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Boosting Enzyme Activity in Biomass Conversion by Modulating the Hydrolysis Process of Cellobiohydrolases

2024; American Chemical Society; Volume: 14; Issue: 21 Linguagem: Inglês

10.1021/acscatal.4c05393

2155-5435

Han Liu, Yu Ding, Scott Mazurkewich, Wenwen Pei, Wei Xu, Johan Larsbrink, Christophe Chipot, Zhangyong Hong, Wensheng Cai, Zhiyou Zong,

Microbial Metabolic Engineering and Bioproduction

Cellobiohydrolases (CBHs) are the most significant cellulose-degrading enzymes, the performance of which determines the cost-effective utilization of renewable lignocellulosic resources. Most engineering strategies for improving CBH hydrolysis are currently focused on accelerating the noncatalytic enzyme–substrate dissociation by increasing the flexibility of eight substrate-enclosing loops (SELs), which does not take the catalysis into account or even deteriorates it. Here, in the model Trichoderma reesei CBHI, we identified a key SEL that affects the dissociation by examining enzyme–enzyme/substrate interactions. Furthermore, through analyzing the hydrogen-bonding network for the catalytic region, we detected a crucial residue D262. Root-mean-square-fluctuation analysis indicates that its replacement with valine (D262V) markedly improves the stability of the catalytic triad. Through QM/MM simulations, we determined that this mutation diminished the free-energy barrier against catalysis by 2.3 kcal/mol and increased kcat by 53.1%, as determined in kinetic experiments. Additionally, the substitution caused a significant enhancement of SEL flexibility and led to a lowered dissociation barrier by 2.1 kcal/mol. The cellobiose yield was increased by 49.8%, owing to the impact of the single valine replacement on the enzyme hydrolysis. This work unlocks a brand-new engineering direction for industrially important CBHs, contributing to more efficient depolymerization of renewable lignocellulose.