Enzymatic hydrolysis of cellulosic materials using synthetic mixtures of purified cellulases bioengineered at N-glycosylation sites.

Mutant forms of recombinant endoglucanase II (EG II, N194A), cellobiohydrolase I (CBH I, N45A) and cellobiohydrolase II (CBH II, N219A) from Penicillium verruculosum with enhanced cellulase activities, achieved by engineering of enzyme N-glycosylation sites in our previous studies, were used as components of the binary and ternary mixtures of cellulases in hydrolysis of Avicel and milled aspen wood. Using the engineered forms of the enzymes at a dosage of 10 mg/g substrate resulted in significant boosting of the glucose release from cellulose in the presence of excess β-glucosidase relative to the performance of the corresponding wild-type mixtures at the same loading. The boosting effects reached 11-40% depending on the reaction time and substrate type. In hydrolysis of both cellulosic substrates by the binary mixtures of cellulases, all the enzyme pairs exhibited synergism. The magnitude of the synergistic effects (Ks) did not depend notably upon the induced mutations in the enzymes, and they were in the range of 1.3-1.8 for the combinations of EG II with CBH I (or CBH II), and 2.3-2.9 for the CBH I-CBH II pair. The results of this study should provide a basis for the development of a more effective fungal strain capable of producing cellulase cocktails with enhanced hydrolytic performance against lignocellulosic materials.