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Cloning, expression and improvement of catalytic activity of alginate lyase by site-directed mutation

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Abstract

Alginate lyase mainly produces active alginate oligosaccharides (AOS) by degrading alginate via β-elimination process. In this study, the Pseudoalteromonas sp. Alg6B alginate lyase-encoding gene alg6B-7 from polysaccharide lyase (PL)-7 family was successfully cloned, sequenced, expressed in Escherichia coli. Based on rational design and amino acid sequence alignment of the alginate lyase from various sources, four positive mutants were obtained. The specific enzyme activities of four mutants I62A, A99K, V132S, and L157T were 38.84%, 42.85%, 75.8% and 51.83% higher than that of the wild enzyme, respectively. The Kcat/Km values of the four mutants were both increased, and the catalytic efficiency of V132S was 1.92-fold higher than that of the wild enzyme, especially. The rational design that was employed in this study achieved the dramatic improvement of catalytic activity, which may provide the application potential in industrial production.

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Acknowledgements

This project was financially supported by the National First-class Discipline Program of the Light Industry Technology and Engineering (LITE2018-11).

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  1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China

    Yu Lu, Jianli Zhou, Qiuya Gu, Wenhua Yang, Liu Yang & Xiaobin Yu

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  1. Yu Lu
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  2. Jianli Zhou
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  3. Qiuya Gu
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Contributions

YL: conceptualization, methodology, software, investigation, data curation, writing—original draft preparation JZ: writing-reviewing and editing. WY: resources, investigation. LY: methodology, software, data curation. QG and XY: funding acquisition, resources, supervision.

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Correspondence to Xiaobin Yu.

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Lu, Y., Zhou, J., Gu, Q. et al. Cloning, expression and improvement of catalytic activity of alginate lyase by site-directed mutation. Syst Microbiol and Biomanuf 2, 555–567 (2022). https://doi.org/10.1007/s43393-022-00084-w

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