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Improving the Thermostability of α-Glucosidase from Xanthomonas campestris through Proline Substitutions Guided by Semi-rational Design

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Abstract

The α-glycosidase from Xanthomonas campestris (XgtA) can specifically catalyze the transglycosylation reactions, thus can be applied for the enzymatic synthesis of α-glycosides. However, the low thermal stability of XgtA has been a bottleneck for its industrial application. In this research, a combined semi-rational design strategy was used based on analysis of the α-helices and β-turns of XgtA, FireProt prediction of thermostable mutants and molecular dynamic simulations for improving the thermostability. The key positions with a significant impact on the thermal stability of XgtA were identified and the effect of proline substitution was tested. Five single-point mutants V167P, A177P, A210P, A220P, T345P, and a combined mutant Mut5 were obtained with improved thermal stability. Mut5 displayed a 3.06-fold increase in time of half-life at 45°C without impairing its initial hydrolytic activity. Molecular dynamics simulations revealed that changes in the flexibility of amino acid residues, newly-forming hydrogen bonding networks and hydrophobic interactions were responsible for the improved thermostability. These results suggest that proline substitution of key flexible positions is an effective strategy for improving enzyme thermostability.

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Acknowledgements

This research was supported by the National Natural Science Foundation of China (82104322) and the Natural Science Foundation of Zhejiang (LQ22H280012).

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Authors and Affiliations

  1. Institute of Fermentation Engineering, Zhejiang University of Technology, Hangzhou, 310014, China

    Luyi Chen, Kunyao Jiang, Yaoyao Zhou, Linjiang Zhu & Xiaolong Chen

  2. College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China

    Luyi Chen, Kunyao Jiang, Yaoyao Zhou, Linjiang Zhu & Xiaolong Chen

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  1. Luyi Chen
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Correspondence to Linjiang Zhu or Xiaolong Chen.

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Chen, L., Jiang, K., Zhou, Y. et al. Improving the Thermostability of α-Glucosidase from Xanthomonas campestris through Proline Substitutions Guided by Semi-rational Design. Biotechnol Bioproc E 27, 631–639 (2022). https://doi.org/10.1007/s12257-022-0129-2

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