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Improving Catalytic Activity, Acid-Tolerance, and Thermal Stability of Glutathione Peroxidase by Systematic Site-Directed Selenocysteine Incorporation

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Abstract

Glutathione peroxidase (GPx) is an important antioxidant enzyme. Selenocysteine (Sec)-containing GPxs (Sec-GPxs) are usually superior to their conventional cysteine-containing counterparts (Cys-GPxs), which make up the majority of the natural GPxs but display unsuitable activity and stability for industrial applications. This study first heterologously expressed and characterized a Cys-GPx from Lactococcus lactis (LlGPx), systematically exchanged all the three Cys to Sec and introduced an extra Sec. The results showed that the insertion of Sec at the active site could effectively increase the enzyme activity and confer a lower optimal pH value on the mutants. The double mutant C36U/L157U increased by 2.65 times (5.12 U/mg). The thermal stability of the C81U mutant was significantly improved. These results suggest that site-directed Sec incorporation can effectively improve the enzymatic properties of LlGPx, which may be also used for the protein engineering of other industrial enzymes containing catalytic or other functional cysteine residues.

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Funding

This work was supported by the National Natural Science Foundation of China General Project (31972050).

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Authors and Affiliations

  1. College of Light Industry and Food Engineering, Guangxi University, Nanning, 530004, China

    Yu-Hui Fang, Yan-Mei Zhang, Shi-Yang Yue, Jing-Jing Peng, Chen-Xing Liu & Cheng-Hua Wang

  2. College of Light Industry and Food Engineering, Guangxi University, 100 Daxue East Road, Nanning, 530004, China

    Cheng-Hua Wang

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  1. Yu-Hui Fang
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Contributions

Conception/design of study—CHW, YHF; data acquisition—YHF, YMZ; data analysis/interpretation—YHF, YMZ, SYY, JJP, CXL, CHW; drafting manuscript—YHF, CHW; critical revision of manuscript—YHF, CHW; final approval and accountability—YHF, CHW. All authors read and approved the final manuscript.

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Correspondence to Cheng-Hua Wang.

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Fang, YH., Zhang, YM., Yue, SY. et al. Improving Catalytic Activity, Acid-Tolerance, and Thermal Stability of Glutathione Peroxidase by Systematic Site-Directed Selenocysteine Incorporation. Mol Biotechnol 65, 1644–1652 (2023). https://doi.org/10.1007/s12033-023-00682-6

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