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Single Amino Acid Substitution in the Pullulanase of Klebsiella variicola for Enhancing Thermostability and Catalytic Efficiency

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Abstract

Based on conserved sites and homology modeling analysis, the residue Phe581 in the Klebsiella variicola SHN-1 pullulanase was selected as the potential thermostability-related site and its role on thermostability and activity was investigated by site-saturated mutagenesis. Compared with the wild-type pullulanase, the optimum temperature of the mutants including F581L, F581Q, F581R, F581T, F581V, and F581Y was increased from 53 to 56 °C, and correspondingly the half lives of these mutants at 55 °C were increased by 4.20, 3.70, 1.90, 7.16, 3.01, and 1.75 min, respectively. By modeling the structure of the pullulanase, formation of more hydrogen bonds by single-site substitution was supposed to be responsible for the improvement of thermostability. Of these mutants, furthermore, F581L and F581V exhibited higher values of V max and k cat/K m, compared with the wild-type enzyme. Therefore, the residue Phe581 was identified as an important site relevant to the activity and thermostability of the pullulanase of K. variicola, and by mutation at this single site, the mutated enzymes with enhanced thermostability and catalytic efficiency were achieved consequently.

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Acknowledgments

Financial supports from the National Key Basic Research and Development Program of China (973 Program) (2011CB710800), the National High Technology Research and Development Program of China (863 Program) (2012AA022207), the National Natural Science Foundation of China (NSFC) (21376107 and 21336009), the 111 Project (111-2-06), the High-end Foreign Experts Recruitment Program (GDW20133200113), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the Jiangsu province “Collaborative Innovation Center for Advanced Industrial Fermentation” industry development program are greatly appreciated.

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Authors and Affiliations

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

    Guo Cui Mu, Yao Nie, Xiao Qing Mu & Yan Xu

  2. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China

    Yan Xu

  3. 2011 Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China

    Yao Nie, Xiao Qing Mu & Yan Xu

  4. Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ, 08854, USA

    Rong Xiao

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  1. Guo Cui Mu
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Correspondence to Yao Nie or Yan Xu.

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Mu, G.C., Nie, Y., Mu, X.Q. et al. Single Amino Acid Substitution in the Pullulanase of Klebsiella variicola for Enhancing Thermostability and Catalytic Efficiency. Appl Biochem Biotechnol 176, 1736–1745 (2015). https://doi.org/10.1007/s12010-015-1675-2

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  • DOI: https://doi.org/10.1007/s12010-015-1675-2

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