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Improved thermostability of maltooligosyl trehalose hydrolase by computer-aided rational design

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Abstract

Trehalose is a widely used and safe natural disaccharide. Maltooligosyl trehalose hydrolase(MTHase) is one of the key enzymes for trehalose preparation by double enzyme method using starch or dextrin as substrate. In industrial production, the thermalstability of MTHase is of great significance. We first heterogeneously expressed MTHase from Arthrobacter in E.coli strains BL21 (DE3). Based on the overall stability of the protein after virtual saturation mutation predicted by FoldX and the evolutionary information from PSSM, 15 mutations were selected and combined. Finally, the combinatorial mutant G589P/A57P was obtained. At 60 ℃, the t1/2 of G589P/A57P and wild type are 37 min and 19 min, respectively, which is 1.9 times higher than that of wild type. The enzyme kinetic parameters of G589P/A57P were analyzed. The KM and kcat are 4.82 mM and 1136 s− 1, respectively, and the results were close to the wild type, indicating that the mutation did not reduce the catalytic efficiency of the enzyme. The molecular dynamics simulation results show that the rigidity and thermal stability of G589P/A57P protein increase in the range of residues 50–100 and 400–500, which may be due to the proline effect caused by the introduction of proline.

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Acknowledgements

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Funding

This work was supported by the National Natural Science Foundation of China (No. 32171471), Key Research and Development Project of Shandong Province, China (2019JZZY020605), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Top-notch Academic Programs Project of Jiangsu Higher Education Institutions.

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  1. Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu Province, PR China

    Rufan Xing, Hengwei Zhang, Qiang Wang, Yanan Hao, Yujue Wang, Jianghua Chen, Xian Zhang & Zhiming Rao

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  1. Rufan Xing
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  2. Hengwei Zhang
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Contributions

Xian Zhang and Zhiming Rao designed the research; Rufan Xing performed the major experiments and wrote the manuscript draft; and Hengwei Zhang, Qiang Wang, Yanan Hao, Yujue Wang, Jianghua Chen analyzed the data and drafted and revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Xian Zhang.

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Zhiming Rao as an editorial board member, the authors declare a conflict of interest for this paper. Zhiming Rao have been excluded from the peer review and decision-making process to ensure unbiased evaluation and strict adherence to ethical guidelines.

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Xing, R., Zhang, H., Wang, Q. et al. Improved thermostability of maltooligosyl trehalose hydrolase by computer-aided rational design. Syst Microbiol and Biomanuf (2024). https://doi.org/10.1007/s43393-024-00260-0

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