergism in hydrolyzing cellulose. Since then, the combined action of cellobiohydrolase ( Ci ) and endoglucanase ( C ) has been identified as the source of the apparent synergism (6,26,55). 

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. 

Total cellulase complex 1.5% oxgall + Phosfon D acid swollen cellulose... 

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... 

Ci activity (1,2), but / -glucosidase and cellobiase activities are probably ubiquitous (e.g., see Ref. 14 and 16). Whether or not these together constitute a cellulase complex as in fungi is unknown. The possibility seems unlikely since there are so few occasions in plants when whole cellulose microfibrils are autolyzed, and there is no need for it on nutritional grounds. Nevertheless, the question remains of why specific plant cells or tissues often elaborate more than one endocellulase. 

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. 

Hemicellulases, similar to the cellulase complex, are seldom found in isolation but are usually present as part of a multicomponent system. 

Corollospora maritima, Cirrenalia pygmea, Varicosporina ramulosa, Pleospora pelagica, Pleospora vagans, Phaeosphaeria typharum, Cellulase complex... 

Cellobiase. Figure 2 outlines the general purification steps used to isolate a pure cellobiase (named for its function in the cellulase complex) and three forms of the hydrocellulase. In purifying cellobiase it was expedient to replace the adsorption or affinity column with a batch separation on DEAE-Sephadex A-50 and to complete the purification with cation exchange chromatography on SP-Sephadex (49, 50). Table IV is a summary of the purification of the cellobiase and co-purification of... 

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. 

The cellulase complex diffuses through the pore system to the microfibrils, attacks the cellulose chains and hydrolyses each chain to the end. The diflerences in the efficacy of cellulases on various fibres are dependent on number of factors such as the amounts of non-cellulosic wood pulp-derived matter, the degree ol polymerisation, the type and degree of crystallinity, and the type and number of chemical substitutions to the cellulose [27-30]. Key features for the cellulose substrate are crystallinity, accessible surface area and pore dimensions [31 ]. Variation of any of these factors, e.g., structural changes of cellulose substrate by pre-treatments, will influence the course of the entire degradation process [32, 33]. 

White BA, Cann IKO, Mackie RI, Morrison M (1997) Cellulase and xylanase genes from ruminal bacteria domain analysis suggests a non-cellulosome-like model for organization of the cellulase complex. In Onodera R, Itabashi H, Ushida K, Yano H,Sasaki Y (eds) Rumen microbes and digestive physiology in ruminants. Jpn Sci Soc Press, Tokyo, p 69... 

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) ... 

T he first part of this paper will be concerned with an experimental approach to the cellulase complex which involves (1) the effect of supramolecular structure on activity of cellulolytic systems, (2) the effect of chemical modification of cellulose on the activity of cellulase, and (3) the use of oligosaccharides in determination of the mode of action of cellulolytic components. 

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