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Computer-aided design of novel cellobiose 2-epimerase for efficient synthesis of lactulose using lactose

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Abstract

Cellobiose 2-epimerase (CE) is ideally suited to synthesize lactulose from lactose, but the poor thermostability and catalytic efficiency restrict enzymatic application. Herein, a non-characterized CE originating from Caldicellulosiruptor morganii (CmCE) was discovered in the NCBI database. Then, a smart mutation library was constructed based on FoldX ΔΔG calculation and modeling structure analysis, from which a positive mutant D226G located within the α8/α9 loop exhibited longer half-lives at 65–75 °C as well as lower Km and higher kcat/Km values compared with CmCE. Molecular modeling demonstrated that the improvement of D226G was largely attributed to the rigidification of the flexible loop, the compactness of the catalysis pocket and the increment of substrate-binding capability. Finally, the yield of synthesizing lactulose catalyzed by D226G reached 45.5%, higher than the 35.9% achieved with CmCE. The disclosed effect of the flexible loop on enzymatic stability and catalysis provides insight to redesign efficient CEs to biosynthesize lactulose.

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The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by the Program of the National Key Research and Development Project (2020YFA0908400), the Pioneer R&D Program of Zhejiang (2023C01113), the Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang, P. R. China (2018R01014), the Fundamental Research Funds for the Provincial Universities of Zhejiang (RF-C2019005), and the Key Research and Development Program of Zhejiang Province (2021C03093).

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Authors and Affiliations

  1. The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, 310014, People’s Republic of China

    Dong-Xu Jia, Hai Yu, Fan Wang, Li-Qun Jin, Zhi-Qiang Liu & Yu-Guo Zheng

  2. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, 310014, People’s Republic of China

    Dong-Xu Jia, Hai Yu, Fan Wang, Li-Qun Jin, Zhi-Qiang Liu & Yu-Guo Zheng

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Contributions

D-XJ: conceptualization, data curation, writing—original draft, and writing—review & editing. HY: methodology, software, validation, investigation, and writing—original draft. FW: methodology, software, validation, investigation, and writing—original draft. L-QJ: visualization, and writing—original draft. Z-QL: visualization and supervision. Y-GZ: supervision and project administration.

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Correspondence to Zhi-Qiang Liu.

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Jia, DX., Yu, H., Wang, F. et al. Computer-aided design of novel cellobiose 2-epimerase for efficient synthesis of lactulose using lactose. Bioprocess Biosyst Eng 46, 1279–1291 (2023). https://doi.org/10.1007/s00449-023-02896-z

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