Dual Enhancement of Thermostability and Activity of Xylanase through Computer-Aided Rational Design

Lu, Meizi; Li, Zhihong; Zhuang, Hui; Yang, Shuanghao; Zhao, Xingchu; Feng, Ruirui; Shen, Haoyu; Kovalevsky, Andrey; Zhang, Shengkai; Xie, Zaipeng; Li, Xin; Shen, Qirong; Wan, Qun

doi:10.1021/acssuschemeng.4c04974

Title: Dual Enhancement of Thermostability and Activity of Xylanase through Computer-Aided Rational Design

Journal Article · 02 October 2024 · ACS Sustainable Chemistry & Engineering

DOI: https://doi.org/10.1021/acssuschemeng.4c04974 · OSTI ID:2472674

^[1]; Li, Zhihong ^[1]; Zhuang, Hui ^[1]; Yang, Shuanghao ^[1]; Zhao, Xingchu ^[1]; Feng, Ruirui ^[1]; Shen, Haoyu ^[2];

^[3]; Zhang, Shengkai ^[4]; Xie, Zaipeng ^[5]; Li, Xin ^[2]; Shen, Qirong ^[1];

^[1]

Nanjing Agricultural University (China)
University of Chinese Academy of Sciences (China); Chinese Academy of Sciences (CAS), Shanghai (China)
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
Institute of Advanced Science Facilities, Shenzhen (China)
Hohai University, Nanjing (China)

In the realm of enzyme engineering, the dual enhancement of thermostability and activity remains a challenge. Herein, we employed a computer-aided approach integrating folding free energy calculations and evolutionary analysis to engineer Paecilomyces thermophila xylanase into a hyperthermophilic enzyme for application in the paper and pulp industry. Through the computational rational design, XynM9 with superior thermostability and enhanced activity was designed. Its optimal reaction temperature increases by 10 °C to 85 °C, its T_m increases by 10 °C to 93 °C, and its half-life increases 11-fold to 5.8 h. Additionally, its catalytic efficiency improves by 57% to 3926 s^–1 mM^–1. Molecular dynamics simulations revealed that XynM9 is stabilized by more hydrogen bonds and salt bridges than wild-type xylanase. The mutant’s narrower catalytic cleft enhances the substrate-binding affinity, thus improving the catalytic efficiency. In harsh conditions at 80 °C and pH 10, using XynM9 significantly reduced both hemicellulose and lignin, which makes it a good candidate for use in the paper and pulp process. Finally, our study presents an accurate and efficient strategy for the dual enhancement of enzyme properties, guiding further improvement of computational tools for protein stabilization.

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Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES)

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 2472674

Journal Information:: ACS Sustainable Chemistry & Engineering, Journal Name: ACS Sustainable Chemistry & Engineering Journal Issue: 41 Vol. 12; ISSN 2168-0485

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
conservation analysis
folding free energy change
rational design
thermostability
xylanase

Title: Dual Enhancement of Thermostability and Activity of Xylanase through Computer-Aided Rational Design

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