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Hotspot-based mutation engineering of MAase from Lactobacillus rhamnosus YXY412 for the improvement of hydrolytic activity

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Abstract

The semi-rational design of enzymes has become a popular and effective modification method to improve their hydrolytic activity and/or thermal stability toward target substrates. Here, the specific activity of a maltogenic amylase from Lactobacillus rhamnosus YXY412 (LrMA) toward soluble starch was exactly enhanced through hotspot-based research. Based on multiple sequence alignment, three-dimensional structure and existed literature, thirty-eight amino acid residues of LrMA were rationally selected for site-directed mutagenesis. After the screening of the mutants, LrMAD172A, LrMAG260A, LrMAK334A and LrMAM477A were selected with the activity accounted for 144–209% of that in wild-type. Among all the mutants, LrMAG260A possessed the highest activity toward soluble starch, reached 133 U/mg, about twice as high as that in the wild-type. Its temperature for optimum activity still maintained at 60 °C, while had no significant loss of thermal stability occurred. In addition, compared with the wild-type in pH stability, the mutant retained over 80% residual activity at a wider pH range of 4.5–8.5. Furthermore, the kcat/Km of LrMAG260A was two times higher than that of the wild-type, indicating that the mutant had a better affinity and a higher conversion efficiency for soluble starch.

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Acknowledgements

This work was financially supported by the Postdoctoral Science Foundation of China (2021M691278) and the National Key Special Project for the 13th National 5-Year Plan Program of China (2016YFD0400500).

Funding

This work was financially supported by the Postdoctoral Science Foundation of China (2021M691278) and the National Key Special Project for the 13th National 5-Year Plan Program of China (2016YFD0400500).

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Authors and Affiliations

  1. Wuxi School of Medicine, Jiangnan University, Wuxi, 214122, China

    Xiaoya Su & Minchen Wu

  2. Key Laboratory of Carbohydrate Chemistry and Biotechnology, School of Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China

    Xiaoya Su, Dong Zhang & Yuqing Lei

  3. State Key Laboratory of Food Science and Technology, Laboratory of Baking and Fermentation Science, Cereals/Sourdough and Ingredient Functionality Research, Jiangnan University, Wuxi, 214122, China

    Jing Huang & Weining Huang

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  1. Xiaoya Su
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Contributions

Experimental design was done by SXY, WMC and HWN; experiments conducted and analyzed by SXY, ZD, HJ and LYQ; SXY wrote and edited the manuscript. All authors finally approved the manuscript.

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Correspondence to Weining Huang or Minchen Wu.

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Su, X., Zhang, D., Huang, J. et al. Hotspot-based mutation engineering of MAase from Lactobacillus rhamnosus YXY412 for the improvement of hydrolytic activity. Syst Microbiol and Biomanuf (2024). https://doi.org/10.1007/s43393-024-00261-z

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