Exo-/3- -Glucanase

Karl-Erik Eriksson (42) reported that he had prepared an enzyme from Chrysosporium lignorum which, when combined with each of three CM-cellulase enzymes, increased the hydrolysis rate of cotton tenfold. When he incubated this enzyme, which he believed to be of the Ci type, with cellohexaose and observed cellobiose as the only product, he concluded that it was an exo-glucanase which split off cellobiose units. Later (27) Eriksson and Rzedowski reported that the three CM-cellulase enzymes from C. lignorum contained 13, 10, and 7% carbohydrate and catalyzed the hydrolysis of cotton and cellodextrins to produce cellobiose and glucose in the approximate ratio of 3 1. 

Arnold (51) partially purified such an enzyme from cell-free extracts of bakers yeast Matile et al, (45) and Cortat et al. (44) demonstrated the existence of glucanase-containing vesicles within the cytoplasm of Sac-charomyces cerevisiae. These vesicles contained exo- as well as endo-glucanases but the enzymes were not studied in detail. Fleet and Phaff (47) obtained qualitative evidence for the occurrence of endo-/ -( 1 — 3) glucanases in the cell walls of Saccharomyces rosei, Kluyveromyces fragilis, Hansenula anomala, Pichia pastoris, and Candida utilis. K. fragilis and H. anomala contained only exo-glucanase in cell extracts (38). 

These enzymes also resemble the exo-glucanases in their specificity —i.e., they form, or split, a single linkage type—e.g., B-l - 3, or B-l - 4. Some, at least, of the phosphorylases show a preference for oligomers over dimers (20) others act more readily (or exclusively) on the dimers (like the glucosidases). Most do not act on the long polymers (laminarin, etc.) and in this respect, they differ from exo-glucanases. 

Further work on exo-0-1 4-glucanase demands a method for its detection and estimation in crude solutions—i.e.y in the presence of both endo-0-1 — 4-glucanase and cellobiase. For simplification and other reasons (see below), let us ignore the contribution of the endo-0-1- 4-glucanases. If our generalizations on exo-glucanases (1, 2) are correct, we may approach the problem in several ways. The one which seems most promising depends upon the differences in hydrolysis rates of dimer... 

Figure I. Activity of glucosidases and of exo-glucanases as a function of degree of polymerization (DP) of substrate (Reference 2). The exo-13-glu canases shown here act only on fi-1 3 linkages...

The values for the ratio G4/G2 for pure cellobiase, and for pure exo-glucanase vary depending upon the source of enzyme (2). For our example (Figure 2), we assign for cellobiase a ratio G4/G2 = 0.2, and for exo-/M - 4 glucanase G4/G2 = 100. These values are based on data with purified enzymes, and approach the low and the high limits which the ratios may reach. The curve (Figure 2) is plotted from the formula ... 

Second, is the proportion of this hydrolysis which can be assigned to the exo-enzyme (here represented by the ratio YT/YD). The product of these two is a measure of exo-glucanase. 

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. 

Dr. Reese This is quite right. The proposed method is to help us find new exo-glucanases. Then we can determine their R G4/G2 vaj and refine the procedure if need be. ... 

Cellobiohydrolase (1,4-D-glucan cellobiohydrolase) is an exo-glucanase that catalyzes the hydrolysis of (l,4)-p-D-glucosidic linkages in cellulose with the release of cellobiose. Various forms of this enzyme are thought to hydrolyze cellulose chains either from the reducing or from the nonreducing end." " ... 

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