Mechanisms and strategies for lignin-targeted pretreatment and β-glucosidase molecular engineering in enhancing cellulase hydrolysis efficiency: A review.

Lignocellulosic biomass (LCB), a second-generation feedstock for cellulosic ethanol production, requires efficient pretreatment. This step is critical in biorefinery processes. During enzymatic hydrolysis, lignin limits cellulase accessibility, whereas β-glucosidase (BGL) functions as a rate-limiting enzyme. This review systematically summarises lignin-targeted pretreatment methods, including acidic, organic solvent, deep eutectic solvent (DES), and biological approaches. Particular emphasis is placed on lignin structural modifications and their implications for enzyme engineering. In addition, recent advances in BGL molecular engineering are discussed, covering genetic engineering such as CRISPR-Cas9-based gene editing, protein engineering including site-directed mutagenesis, directed evolution, and surface charge engineering, and enzyme immobilisation strategies. The potential application of artificial intelligence (AI)-assisted protein design for enzyme optimisation is also briefly discussed. The synergistic relationship between pretreatment and enzyme engineering is examined, together with key factors influencing process efficiency and operational conditions. In conclusion, pretreatment-induced lignin structural modifications and BGL engineering can exhibit synergistic effects in improving enzymatic hydrolysis efficiency. Their coordinated optimisation improves hydrolysis performance and reduces overall process costs. This review suggests that the coordinated optimisation of pretreatment and enzyme engineering provides a promising strategy for achieving cost-effective enzymatic hydrolysis and sustainable biofuel production.