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Enhanced thermostability and catalytic efficiency of glucose oxidase in Pichia Pastoris

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Abstract

Glucose oxidase (GOD) has many practical applications, but its poor thermostability limits its broader use. In this research, three primary mutants of wild-type GOD were designed using rational mutagenesis, and the GODm mutant was constructed by combinatorial design. The expression, purification, and enzymatic properties of the mutants were studied. The specific enzyme activity of GODm was 2.10-fold higher than that of wild type, and the (kcat/Km) value was increased by 1.45-fold. After treatment at 55 ℃ for 3 h, GODm retained 37.5% of its enzymatic activity, and the half-life (t1/2) of GODm at 55 ℃ and 65 ℃ was 2.28-fold and 3.36-fold higher than that of wild type, respectively. By analyzing the three-dimensional structure of wild type and the GODm mutant, it was found that T30V formed a new hydrogen bond with FAD and strengthened the hydrophobic interaction, D315K optimized the surface electrostatic interaction, and A162T improved the efficiency of the electron pathway. Thus, a novel mutant with improved thermostability and catalytic efficiency was obtained in this research.

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Acknowledgements

The authors are grateful for the financial support from the National First-class Discipline Program of Light Industry Technology and Engineering (Grant No. LITE2018-04), the Topnotch Academic Programs Project of Jiangsu Higher Education Institutions (TAPP), and the National Natural Science Foundation of China (NSFC) (32072162).

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Authors and Affiliations

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

    Hanlei Zhou, Pu Zheng, Pengcheng Chen, Xiaowei Yu & Dan Wu

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  1. Hanlei Zhou
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  2. Pu Zheng
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  3. Pengcheng Chen
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  4. Xiaowei Yu
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  5. Dan Wu
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Contributions

HZ: conceptualization, methodology, formal analysis, investigation, data curation, writing—original draft, and writing—review and editing. DW: conceptualization, resources, project administration, funding acquisition, and writing—review and editing. PZ: resources and supervision. PC and XY: supervision and validation.

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Correspondence to Dan Wu.

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Zhou, H., Zheng, P., Chen, P. et al. Enhanced thermostability and catalytic efficiency of glucose oxidase in Pichia Pastoris. Syst Microbiol and Biomanuf 2, 296–304 (2022). https://doi.org/10.1007/s43393-021-00057-5

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  • DOI: https://doi.org/10.1007/s43393-021-00057-5

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