Boosting Enzyme Activity in Biomass Conversion by Modulating the Hydrolysis Process of Cellobiohydrolases
Artikel i vetenskaplig tidskrift, 2024

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.

catalysis

lignocellulose

bioconversion

simulation

dissociation

cellobiohydrolase

Författare

Han Liu

Tianjin Institute of Industrial Biotechnology

National Technology Innovation Center of Synthetic Biology

Yu Ding

Nankai University

Scott Mazurkewich

Chalmers, Life sciences, Industriell bioteknik

Wenwen Pei

Tianjin Institute of Industrial Biotechnology

National Technology Innovation Center of Synthetic Biology

Xiuxin Wei

Nankai University

Johan Larsbrink

Chalmers, Life sciences, Industriell bioteknik

Christophe Chipot

University of Chicago

University of Illinois

Zhangyong Hong

Nankai University

Wensheng Cai

Nankai University

Zhiyou Zong

National Technology Innovation Center of Synthetic Biology

Tianjin Institute of Industrial Biotechnology

ACS Catalysis

21555435 (eISSN)

Vol. 14 16044-16054

Ämneskategorier

Biokemi och molekylärbiologi

Biokatalys och enzymteknik

DOI

10.1021/acscatal.4c05393

Mer information

Senast uppdaterat

2024-12-10