p Cellobiohydrolase II (CBHII) is the second most abundant enzyme in cellulase cocktails derived from Trichoderma reesei. Compared to CBHI, the linker region of CBHII is longer and contains more arginine residues. However, the role of these features in regulating CBHII catalytic activity and lignin tolerance remains poorly understood. In this study, linker-engineered CBHII variants were created by gradually shortening the central part or inserting or substituting arginine residues in linker region, aiming to improve hydrolytic activity and lignin resistance. The presence of the first arginine residue near CBM1 was essential for the proper folding and expression of CBHII. Reducing the length of the linker, as well as decreasing the number of arginine residues and O-glycosylation sites, significantly influenced the catalytic properties, cellulose and lignin-binding capabilities, and solution-phase enzyme conformation. Two engineered variants, CBHII-L-14 and CBHII-L-17, showed notable enhancements in both catalytic performance and lignin resistance, achieving 67.7 % and 60.5 % greater hydrolysis efficiency, respectively, compared with the wild-type CBHII enzyme when acting on filter paper (FP) in lignin-rich environments. Small-angle X-ray scattering (SAXS) experiments indicated these engineered variants possessed shortened yet structurally more rigid linker regions. The combination effect of the more rigidity of the shorter linker, along with the reduced arginine residues and O-glycosylation sites likely accounts for their improved catalytic activities and reduced lignin inhibition. The findings suggest that engineering shorter linker regions in CBHII represents a viable approach for increasing catalytic efficiency, thereby offering potential advancements in enzyme robustness and enhanced hydrolysis of lignocellulosic biomass.

Cellobiohydrolase II (CBHII) is the second most abundant enzyme in cellulase cocktails derived from Trichoderma reesei. Compared to CBHI, the linker region of CBHII is longer and contains more arginine residues. However, the role of these features in regulating CBHII catalytic activity and lignin tolerance remains poorly understood. In this study, linker-engineered CBHII variants were created by gradually shortening the central part or inserting or substituting arginine residues in linker region, aiming to improve hydrolytic activity and lignin resistance. The presence of the first arginine residue near CBM1 was essential for the proper folding and expression of CBHII. Reducing the length of the linker, as well as decreasing the number of arginine residues and O-glycosylation sites, significantly influenced the catalytic properties, cellulose and lignin-binding capabilities, and solution-phase enzyme conformation. Two engineered variants, CBHII-L-14 and CBHII-L-17, showed notable enhancements in both catalytic performance and lignin resistance, achieving 67.7 % and 60.5 % greater hydrolysis efficiency, respectively, compared with the wild-type CBHII enzyme when acting on filter paper (FP) in lignin-rich environments. Small-angle X-ray scattering (SAXS) experiments indicated these engineered variants possessed shortened yet structurally more rigid linker regions. The combination effect of the more rigidity of the shorter linker, along with the reduced arginine residues and O-glycosylation sites likely accounts for their improved catalytic activities and reduced lignin inhibition. The findings suggest that engineering shorter linker regions in CBHII represents a viable approach for increasing catalytic efficiency, thereby offering potential advancements in enzyme robustness and enhanced hydrolysis of lignocellulosic biomass.