Enzymatic hydrolysis of cellulosic materials using synthetic mixtures of purified cellulases bioengineered at N-glycosylation sites
- 66 Downloads
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.
KeywordsCellulase Protein engineering N-glycosylation Enzymatic hydrolysis of cellulose Boosting effect Synergism
This work was supported by the Russian Science Foundation (Grant No. 16-14-00163).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- dos Santos LV, de Barros Grassi MC, Gallardo JCM, Pirolla RAS, Calderón LL, de Carvalho-Netto OV, Parreiras LS, Camargo ELO, Drezza AL, Missawa SK, Teixeira GS, Lunardi I, Bressiani J, Pereira GAG (2016) Second-generation ethanol: the need is becoming a reality. Ind Biotechnol 12:40–57CrossRefGoogle Scholar
- Proskurina OV, Korotkova OG, Rozhkova AM, Matys VY, Koshelev AV, Okunev ON, Nemashkalov VA, Sinitsyna OA, Sinitsyn AP (2013) Application of the “fusion” approach for the production of highly efficient biocatalysts based on recombinant strains of the fungus Penicillium verruculosum for the conversion of cellulose-containing biomass. Catal Ind 5:327–334CrossRefGoogle Scholar
- Sinitsyn AP, Korotkova OG, Sinitsyna OA, Rozhkova AM, Dotsenko GS, Proskurina OV, Osipov DO, Kondrat’eva EG, Chekushina AV (2016) Optimizing the composition of cellulase enzyme complex from Penicillium verruculosum: enhancing hydrolytic capabilities via genetic engineering. Catal Ind 8:101–106CrossRefGoogle Scholar