Abstract
Xylanases have a broad range of applications in industrial biotechnologies, which require the enzymes to resist the high-temperature environments. The majority of xylanases have maximum activity at moderate temperatures, which limited their potential applications in industries. In this study, a thermophilic GH10 family xylanase XynAF1 from the high-temperature composting strain Aspergillus fumigatus Z5 was characterized and engineered to further improve its thermostability. XynAF1 has the optimal reaction temperature of 90 °C. The crystal structure of XynAF1 was obtained by X-ray diffraction after heterologous expression, purification, and crystallization. The high-resolution X-ray crystallographic structure of the protein-product complex was obtained by soaking the apo-state crystal with xylotetraose. Structure analysis indicated that XynAF1 has a rigid skeleton, which helps to maintain the hyperthermophilic characteristic. The homologous structure analysis and the catalytic center mutant construction of XynAF1 indicated the conserved catalytic center contributed to the high optimum catalytic temperature. The amino acids in the surface of xylanase XynAF1 which might influence the enzyme thermostability were identified by the structure analysis. Combining the rational design with the saturation mutation at the high B-value regions, the integrative mutant XynAF1-AC with a 6-fold increase of thermostability was finally obtained. This study efficiently improved the thermostability of a GH10 family xylanase by semi-rational design, which provided a new biocatalyst for high-temperature biotechnological applications.
Key points
• Obtained the crystal structure of GH10 family hyperthermophilic xylanase XynAF1.
• Shed light on the understanding of the GH10 family xylanase thermophilic mechanism.
• Constructed a 6-fold increased thermostability recombinant xylanase.
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Data availability
The atomic coordinates and structure factors have been deposited in the Protein Data Bank (http://www.wwpdb.org/).
PDB # 6JDT: the apo-structure of GH10 family xylanase XynAF1;
PDB # 6JDZ: the ligand complex structure of XynAF1, with 120 minutes soaking in 100 mM XTE.
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Acknowledgements
We thank Dr. Feng Wang and Dr. Rui Wang in Wuxi Biortus Biosciences Co. Ltd. for their technical assistance. We thank the Shanghai Synchrotron Radiation Facility for X-ray data collection.
Funding
This work was financially supported by the National Key Research and Development Program (2018YFD0500201) and the National Natural Science Foundation of China (31801935). Q.W was supported by the National Natural Science Foundation of China (31670790) and the Fundamental Research Funds for the Central Universities (KYXK202009). Y.M was supported by the Jiangsu Provincial Natural Science Foundation of China (BK20180538).
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G.L performed the experiments and analyzed the data. R.Z and Q.W designed and coordinated the study. Y.L and D.L prepared and characterized the enzyme. X.Z and Z.L conducted the X-ray crystallography experiments. G.L, D.L and Y.M contributed to the writing of this manuscript. All authors reviewed the results and approved the final version of the manuscript.
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Li, G., Zhou, X., Li, Z. et al. Significantly improving the thermostability of a hyperthermophilic GH10 family xylanase XynAF1 by semi-rational design . Appl Microbiol Biotechnol 105, 4561–4576 (2021). https://doi.org/10.1007/s00253-021-11340-9
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DOI: https://doi.org/10.1007/s00253-021-11340-9