Detection and characterization of the specific and nonspecific endoglucanases of Trichoderma reesei: Evidence demonstrating endoglucanase activity by cellobiohydrolase II

The cellulase enzyme system of Trichoderma reesei RUT C-30 has been separated by DEAE ion exchange chromatography into four fractions. Their specificity towards substituted cellulose and cellooligosaccharides was revealed by analytical IEF and activity stains. Fraction EGI (26% of the total protein) exhibited mainly endoglucanase activity on carboxymethylcellulose (CMC) whereas endoglucanases EGII and EGIII (15% of the total protein) showed high activity towards CMC as well as xylan, 4-methylumbelliferyl cellobioside MeUmb(Glc)₂] and p-nitrophenyl lactoside (pNPL). A subfraction of EGI (pI 5.9) which has been described in the literature as a cellobiohydrolase (CBHII) was isolated by preparative isoelectric focusing, and was shown to have only 3 U CMCase activity per milligram. Turbidimetric measurements and phase contrast microscopy demonstrated differences between endoglucanase and cellobiohydrolase behaviour during the hydrolysis of purified cellulose (Solka Floc BW-40). Treatment of the purified cellulose with endoglucanases resulted in fibre breakdown into small particles. This was contrasted with no morphological change to the fibres when contacted with the cellobiohydrolase. By this technique it was revealed that the EGI subfraction (pI 5.9) behaves as an endoglucanase and not as a cellobiohydrolase. Incubation of this enzyme with acid-swollen cellulose resulted in cellotriose production, as it did with other endoglucanases which exhibited CMCase activities >; 100 U mg^?1. Cellotriose was not present during the hydrolysis of acid-swollen cellulose with the CBHI fraction.