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

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    Research Article

    Dual Enhancement of Thermostability and Activity of Xylanase through Computer-Aided Rational Design
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    • Meizi Lu
      Meizi Lu
      Jiangsu Provincial Key Lab for Solid Organic Waste Utilization, Key Lab of Organic-Based Fertilizers of China, Jiangsu Collaborative Innovation Center for Solid Organic Wastes, Educational Ministry Engineering Center of Resource-Saving fertilizers, Nanjing Agricultural University, Nanjing 210095, China
      College of Science, Nanjing Agricultural University, Nanjing 210095, China
      More by Meizi Lu
      Orcidhttps://orcid.org/0000-0002-5780-0151
    • Zhihong Li
      Zhihong Li
      College of Science, Nanjing Agricultural University, Nanjing 210095, China
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    • Hui Zhuang
      Hui Zhuang
      College of Science, Nanjing Agricultural University, Nanjing 210095, China
      More by Hui Zhuang
    • Shuanghao Yang
      Shuanghao Yang
      College of Science, Nanjing Agricultural University, Nanjing 210095, China
      More by Shuanghao Yang
    • Xingchu Zhao
      Xingchu Zhao
      Jiangsu Provincial Key Lab for Solid Organic Waste Utilization, Key Lab of Organic-Based Fertilizers of China, Jiangsu Collaborative Innovation Center for Solid Organic Wastes, Educational Ministry Engineering Center of Resource-Saving fertilizers, Nanjing Agricultural University, Nanjing 210095, China
      More by Xingchu Zhao
    • Ruirui Feng
      Ruirui Feng
      College of Science, Nanjing Agricultural University, Nanjing 210095, China
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    • Haoyu Shen
      Haoyu Shen
      CAS Key Laboratory of Computational Biology, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China
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    • Andrey Kovalevsky
      Andrey Kovalevsky
      Neutron Scattering Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States
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      Orcidhttps://orcid.org/0000-0003-4459-9142
    • Shengkai Zhang
      Shengkai Zhang
      Institute of Advanced Science Facilities, Shenzhen 518107 China
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    • Zaipeng Xie
      Zaipeng Xie
      Key Laboratory of Water Big Data Technology of Ministry of Water Resources, Hohai University, Nanjing 211100 China
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    • Xin Li
      Xin Li
      CAS Key Laboratory of Computational Biology, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China
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    • Qirong Shen
      Qirong Shen
      Jiangsu Provincial Key Lab for Solid Organic Waste Utilization, Key Lab of Organic-Based Fertilizers of China, Jiangsu Collaborative Innovation Center for Solid Organic Wastes, Educational Ministry Engineering Center of Resource-Saving fertilizers, Nanjing Agricultural University, Nanjing 210095, China
      More by Qirong Shen
    • Qun Wan*
      Qun Wan
      Jiangsu Provincial Key Lab for Solid Organic Waste Utilization, Key Lab of Organic-Based Fertilizers of China, Jiangsu Collaborative Innovation Center for Solid Organic Wastes, Educational Ministry Engineering Center of Resource-Saving fertilizers, Nanjing Agricultural University, Nanjing 210095, China
      *Email: [email protected]
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      Orcidhttps://orcid.org/0000-0002-8309-0341
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    ACS Sustainable Chemistry & Engineering

    Cite this: ACS Sustainable Chem. Eng. 2024, 12, 41, 15114–15124
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acssuschemeng.4c04974
    Published October 2, 2024
    Copyright © 2024 American Chemical Society
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    Abstract

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    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 Tm 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. Our study presents an accurate and efficient strategy for the dual enhancement of enzyme properties, guiding further improvement of computational tools for protein stabilization.

    Copyright © 2024 American Chemical Society

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    Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssuschemeng.4c04974.

    • Material and methods, evaluation of the AF2-built model of XynM2 using MolProbity (Table S1), characteristics of mutants with substantially reduced ΔGfold and low frequencies (Table S2), the relative activities and thermal stabilities of the proposed mutants (Table S3), calculated ΔΔGfold and characterization of the combined mutants (Table S4), comparing the binding free energy of XynM2-X6 and XynM9-X6 (Table S5), examples of xylanase engineering using various strategies (Table S6), primers used in this study (Table S7), Ramachandran plot of the AF2-built model for XynM2 (Figure S1), the ΔΔGfold heatmaps of all possible mutations calculated using Rosetta Cartesian_ddg, Rosetta ddg_monomer, and FoldX (Figure S2), the frequency heatmap of XynM2 analyzed using the Hotspot Wizard (Figure S3), RMSD analysis for XynM2, XynM9, the XynM2-X6 complex, and the XynM9-X6 complex after MD simulations at 353 K (Figure S4), the built structure of XynM2 (Figure S5), electrostatics comparison between XynM2 and XynM9 (Figure S6), comparison of the substrate-binding pocket between XynM2 and the mutant S16Y (Figure S7), the melt curves of all selected single-point mutants and the combined mutants (Figure S8), the half-lives (t1/2) of XynM2 and its mutants at 80 and 85 °C (Figure S9), and SDS-PAGE analysis of the purified mutants (Figure S10) (PDF)

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    Cited By

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    This article is cited by 1 publications.

    1. Xingyu Liu, Yuxi Zhang, Xiaoya Qi, Dandan Zhao, Huan Rao, Xia Zhao, Yanxiao Li, Jun Liu, Zhen Qin, Jianxiong Hao, Xueqiang Liu. Advances of microbial xylanases in the application of flour industries: A comprehensive review. International Journal of Biological Macromolecules 2024, 282 , 137205. https://doi.org/10.1016/j.ijbiomac.2024.137205
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    ACS Sustainable Chemistry & Engineering

    Cite this: ACS Sustainable Chem. Eng. 2024, 12, 41, 15114–15124
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acssuschemeng.4c04974
    Published October 2, 2024
    Copyright © 2024 American Chemical Society
    Request reuse permissions

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