Complexed cellulases

Cellulase enzyme complexes consist of three major types of proteins that synergistically catalyze the breakdown of a cellulosic substrate. Because the enzymes are strictly substrate-specific in their action, any change in the structure or accessibility of the substrate can have a considerable influence on the course of the hydrolysis reaction. A pretreatment method based on exposing cellulosic substrate to phosphoric acid solution [9] and addition of the nonionic... 

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). 

The anaerobic biological conversion of the major polymeric components of MSW identified require appropriate microorganisms and hydrolytic enzyme systems. Extracellular hydrolytic enzymes, such as cellulases and lipases, have been shown to be effective in the post hydrolysis of anaerobic digester efQuent solids 34) or pretreatment of complex organic polymers before the digestion process 48),... 

Figures 1 and 2 show positive correlation, although of varying degrees of goodness of fit, between the specific activities of endo ucanase, exoglucanase, and -glucosidase activities with the cellulose composition and degradation in digester feed, respectively. These data indicate a direct relationship between the cellulose content in the feed and cellulase enzyme complex production by the digester consortia. In this system, as in many others studied, cellulose is an effective inducer of cellulase secretion (67,68),...

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... 

T. harzianum IMI 275950 isolated from wheat straw grew better on lignocellulose (Table I). Despite these differences, the enzyme yields of the cellulase/xylanase complexes did not correlate with growth rate. These observations from this empirical study indicate that strains isolated or derived for growth on extracted substances may not necessarily be the most useful strains to exploit natural substrates. 

Selective inactivation of cellulase activities is another method which has been applied to fungal culture filtrates to produce cellulase-free xylanase preparations. Cellulase inactivation of a crude enzymatic complex was achieved by Barnoud et al. (15) using a 1 mM mercuric chloride solution. In the presence of this sulfhydryl binding metal, complete inactivation of endocellulases was observed whereas the xylanases retained 80% of their activity. 

Because of the complex structure and the insolubility of cellulose, the precise mechanism of recognition and action of cellulases have been examined by the use of inhibitors and artificial substrates. Cellulases have been found, so far, in 11 families of glycosyl hydrolases [65]. 

The same researchers studied glucose production using beta-glucosidase immobilized with the same hydrophilic foam (Hypol 2002). " The immobilized enzyme showed 95% retention of activity after 1000 hours of continuous use at 23°C. Co-immobilization with cellulase yielded a cellulose-hydrolyzing complex with a... 

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. 

Assay of Endocellulase Activity. Cellulase is an enzyme complex a synergistic action between the components is required for a complete hydrolysis of the insoluble cellulose. There is no consensus about the substrate to be used for the cellulase activity measurements. 

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. 

Thus the mechanism of cellulase action is a complex one, involving three different types of enzyme and two types of synergism. In the first stage of the reaction, an enzyme A is producing a reactive product for enzyme B, while in the second stage an enzyme C is removing an inhibitory product of the action of the other enzyme. Only synergism of the first type will be discussed further. 

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