Cellulase multiplicity

The effects of feedstock cellulose content on cellulase enzyme activities in the digester system were examined in multiple laboratory-scale CSTR digesters operated under similar conditions with identical levels of feedstock organic loading (g VS/reactor d) but different levels of cellulose (Solka Floe). In general, all celli se enzyme... 

The classification of cellulase sequences into domains allows the further recognition of distinct sequences that characterize each family. To make these sequences evident, one must use multiple sequence alignments of the members of each individual family. We have concentrated on the Microhispora bispora endoglucanase, and present some initial multiple sequence alignments of the catalytic and binding domain families of which it is a member. 

Cx enzymes occur in multiple forms in most cellulose systems, and they appear to be different enzymes with different relative activities on a number of cellulose substrates. It was always possible, then, but not necessarily likely, that a completely different Cx system was being synthesized in each case and that this would account for the difference in synergism. This possibility was excluded, however, when it was found that each Cx system was identical in terms of CM-cellulase activity in each case the number of reducing end groups produced per unit decrease in viscosity was the same. 

As mentioned earlier, Cx enzymes exist in multiple forms in most cellulase systems. As a rule they vary in the degree of randomness of their attack on CM-cellulose, and they may be distinguished by plotting... 

Five endoglucanases have been isolated from S. pulverulentum cellu-lase (23), four from T. viride Onozuka cellulase (37), and three from T. viride Meicelase (31), but there are many other reports of the fractionation of multiple Cx components. P. funiculosum cellulase contains three and possibly four endoglucanases (Figure 7), while we have reported T. koningii cellulase to contain five (17) and recently six (46). 

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. 

The multiplicity of cellulases is of fundamental interest because of its implications on the basic understanding of cellulose hydrolysis as well as the regulation of cellulase biosynthesis. 

This data, together with the observation of prolific protease activity in crude commercial cullulase preparations that are probably obtained from older cultures, has led us to speculate that the multiple enzyme peaks in the older cultures could have resulted from protease modification of one parent endoglucanase. This prompted us to discontinue the use of commercial cellulase preparations. 

This chapter deals with three aspects of the cellulolytic enzyme system of Thermoactinomyces sp. the location of the CM-cellulase, Avicelase, and / -glucosidase (cellobiase) activities in the culture, the multiplicity of the extracellular enzyme system, and the stability of the different activities as a function of pH, temperature, and time. The results are discussed with reference to saccharification of cellulosic materials. 

When cellulase activity is induced in higher plants, analysis shows it to be present in multiple forms, which differ in electrophoretic and/or chromatographic mobility (3,13,15-18). These forms are analogous to those found in fungi in at least one respect, namely, that they include two or more endohydrolases which can attack CMC (CMCases). There is only one report of a plant exocellulase (16) and none of the synergistic... 

On the basis of these observations, it was tentatively concluded (3) that auxin-treated pea tissue elaborates two cellulases which are physically so distinct that it is unlikely that one could have derived from the other. Of course, if two forms of cellulase arise from genetically determined differences in protein structure, it would be legitimate to refer to them as isozymes (21). But in the absence of proof that the pea or any other plant cellulases are under separate genetic control, we will continue to refer to them as multiple forms. 

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. 

In contrast to cellulases, the hemicellulases encompass a much broader suite of activities. In addition to analogue versions of endo-, exo-, and glycosidase cellulase activities, multiple debranching activities are needed to handle the high complexity of the heterogeneous hemicelluloses (Table 33.3). The varied backbone composition of hemicelluloses also adds complexity. Xylans, xyloglucans, man-nans, and numerous other minor polysaccharide chains form the backbone for different hemicelluloses. 

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). 

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