Engineering a Cold-Active Cellulase Complex with a Novel Mushroom Cellobiohydrolase for Efficient Biomass...

Cold-active cellulases are highly desirable for temperature-sensitive biomass valorization and food processing, yet they remain scarce in conventional industrial fungal platforms. In this study, a novel cold-induced cellobiohydrolase, VvCBHI-II, was mined from the mushroom Volvariella volvacea and successfully engineered into the industrial workhorse Trichoderma reesei via site-specific homologous replacement. Structural homology modeling revealed that the substitution of the flexible B3 loop with a β-sheet creates a more open substrate-binding cleft in VvCBHI-II. Consequently, the purified VvCBHI-II exhibited robust endoglucanase-like characteristics with superior catalytic efficiency on amorphous cellulose. At 10 °C, the engineered cellulase complex demonstrated an 8.1-fold increase in filter paper activity compared to the wild-type strain. Mechanistic structural analyses indicated that the open cleft architecture elongates and weakens the hydrogen-bonding network with the cellobiose product, facilitating rapid product dissociation and alleviating severe cold-induced product inhibition. In practical applications, the engineered cold-active enzyme complex exhibited an exceptional saccharification capacity on natural pear pomace at 10 °C. Furthermore, when applied to simulated fruit juice processing, it significantly maximized the extraction yield, elevated the sweetness response, and substantially mitigated undesirable bitterness and astringency. This study elucidates the structural-functional paradigm of cold-adapted cellobiohydrolases and provides a promising strategy for formulating highly efficient, energy-saving biocatalysts for the food and biorefinery industries.