Purification cellobiase

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. 

These methods for enzyme activity determination are easy and convenient for screening enzymes during purification procedures, especially for cellobiase and endoglucanase. Up to 72 of these types of samples can be run at the same time. For cellobiohydrolase it is more difficult, but it is still possible to run up to 24 samples at the same time. In this procedure, the samples in Corex tubes are continuously stirred while kept in a constant-temperature water bath. After incubation the tubes are removed, put on ice, and 5% TCA is added to stop the reaction. The solid substrate is subsequently removed by centrifugation. 

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... 

Two years later Selby and Maitland (17) resolved the T. viride system into three components which they called Ci, CM-cellulase, and cellobiase. The principal advance was the purification of the Ci to the point that it had little effect on cotton. This permitted a large increase in total solubilization as the result of recombination of individually ineffective components. In a similar fashion in 1969 Wood (54) reported resolution of the Fusarium solani complex into three components which he called Ci, Cx, and / -glucosidase. 

Gong C-S, Ladisch MR, Tsao GT (1977) Cellobiase from Trichoderma viride-. purification, properties,kinetics, and mechanism. Biotechnology and Bioengineering 19 959-981 Gong C-S, Ladisch MR, Tsao GT (1979) Biosynthesis, purification and mode of action of cellulase of Trichoderma reesei-. hydrolysis of cellulose mechanisms of enzymatic and acid catalysis. Advances in Chemistry Series vol. 181, American Chemical Society, Washington, DC, pp 261-287... 

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