Toward Efficient Cellulosic Biomass Breakdown: Enzymatic Innovation and Kinetic Insights

ABSTRACT

Efficient hydrolysis of crystalline cellulose necessitates a coordinated approach to substrate targeting, depolymerization, and product conversion. In this study, we present a tri‐domain GH7‐CBM2a‐GH3 fusion architecture that facilitates intramolecular synergy among processive cellulose depolymerization, substrate binding, and oligosaccharide hydrolysis within a single modular enzyme. The covalent integration of cellobiohydrolase (GH7) and β‐glucosidase (GH3) via a CBM2a targeting domain enhances localized catalytic coordination at the cellulose surface, thereby improving binding to crystalline substrates and minimizing diffusional constraints. In comparison to individual or merely mixed enzymes, the fusion construct demonstrates enhanced hydrolytic activity and greater stability under operational conditions. This research establishes enforced domain integration as an effective strategy for optimizing interfacial catalysis and advancing the design of efficient biocatalysts for sustainable biomass conversion.