Advertisement

Applied Biochemistry and Biotechnology

, Volume 187, Issue 4, pp 1255–1271 | Cite as

Characterization of a Recombinant Trehalose Synthase from Arthrobacter chlorophenolicus and its Unique Kinetics Indicating a Substrate Cooperativity

  • Xue Cai
  • Ines Seitl
  • Wanmeng Mu
  • Tao Zhang
  • Timo Stressler
  • Lutz FischerEmail author
  • Bo JiangEmail author
Article
  • 132 Downloads

Abstract

Trehalose is a non-reducing disaccharide with beneficial physiological properties and commercial potential. Trehalose synthase (EC 5.4.99.16) catalyzes the reversible conversion between maltose and trehalose. A recombinant trehalose synthase from Arthrobacter chlorophenolicus SK 33.001 (ACTS) was cloned, expressed, and characterized. The recombinant enzyme encoded a protein of 598 amino acids with a molecular mass of 66 kDa. Gel filtration showed that ACTS is a tetramer in sodium phosphate buffer. The enzyme was metal ion independent and exhibited maximal activity in sodium phosphate buffer (pH 7.5) at 30 °C. The kinetic investigations resulted in a KM value of 120.5 ± 4.5 mM for maltose and a KM value of 343.1 ± 13.8 mM for trehalose. The catalytic efficiency (Vmax/KM) for maltose and trehalose were 0.2 and 0.15 U mg−1 mM−1, respectively. In addition, a cooperative substrate binding was found displayed by the determined Hill coefficients (nH) of 2.8 for maltose and 2.1 for trehalose as a substrate, respectively. The final trehalose yield of various maltose concentrations (50–1000 mM) was constant between 58 and 59%, implying that substrate concentration had no inhibitory influence on ACTS activity.

Keywords

Trehalose Trehalose synthase Maltose Arthrobacter chlorophenolicus Biotechnological production Substrate cooperativity 

Notes

Acknowledgments

We would like to thank Beatrice Kuschel and Jacob Ewert (University of Hohenheim, Germany) for their help in analytical experiments and instructive discussions about kinetics. We also thank Wolfgang Claaßen (University of Hohenheim, Germany) for his support during the cultivation experiments.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

References

  1. 1.
    Elbein, A. D., Pan, Y. T., Pastuszak, I., & Carroll, D. (2003). New insights on trehalose: a multifunctional molecule. Glycobiology, 13(4), 17R–27R.  https://doi.org/10.1093/glycob/cwg047.CrossRefGoogle Scholar
  2. 2.
    Magazù, S., Migliardo, F., Gonzalez, M. A., Mondelli, C., Parker, S. F., & Vertessy, B. G. (2012). Molecular mechanisms of survival strategies in extreme conditions. Life, 2(4), 364–376.  https://doi.org/10.3390/life2040364.CrossRefGoogle Scholar
  3. 3.
    Ohtake, S., & Wang, Y. J. (2011). Trehalose: current use and future applications. Journal of Pharmaceutical Sciences, 100(6), 2020–2053.  https://doi.org/10.1002/jps.22458.CrossRefGoogle Scholar
  4. 4.
    Schiraldi, C., Di Lernia, I., & De Rosa, M. (2002). Trehalose production: exploiting novel approaches. Trends in Biotechnology, 20(10), 420–425.  https://doi.org/10.1016/S0167-7799(02)02041-3.CrossRefGoogle Scholar
  5. 5.
    Nakada, T., Maruta, K., Tsusaki, K., Kubota, M., Chaen, H., Sugimoto, T., Kurimoto, M., & Tsujisaka, Y. (1995). Purification and properties of a novel enzyme, maltooligosyl trehalose synthase, from Arthrobacter sp. Q36. Bioscience, Biotechnology, and Biochemistry, 59(12), 2210–2214.  https://doi.org/10.1271/bbb.59.2210.CrossRefGoogle Scholar
  6. 6.
    Nakada, T., Maruta, K., Mitsuzumi, H., Kubota, M., Chaen, H., Sugimoto, T., Kurimoto, M., & Tsujisaka, Y. (1995). Purification and characterization of a novel enzyme, maltooligosyl trehalose trehalohydrolase, from Arthrobacter sp. Q36. Bioscience, Biotechnology, and Biochemistry, 59(12), 2215–2218.  https://doi.org/10.1271/bbb.59.2215.CrossRefGoogle Scholar
  7. 7.
    Nishimoto, T., Nakano, M., Nakada, T., Chaen, H., Fukuda, S., Sugimoto, T., Kurimoto, M., & Tsujisaka, Y. (1996). Purification and properties of a novel enzyme, trehalose synthase, from Pimelobacter sp. R48. Bioscience, Biotechnology, and Biochemistry, 60(4), 640–644.  https://doi.org/10.1271/bbb.60.640.CrossRefGoogle Scholar
  8. 8.
    Cai, X., Seitl, I., Mu, W., Zhang, T., Stressler, T., Fischer, L., & Jiang, B. (2018). Biotechnical production of trehalose through the trehalose synthase pathway: current status and future prospects. Applied Microbiology and Biotechnology, 102(7), 2965–2976.  https://doi.org/10.1007/s00253-018-8814-y.CrossRefGoogle Scholar
  9. 9.
    Cho, C. B., Park, D. Y., & Lee, S. B. (2017). Effect of C-terminal domain truncation of Thermus thermophilus trehalose synthase on its substrate specificity. Enzyme and Microbial Technology, 96, 121–126.  https://doi.org/10.1016/j.enzmictec.2016.10.006.CrossRefGoogle Scholar
  10. 10.
    Kuschel, B., Seitl, I., Gluck, C., Mu, W., Jiang, B., Stressler, T., & Fischer, L. (2017). Hidden reaction: mesophilic cellobiose 2-epimerases produce lactulose. Journal of Agricultural and Food Chemistry, 65(12), 2530–2539.  https://doi.org/10.1021/acs.jafc.6b05599.CrossRefGoogle Scholar
  11. 11.
    Robert, X., & Gouet, P. (2014). Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Research, 42(W1), W320–W324.  https://doi.org/10.1093/nar/gku316.CrossRefGoogle Scholar
  12. 12.
    Roy, R., Usha, V., Kermani, A., Scott, D. J., Hyde, E. I., Besra, G. S., Alderwick, L. J., & Fütterer, K. (2013). Synthesis of α-glucan in Mycobacteria involves a hetero-octameric complex of trehalose synthase TreS and maltokinase Pep2. ACS Chemical Biology, 8(10), 2245–2255.  https://doi.org/10.1021/cb400508k.CrossRefGoogle Scholar
  13. 13.
    Wang, Y. L., Chow, S. Y., Lin, Y. T., Hsieh, Y. C., Lee, G. C., & Liaw, S. H. (2014). Structures of trehalose synthase from Deinococcus radiodurans reveal that a closed conformation is involved in catalysis of the intramolecular isomerization. Acta Crystallographica Section D: Biological Crystallography, 70(12), 3144–3154.  https://doi.org/10.1107/S1399004714022500.CrossRefGoogle Scholar
  14. 14.
    Wang, J., Ren, X., Wang, R., Su, J., & Wang, F. (2017). Structural characteristics and function of a new kind of thermostable trehalose synthase from Thermobaculum terrenum. Journal of Agricultural and Food Chemistry, 65(35), 7726–7735.  https://doi.org/10.1021/acs.jafc.7b02732.CrossRefGoogle Scholar
  15. 15.
    Caner, S., Nguyen, N., Aguda, A., Zhang, R., Pan, Y. T., Withers, S. G., & Brayer, G. D. (2013). The structure of the Mycobacterium smegmatis trehalose synthase reveals an unusual active site configuration and acarbose-binding mode. Glycobiology, 23(9), 1075–1083.  https://doi.org/10.1093/glycob/cwt044.CrossRefGoogle Scholar
  16. 16.
    Janeček, Š., Svensson, B., & MacGregor, E. A. (2014). α-Amylase: an enzyme specificity found in various families of glycoside hydrolases. Cellular and Molecular Life Sciences, 71(7), 1149–1170.  https://doi.org/10.1007/s00018-013-1388-z.CrossRefGoogle Scholar
  17. 17.
    Wu, X. L., Ding, H. B., Yue, M., & Qiao, Y. (2009). Gene cloning, expression, and characterization of a novel trehalose synthase from Arthrobacter aurescens. Applied Microbiology and Biotechnology, 83(3), 477–482.  https://doi.org/10.1007/s00253-009-1863-5.CrossRefGoogle Scholar
  18. 18.
    Gao, Y., Xi, Y., Lu, X. L., Zheng, H., Hu, B., Liu, X. Y., & Jiao, B. H. (2013). Cloning, expression and functional characterization of a novel trehalose synthase from marine Pseudomonas sp. P8005. World Journal of Microbiology and Biotechnology, 29(11), 2195–2206.  https://doi.org/10.1007/s11274-013-1385-2.CrossRefGoogle Scholar
  19. 19.
    Lee, J.-H., Lee, K.-H., Kim, C.-G., Lee, S.-Y., Kim, G.-J., Park, Y.-H., & Chung, S.-O. (2005). Cloning and expression of a trehalose synthase from Pseudomonas stutzeri CJ38 in Escherichia coli for the production of trehalose. Applied Microbiology and Biotechnology, 68(2), 213–219.  https://doi.org/10.1007/s00253-004-1862-5.CrossRefGoogle Scholar
  20. 20.
    Filipkowski, P., Pietrow, O., Panek, A., & Synowiecki, J. (2012). Properties of recombinant trehalose synthase from Deinococcus radiodurans expressed in Escherichia coli. Acta Biochimica Polonica, 59(3), 425–431.CrossRefGoogle Scholar
  21. 21.
    Zhu, Y., Wei, D., Zhang, J., Wang, Y., Xu, H., Xing, L., & Li, M. (2010). Overexpression and characterization of a thermostable trehalose synthase from Meiothermus ruber. Extremophiles, 14(1), 1–8.  https://doi.org/10.1007/s00792-009-0281-z.CrossRefGoogle Scholar
  22. 22.
    Ohguchi, M., Kubota, N., Wada, T., Yoshinaga, K., Uritani, M., Yagisawa, M., Ohishi, K., Yamagishi, M., Ohta, T., & Ishikawa, K. (1997). Purification and properties of trehalose-synthesizing enzyme from Pseudomonas sp. F1. Journal of Fermentation and Bioengineering, 84(4), 358–360.  https://doi.org/10.1016/S0922-338X(97)89260-4.CrossRefGoogle Scholar
  23. 23.
    Hans Bisswanger. (2008). Enzyme kinetics: principles and methods. Wiley-Blackwell. http://as.wiley.com/WileyCDA/WileyTitle/productCd-3527319573.html
  24. 24.
    Chen, Y. S., Lee, G. C., & Shaw, J. F. (2006). Gene cloning, expression, and biochemical characterization of a recombinant trehalose synthase from Picrophilus torridus in Escherichia coli. Journal of Agricultural and Food Chemistry, 54(19), 7098–7104.  https://doi.org/10.1021/jf060828q.CrossRefGoogle Scholar
  25. 25.
    Koh, S., Kim, J., Shin, H. J., Lee, D., Bae, J., Kim, D., & Lee, D. S. (2003). Mechanistic study of the intramolecular conversion of maltose to trehalose by Thermus caldophilus GK24 trehalose synthase. Carbohydrate Research, 338(12), 1339–1343.  https://doi.org/10.1016/S0008-6215(03)00172-1.CrossRefGoogle Scholar
  26. 26.
    Kim, T. K., Jang, J. H., Cho, H. Y., Lee, H. S., & Kim, Y. W. (2010). Gene cloning and characterization of a trehalose synthase from Corynebacterium glutamicum ATCC13032. Food Science and Biotechnology, 19(2), 565–569.  https://doi.org/10.1007/s10068-010-0079-x.CrossRefGoogle Scholar
  27. 27.
    Yue, M., Wu, X. L., Gong, W. N., & Ding, H. B. (2009). Molecular cloning and expression of a novel trehalose synthase gene from Enterobacter hormaechei. Microbial Cell Factories, 8(1), 34.  https://doi.org/10.1186/1475-2859-8-34.CrossRefGoogle Scholar
  28. 28.
    Yan, J., Qiao, Y., Hu, J., & Ding, H. (2013). Cloning, expression and characterization of a trehalose synthase gene from Rhodococcus opacus. Protein Journal, 32(3), 223–229.  https://doi.org/10.1007/s10930-013-9476-3.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.State Key Laboratory of Food Science and TechnologyJiangnan UniversityWuxiChina
  2. 2.Department of Biotechnology and Enzyme Science, Institute of Food Science and BiotechnologyUniversity of HohenheimStuttgartGermany

Personalised recommendations