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Simultaneously Improved Thermostability and Hydrolytic Pattern of Alpha-Amylase by Engineering Central Beta Strands of TIM Barrel

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Abstract

This study reported simultaneously improved thermostability and hydrolytic pattern of α-amylase from Bacillus subtilis CN7 by rationally engineering the mostly conserved central beta strands in TIM barrel fold. Nine single point mutations and a double mutation were introduced at the 2nd site of the β7 strand and 3rd site of the β5 strand to rationalize the weak interactions in the beta strands of the TIM barrel of α-amylase. All the five active mutants changed the compositions and percentages of maltooligosaccharides in final hydrolytic products compared to the product spectrum of the wild-type. A mutant Y204V produced only maltose, maltotriose, and maltopentaose without any glucose and maltotetraose, indicating a conversion from typical endo-amylase to novel maltooligosaccharide-producing amylase. A mutant V260I enhanced the thermal stability by 7.1 °C. To our best knowledge, this is the first report on the simultaneous improvement of thermostability and hydrolytic pattern of α-amylase by engineering central beta strands of TIM barrel and the novel “beta strands” strategy proposed here may be useful for the protein engineering of other TIM barrel proteins.

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Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China [grant number 21868003]; the Chinese National Basic Research Program (“973”) [grant number 2009CB724703]; the Major Program of Natural Science Foundation of Guangxi [grant number 2016GXNSFEA380003]; and the Guangxi BaGui Scholars of Guangxi Zhuang Autonomous Region of China.

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Authors and Affiliations

  1. College of Light Industry and Food Engineering, Guangxi University, 100 Daxue East Road, Nanning, 530004, People’s Republic of China

    Cheng-Hua Wang, Liang-Hua Lu & Cheng Huang

  2. College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 210009, China

    Bing-Fang He

  3. College of Life Science and Technology, Guangxi University, Nanning, 530004, China

    Ri-Bo Huang

  4. State Key Laboratory of Non-Food Biomass and Enzyme Technology, National Engineering Research Center for Non-food Biorefinery, Guangxi Key Laboratory of Biorefinery, Guangxi Academy of Sciences, Nanning, 530007, China

    Ri-Bo Huang

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  1. Cheng-Hua Wang
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Correspondence to Cheng-Hua Wang.

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Wang, CH., Lu, LH., Huang, C. et al. Simultaneously Improved Thermostability and Hydrolytic Pattern of Alpha-Amylase by Engineering Central Beta Strands of TIM Barrel. Appl Biochem Biotechnol 192, 57–70 (2020). https://doi.org/10.1007/s12010-020-03308-8

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