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

Abstract

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.

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

Keywords: Alkaline stability; Cellobiohydrolase; Pulp and paper; Structure-based protein engineering.

Fig. 1

Schematic diagram of CtCel6A mutants construction. The CBM1 was removed from wild-type CtCel6A (FL) resulting in the CBM-deleted mutant (CD) containing only the catalytic domain. The mutant with single (L173E, Y200F) and double mutation (L173E/Y200F) were generated by PCR-based mutagenesis. The wild-type and all mutants were constructed and expressed in yeast P. pastoris

Fig. 2

3D structures of CtCel6A complexed with cellooctaose from a cellotetraose at subsites + 1 to + 4 (PDB: 4A05) and a docked cellotetraose at the subsites − 1 to − 4. A Wild-type CtCel6A showing H-bond interactions (Å) at the subsites − 1 to − 4, and distances (Å) between Leu173 and Tyr200 with the substrate. B Overlaid of Leu173 to Glu and Tyr200 to Phe in mutants to the wild-type CtCel6A revealing 3.0 Å hydrogen bond of Leu173Glu and the saccharide unit at subsite − 4 and a release of steric clash of Tyr200Phe to the cellooctaose substrate at subsite − 1. Figures were generated with PyMol2.1

Fig. 3

Temperature and pH activity profiles of wild-type full-length CtCel6A (FL) and mutants. Mutants included deletion of residues 19–115 of the CBM (CD) and substitution of residues 173 and/or 200 in FL and CD. A Cellulase activities on PASC substrate were determined at temperatures ranging from 40 to 90 °C relative to the mean maximum value observed for each enzyme (100%). B pH profile was determined in buffer ranging from pH 3.0 to 10.0. Reducing sugar production was assessed using DNS reagent. Mean values are shown (n = 3) and error bars indicate standard deviation

Fig. 4

Stability of CtCel6A and its mutants. Reactions on PASC substrate were performed by incubating enzyme in buffer pH 5.0 and pH 8.0 at 60 °C from 0 to 48 h. A Sodium acetate buffer pH 5.0. B Tris–HCl buffer pH 8.0. The remaining activity was determined using DNS reagent. Mean values are shown (n = 3) and error bars indicate standard deviation

Fig. 5

Enzymatic hydrolysis of CtCel6A and its mutants. Reactions were performed with 10 mg of pretreated sugarcane trash as the substrate in buffer pH 5.0 and pH 8.0 at 60 °C for 72 h. The product yield was determined by comparison of HPLC profiles with C₁–C₆ standards. Mean values are shown (n = 3) and error bars indicate standard deviation. Asterisks indicate significant differences (p < 0.05) compared to FT in each pH

Fig. 6

Hydrolysis of pretreated sugarcane trash with different substrate concentration by FL, FL-L173E, CD and CD-L173E. The reactions were performed with pretreated sugarcane trash (10 and 50 mg) in buffer pH 5.0 and pH 8.0 at 60 °C for 72 h. A Sodium acetate buffer pH 5.0. B Tris–HCl buffer pH 8.0. The product yield was determined by comparison of HPLC profiles with C₁–C₆ standards. Mean values are shown (n = 3) and error bars indicate standard deviation. Asterisks indicate significant differences (p < 0.05) compared to FT in each condition