Catalytic Efficiency Improvement in Cellobiohydrolase I by Cross-Species Domain Exchange Engineering

Understanding the molecular mechanisms of cellobiohydrolase I (CBHI), a key enzyme in cellulase complexes, is crucial for developing efficient enzymes for the degradation of lignocellulosic biomasses (LCB). Building on our previous discovery that <i>Chaetomium thermophilum</i> CBHI (C-CBH) exhibits significantly higher specific activity than <i>Trichoderma reesei</i> CBHI (T-CBH), systematic domain-swapping experiments were conducted to elucidate the structural determinants of catalytic efficiency in CBHI. Herein, the carbohydrate-binding modules (CBM) of the CBHIs from <i>Trichoderma reesei</i> (T-CBH) and <i>Chaetomium thermophilum</i> (C-CBH) were interchanged and to obtain two chimeric mutants TC-CBH and CT-CBH. These four CBHs were expressed in <i>T. reesei</i>, and the enzyme properties were analyzed. Comparative characterization revealed that while module exchange preserved native temperature/pH adaptability, it significantly altered substrate specificity and catalytic performance. The CT-CBH variant was identified as the most efficient biocatalyst, exhibiting four key advantages over T-CBH: (1) protein expression levels that far exceed those of T-CBH, (2) specific activity enhanced by 2.6-fold (734.5 U/μM vs. 282.5 U/μM on MU-cellobiose), (3) superior degradation capacities for filter paper (1.6-fold) and xylan, and (4) improved binding affinity for crystalline cellulose. These findings establish cross-species domain engineering as a viable strategy for creating high-performance cellulases, providing both mechanistic insights and practical solutions for lignocellulose degradation.