Enhancement of catalytic activity and alkaline stability of cellobiohydrolase by structure-based protein engineering

Alkaline cellobiohydrolases have the potential for application in various industries, including pulp processing and laundry where operation under high pH conditions is preferred. In this study, variants of CtCel6A cellobiohydrolase from Chaetomium thermophilum were generated by structural-based protein engineering with the rationale of increasing catalytic activity and alkaline stability. The variants included removal of the carbohydrate-binding module (CBM) and substitution of residues 173 and 200. The CBM-deleted enzyme with Y200F mutation predicted to mediate conformational change at the N-terminal loop demonstrated increased alkaline stability at 60 °C, pH 8.0 for 24 h up to 2.25-fold compared with the wild-type enzyme. Another CBM-deleted enzyme with L173E mutation predicted to induce a new hydrogen bond in the substrate-binding cleft showed enhanced hydrolysis yield of pretreated sugarcane trash up to 4.65-fold greater than that of the wild-type enzyme at the pH 8.0. The variant enzymes could thus be developed for applications on cellulose hydrolysis and plant fiber modification operated under alkaline conditions.

The online version contains supplementary material available at 10.1007/s13205-022-03339-4.