| Résumé | The β-glucosidase plays an important role in cellulose degradation by hydrolyzing soluble oligosaccharides, including cellobiose, with a concomitant release of glucose. This prevents the accumulation of cellobiose, an inhibitor of cellobiohydrolase, thus increasing the rate of cellulose hydrolysis. To study the kinetics and enzyme hydrolysis mechanism of the β-glucosidase of the Basidiomycete Schizophyllum commune, this chapter describes procedures that are developed for the purification and the detection of β-glucosidase. Rapid techniques have also been utilized for the analysis of oligosaccharides of cellulose by HPLC chromatography. Two procedures are adopted to measure the activity of β-glucosidase in this chapter; the p-nitrophenyl-β-D-glucosidase (PNPGase) and a commercial glucose detection kit. The PNPGase activity is used in the enzyme purification steps and the glucose determination kit for studying enzyme kinetics. However, both assays have been adapted to microtiter dish scale so that as many as 96 samples can be assayed and scanned by the plate reader rapidly. These microassays also offer another advantage in that only a minute amount of sample is required for accurate measurement. |
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