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High-temperature behavior of hyperthermostable Thermotoga maritima xylanase XYN10B after designed and evolved mutations

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Abstract

A hyperthermostable xylanase XYN10B from Thermotoga maritima (PDB code 1VBR, GenBank accession number KR078269) was subjected to site-directed and error-prone PCR mutagenesis. From the selected five mutants, the two site-directed mutants (F806H and F806V) showed a 3.3–3.5-fold improved enzyme half-life at 100 °C. The mutant XYNA generated by error-prone PCR showed slightly improved stability at 100 °C and a lower Km. In XYNB and XYNC, the additional mutations over XYNA decreased the thermostability and temperature optimum, while elevating the Km. In XYNC, two large side-chains were introduced into the protein’s interior. Micro-differential scanning calorimetry (DSC) showed that the melting temperature (Tm) dropped in XYNB and XYNC from 104.9 °C to 93.7 °C and 78.6 °C, respectively. The detrimental mutations showed that extremely thermostable enzymes can tolerate quite radical mutations in the protein’s interior and still retain high thermostability. The analysis of mutations (F806H and F806V) in a hydrophobic area lining the substrate-binding region indicated that active site hydrophobicity is important for high activity at extreme temperatures. Although polar His at 806 provided higher stability, the hydrophobic Phe at 806 provided higher activity than His. This study generates an understanding of how extreme thermostability and high activity are formed in GH10 xylanases.

Key points

• Characterization and molecular dynamics simulations of TmXYN10B and its mutants

• Explanation of structural stability of GH10 xylanase

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Data availability

All data generated or analyzed during this study are included in this published article (and its supplementary information files).

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Acknowledgements

This work was financially supported by the project (no. DY135-B2-07) from the China Ocean Mineral Resources R&D Association, Hubei Provincial Technical Innovation Program (no. 2018ABA093).

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Authors and Affiliations

  1. College of Life Science and Technology, Wuhan Polytechnic University, Wuhan, 430048, China

    Yawei Wang & Jiangke Yang

  2. College of Life Science, South-central University for Nationalities, Wuhan, 430074, China

    Jing Wang, Zhongqiang Zhang & Hairong Xiong

  3. School of Forest Sciences, University of Eastern Finland, FI-80101, Joensuu, Finland

    Ossi Turunen

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  1. Yawei Wang
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  2. Jing Wang
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  3. Zhongqiang Zhang
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Contributions

YW, OT, and HX conceived and designed research. YW, JW, and ZZ conducted experiments. JY and OT analyzed data. YW, OT, and HX wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Ossi Turunen or Hairong Xiong.

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Wang, Y., Wang, J., Zhang, Z. et al. High-temperature behavior of hyperthermostable Thermotoga maritima xylanase XYN10B after designed and evolved mutations. Appl Microbiol Biotechnol 106, 2017–2027 (2022). https://doi.org/10.1007/s00253-022-11823-3

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