Cellobiase production

Much effort has been expended over the years on increasing enzyme production of T. reesei by isolation of high yielding mutants and optimizing media and fermentation conditions. Strains have been isolated that produce 2-6 times the cellulase productivity of the parent wild strain (QM 6a) in batch culture. The mutants produce higher levels of cellulase protein but the specie activity of the enzymes and the proportions of the individual components (ca. 30% endo- -glucanase, 70% o- -glucanase, and less 1% cellobiase) are similar to those of the parent. 

Cellobiase can be measured by following the glucose production from cellobiose and cellodextrins, the saligenin from salicin, and the p-nitrophenol formation from its / -glucoside (2). 

Previously, both in our laboratory and elsewhere, cellulases subjected to purification procedures were obtained from commercial sources (5,6, 8,9,10,13,39,46). Three cellobiases and several endoglucanases and cellobiohydrolases from commercial preparations were purified in our laboratory. While use of protease inhibitors in the fractionation procedures minimized proteolysis during enzyme purification, the existence of enzymes proteolytically modified, presumedly during prolonged fermentation (required for obtaining high titres for commercial production), was a source of confusion, as previously explained. Therefore, we prepared T. reesei cellulase harvested from young culture broth. This was used to carry out the enzyme purification procedures described below. 

During purification procedures cellobiase activity was monitored by measuring nitrophenol (at A42onm) release for p-nitrophenyl-/ -D-glucoside (JO). Kinetic studies and enzyme characterization were carried out using / -D-cellobiose as substrate with the product, glucose, measured with a Beckman Glucose Analyzer (JO). Assay conditions were pH 4.8 and 50°C. 

Preliminary experiments done in our laboratory showed that antibodies specific for cellobiohydrolase failed to cross react with either purified cellobiase, purified endoglucanase, or crude endoglucanase. These results, together with data reported in the literature, which show that endoglucanase and cellobiohydrolase have different physical structures, indicate that the three cellulases could be transcribed and translated by different genomes. In this context, then, the question arises as to whether cellulase production is regulated by a common regulatory circuit or by different circuits. 

In addition to catabolite repression, the cellulase enzymes themselves are subject to end-product inhibition. For example, as glucose accumulates during saccharification, it interacts noncompetively with cellobiase to inhibit further activity of this enzyme (6). Similar inhibition of endoglucanases occurs when cellobiose accumulates in a saccharification reactor (18,19,20). 

Commercial cellulase and P-glucosidase (Novo Nordisk, Bagvaerd, Denmark) supplied from Novozymes Korea were used. A mixture of Celluclast (80 IU or international filter paper units [IFPU]/mL) and Novozym 188 (792 cellobiase units [CBU]/mL) was used with a ratio of 4 IU of Celluclast/CBU of Novozym to alleviate end-product inhibition by cellobiose. 

The principal application of lectins in bioanalytical systems involves the reversible immobilization of glucose oxidase, invertase, and peroxidase on Con A-Sepharose. Such lectin-based affinity media have also been utilized for immobilization of glycoenzymes. Woodward (18) shows that cellobiase is not desorbed by its substrate cellobiose and product glucose from the support matrix. 

In 1971 and 1972 Halliwell and co-workers (21, 57) separated the T. viride cellulase into four fractions—Ci, C2, CM-cellulase, and cello-biase. It was suggested that the Ci enzyme was a cellobiohydrolase since the principal product of its action was cellobiose. Addition of cellobiase with the Cl enzyme permitted 70% solubilization of cotton after 21 days. No evidence of the enzyme purity was presented. 

Fig. 3. p-Glucosidase inhibition shown by Lineweaver-Burk plot (reproduced from [2]). Lineweaver-Burk plot of kinetic data from peak 2 cellobiase ((3-glucosidase) at several product inhibitor levels. This is an example of noncompetitive inhibition where the product is not only completing for binding in the active site but also binding to a secondary site on the enzyme that alters the enzyme catalytic ability... 

Hence, the presence of glucose as the final product arises in some instances from the presence of a cellobiase, distinct from the cellulase, in the crude preparation. Evidence has been offered also for a random attack at any position in the chain rather than a splitting-off of successive small units from the end of the chains (J52). Reese and co-workers 63) suggest that several enz3ones take part in the degradation, the first of which is demonstrated, in the case of cotton, by its ability to attack the primary wall and, thus, to make the cellulose chains more available to hydrolytic attack 64). 

The immediate product of cellulose hydrolysis by cellulase is believed to be cellobiose, but in crude extracts a cellobiase is generally present and thus D-glucose is the product. The successful separation of cellobiase from cellulase indicates that the two enzymes are not identical. Pigman has reviewed the literature on cellulases. ... 

As shown in Table 4.29, the Ci and Cx factors, which were found to be endo- and exo-l,4-P-glucanases respectively, hydrolyze cellulose to cellobiose. Since the Ci factor is increasingly inhibited by its product, a cellobiase is needed so that cellulose breakdown is not rapidly brought to a standstill. However, cellobiase is also subject to product inhibition. Therefore, complete cellulose degradation is possible only if cellobiase is present in large excess or the glucose formed is quickly eliminated. 

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