Domain Compatibility and Linker Design Dictate the Success of Chimeric Cellulase Engineering

Efficient conversion of lignocellulosic biomass into fermentable sugars remains a major challenge due to individual cellulases’ limited synergy and catalytic efficiency. Engineering chimeric enzymes provides a promising strategy to streamline biomass hydrolysis by combining complementary catalytic activities in a single protein, thereby enhancing efficiency and lowering process costs. In this study, we constructed chimeric cellulases by fusing a thermophilic GH1 β-glucosidase ( Ts BG) with endoglucanases from the GH5 ( Bs EG2) or GH9 ( Bl EG) families through flexible peptide linkers. Constructs containing BsEG2 exhibited a pronounced loss of β-glucosidase activity and reduced endoglucanase activity, whereas substitution with the full-length BlEG restored dual functionality under identical design conditions. The optimized chimera ( Bl EG+(G ₄ S) ₂ + Ts BG) demonstrated enhanced catalytic performance, with a 4.8-fold lower K _m , a 1.7-fold higher V _max , and an increased k _cat (from 1088 to 1454 s ^-1 ). The chimera also exhibited enhanced stability, retaining ∼10 % higher activity under elevated cellobiose (up to 300 mM) and >90 % specific activity in 2.5 M NaCl. Molecular dynamics simulations further revealed that activity loss in non-optimized constructs arose from C-terminal structural instability and steric clashes, underscoring the critical role of domain orientation and linker flexibility in chimera design. These findings establish a chimeric cellulase that integrates endoglucanase and β-glucosidase activities in a single polypeptide, offering a robust and cost-effective biocatalyst for lignocellulosic biomass conversion.