Improvement of the Thermostability and Activity of Pullulanase from Anoxybacillus sp. WB42

  • Bo Pang
  • Li Zhou
  • Wenjing Cui
  • Zhongmei Liu
  • Zhemin ZhouEmail author


Pullulanase is a commonly used starch-debranching enzyme with broad application in food, chemical and pharmaceutical industries. Since the starch-debranching process requires a high temperature, a thermostable pullulanase is desirable. In this study, based on the strategy of surficial residue replacement and disulfide bond introduction, a mutant pullulanase (PulAC) derived from the pullulanase (PulA) of Anoxybacillus sp. WB42 with higher thermostability and activity was isolated. The surficial residue Lys419 from the wild-type PulA was replaced by arginine, and two disulfide bonds were introduced between Thr245 and Ala326 and Trp651 and Val707. The specific activity and kcat/Km value of the PulAC reached 98.20 U/mg and 12.22 mL/mg/s respectively, 1.5 times greater than that of wild-type PulA. The optimum temperature of the mutant PulAC was 65 °C. The PulAC retained more than 85% activity after incubation at 65 °C for 30 min, which is much higher than the activity maintained by wild-type PulA. Due to its high optimum temperature, thermostability, and specific activity, the mutant PulAC reported here could play an important role in improving hydrolytic efficiency in the starch-debranching process.


Anoxybacillus sp. WB42 Cysteine Disulfide bond Pullulanase Surficial residue Starch-debranching process 


Funding Information

This work is financially supported by a Project Funded by the International S&T Innovation Cooperation Key Project (2017YFE0129600), the National Natural Science Foundation of China (21878125), the Natural Sciences Foundation of Jiangsu (BK20181206), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project (No. 111-2-06), the Jiangsu Province “Collaborative Innovation Center for Advanced Industrial Fermentation” Industry Development Program, and First-Class Discipline Program of Light Industry Technology and Engineering (LITE2018-04).

Compliance with Ethical Standards

We confirm that this manuscript has not been published elsewhere and is not under consideration by another journal. All authors have approved the manuscript and agree with submission to Applied Biochemistry and Biotechnology.

Conflict of Interest

The authors declare that they have no competing interests.

Supplementary material

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  1. 1.
    Møller, M. S., Henriksen, A., & Svensson, B. (2016). Structure and function of α-glucan debranching enzymes. Cellular & Molecular Life Sciences Cmls, 73, 1–23.CrossRefGoogle Scholar
  2. 2.
    Hii, S. L., Tan, J. S., Ling, T. C., & Ariff, A. B. (2012). Pullulanase: role in starch hydrolysis and potential industrial applications. Enzyme Research, 2012, 921362.CrossRefGoogle Scholar
  3. 3.
    Nisha, M., & Satyanarayana, T. (2013). Recombinant bacterial amylopullulanases developments and perspectives. Bioengineered, 4(6), 388–400.CrossRefGoogle Scholar
  4. 4.
    Linko, Y. Y., & Wu, X. Y. (1993). Improvement and estimation of enzymic starch saccharification process. Biotechnology Techniques, 7, 551–556.CrossRefGoogle Scholar
  5. 5.
    Chen, A. N., Li, Y. M., & Nie, J. Q. (2015). Protein engineering of Bacillus acidopullulyticus pullulanase for enhanced thermostability using in silico data driven rational design methods. Enzyme and Microbial Technology, 78, 74–83.CrossRefGoogle Scholar
  6. 6.
    Lu, Z., Hu, X., & Shen, P. (2017). A pH-stable, detergent and chelator resistant type I pullulanase from Bacillus pseudofirmus 703 with high catalytic efficiency. International Journal of Biological Macromolecules, 109, 1302–1310.CrossRefGoogle Scholar
  7. 7.
    Elleuche, S., Qoura, F. M., & Lorenz, U. (2015). Cloning, expression and characterization of the recombinant cold-active type-I pullulanase from Shewanella arctica. Journal of Molecular Catalysis B: Enzymatic, 116, 70–77.CrossRefGoogle Scholar
  8. 8.
    Tekaia, F., Yeramian, E., & Dujon, B. (2002). Amino acid composition of genomes, lifestyles of organisms, and evolutionary trends: a global picture with correspondence analysis. Gene, 297(1-2), 51–60.CrossRefGoogle Scholar
  9. 9.
    Vieille, C., & Zeikus, G. J. (2001). Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiology & Molecular Biology Reviews, 65, 1.CrossRefGoogle Scholar
  10. 10.
    Zheng, J. X., Yang, T. W., & Zhou, J. P. (2017). Elimination of a free cysteine by creation of a disulfide bond increases the activity and stability of Candida boidinii formate dehydrogenase. Applied and Environmental Microbiology, 83, e02624–e02616.PubMedGoogle Scholar
  11. 11.
    Liu, L., Deng, Z. M., Du, G. C., & Chen, J. (2014). In silico rational design and systems engineering of disulfide bridges in the catalytic domain of an alkaline α-amylase from Alkalimonas amylolytica to improve thermostability. Applied and Environmental Microbiology, 80(3), 798–807.CrossRefGoogle Scholar
  12. 12.
    Teng, C., Jiang, Y. F., & Wang, C. T. (2019). Improving the thermostability and catalytic efficiency of GH11 xylanase PjxA by adding disulfide bridges. International Journal of Biological Macromolecules, 128, 354–362.CrossRefGoogle Scholar
  13. 13.
    Wang, J., Liu, Z., & Zhou, Z. (2017). Cloning and characterization of a novel thermophilic amylopullulanase with a type I pullulanase structure from Anoxybacillus sp. WB42. Starch/Stärke, 70, 1700265.CrossRefGoogle Scholar
  14. 14.
    Jeong, M. Y., Kim, S., & Cho, S. G. (2007). Engineering a de novo internal disulfide bridge to improve the thermal stability of xylanase from Bacillus stearothermophilus no. 236. Journal of Biotechnology, 127(2), 300–309.CrossRefGoogle Scholar
  15. 15.
    Xu, J., Ren, F., & Huang, C. H. (2014). Functional and structural studies of pullulanase from Anoxybacillus sp. LM18-11. Proteins-Structure Function & Bioinformatics, 82, 1685–1693.CrossRefGoogle Scholar
  16. 16.
    Huang, Y. J., Acton, T. B., & Montelione, G. T. (2014). DisMeta: a meta server for construct design and optimization. Methods in Molecular Biology, 1091, 3.CrossRefGoogle Scholar
  17. 17.
    Craig, D. B., & Dombkowski, A. A. (2013). Disulfide by Design 2.0: a web-based tool for disulfide engineering in proteins. BMC Bioinformatics, 14, 346.CrossRefGoogle Scholar
  18. 18.
    Wang, K., Luo, H., & Yao, B. (2014). Thermostability improvement of a streptomyces xylanase by introducing proline and glutamic acid residues. Applied and Environmental Microbiology, 80(7), 2158–2165.CrossRefGoogle Scholar
  19. 19.
    Dong, Y. W., Liao, M. L., & Meng, X. L. (2018). Structural flexibility and protein adaptation to temperature: molecular dynamics analysis of malate dehydrogenases of marine molluscs. Proceedings of the National Academy of Sciences, 115, 1274–1279.CrossRefGoogle Scholar
  20. 20.
    Chen, J., Yu, H., Liu, C., & Shen, Z. (2012). Improving stability of nitrile hydratase by bridging the salt-bridges in specific thermal-sensitive regions. Journal of Biotechnology, 164(2), 354–362.CrossRefGoogle Scholar
  21. 21.
    Tu, T., Luo, H., Meng, K., & Zhang, L. (2015). Improvement in thermostability of an Achaetomium sp. strain Xz8 endopolygalacturonase via the optimization of charge-charge interactions. Applied and Environmental Microbiology, 81(19), 6938–6944.CrossRefGoogle Scholar
  22. 22.
    Kuriki, T., & Imanaka, T. (1999). The concept of the α-amylase family: structural similarity and common catalytic mechanism. Journal of Bioscience and Bioengineering, 87(5), 557–565.CrossRefGoogle Scholar
  23. 23.
    Mu, G. C., Nie, Y., & Xu, Y. (2015). Single amino acid substitution in the pullulanase of Klebsiella variicola for enhancing thermostability and catalytic efficiency. Applied Biochemistry and Biotechnology, 176(6), 1736–1745.CrossRefGoogle Scholar
  24. 24.
    Rajaei, S., Noghabi, K. A., & Sadeghizadeh, M. (2015). Characterization of a pH and detergent-tolerant, cold-adapted type I pullulanase from Exiguobacterium sp. SH3. Extremophiles, 19(6), 1145–1155.CrossRefGoogle Scholar
  25. 25.
    Wei, W., Ma, J., & Guo, S. (2014). A type I pullulanase of Bacillus cereus Nws-bc5 screening from stinky tofu brine: functional expression in Escherichia coli and Bacillus subtilis and enzyme characterization. Process Biochemistry, 49, 1893–1902.CrossRefGoogle Scholar
  26. 26.
    Rehman, H., Siddiqui, M., & Qayyum, A. (2018). Gene expression in Escherichia coli and purification of recombinant type II pullulanase from a hyperthermophilic archaeon, Pyrobaculum calidifontis. Pakistan Journal of Zoology, 50, 1381–1386.Google Scholar
  27. 27.
    Malle, D., Itoh, T., & Hashimoto, W. (2006). Overexpression, purification and preliminary X-ray analysis of pullulanase from Bacillus subtilis strain 168. Structural Biology and Crystallization Communications, 62(Pt 4), 381–384.CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of BiotechnologyJiangnan UniversityWuxiChina

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