Laboratory Evolution of Bacillus circulans Xylanase Inserted into Pyrococcus furiosus Maltodextrin-Binding Protein for Increased Xylanase Activity and Thermal Stability Toward Alkaline pH


High xylanase activity and stability toward alkaline pH is strongly desired for pulping and bleaching processes. We previously enhanced thermal stability of Bacillus circulans xylanase (BCX) by inserting into a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) (the resulting complex named as PfMBP-BCX165). In the present study, we aimed to evolve the inserted BCX domain within PfMBP-BCX165 for greater xylanase activity toward alkaline pH while maintaining enhanced thermal stability. No BCX sequence variation was required for the thermal stabilization, thus allowing us to explore the entire BCX sequence space for the evolution. Specifically, we randomized the BCX sequence within PfMBP-BCX165 and then screened the resulting libraries to identify a PfMBP-BCX165 variant, PfMBP-BCX165T50R. The T50R mutation enhanced xylanase activity of PfMBP-BCX165 toward alkaline pH without compromising thermal stability. When compared to PfMBP-BCX165T50R, the corresponding unfused BCX mutant, BCXT50R, exhibited similar pH dependence of xylanase activity, yet suffered from limited thermal stability. In summary, we showed that one can improve thermal stability and xylanase activity of BCX toward alkaline pH by inserting into PfMBP followed by sequence variation of the BCX domain. Our study also suggested that insertional fusion to PfMBP would be a useful stabilizing platform for evolving many proteins.

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  1. 1.

    Collins, T., Gerday, C., & Feller, G. (2005). Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiology Reviews, 29(1), 3–23.

    CAS  Article  Google Scholar 

  2. 2.

    Clarke, J. H., Rixon, J. E., Ciruela, A., Gilbert, H. J., & Hazlewood, G. P. (1997). Family-10 and family-11 xylanases differ in their capacity to enhance the bleachability of hardwood and softwood paper pulps. Applied Microbiology and Biotechnology, 48(2), 177–183.

    CAS  Article  Google Scholar 

  3. 3.

    Huang, Y. C., Chen, Y. F., Chen, C. Y., Chen, W. L., Ciou, Y. P., Liu, W. H., & Yang, C. H. (2011). Production of ferulic acid from lignocellulolytic agricultural biomass by Thermobifida fusca thermostable esterase produced in Yarrowia lipolytica transformant. Bioresource Technology, 102(17), 8117–8122.

    CAS  Article  Google Scholar 

  4. 4.

    Zhang, J., Tuomainen, P., Siika-Aho, M., & Viikari, L. (2011). Comparison of the synergistic action of two thermostable xylanases from GH families 10 and 11 with thermostable cellulases in lignocellulose hydrolysis. Bioresource Technology, 102(19), 9090–9095.

    CAS  Article  Google Scholar 

  5. 5.

    Bajpai, P. (1999). Application of enzymes in the pulp and paper industry. Biotechnology Progress, 15(2), 147–157.

    CAS  Article  Google Scholar 

  6. 6.

    Kulkarni, N., Shendye, A., & Rao, M. (1999). Molecular and biotechnological aspects of xylanases. FEMS Microbiology Reviews, 23(4), 411–456.

    CAS  Article  Google Scholar 

  7. 7.

    Beg, Q. K., Kapoor, M., Mahajan, L., & Hoondal, G. S. (2001). Microbial xylanases and their industrial applications: a review. Applied Microbiology and Biotechnology, 56(3–4), 326–338.

    CAS  Article  Google Scholar 

  8. 8.

    Polizeli, M. L., Rizzatti, A. C., Monti, R., Terenzi, H. F., Jorge, J. A., & Amorim, D. S. (2005). Xylanases from fungi: properties and industrial applications. Applied Microbiology and Biotechnology, 67(5), 577–591.

    CAS  Article  Google Scholar 

  9. 9.

    Viikari, L., Kantelinen, A., Sundquist, J., & Linko, M. (1994). Xylanases in bleaching: from an idea to the industry. FEMS Microbiology Reviews, 13(2–3), 335–350.

    CAS  Article  Google Scholar 

  10. 10.

    Gibbs, M. D., Reeves, R. A., Choudhary, P. R., & Bergquist, P. L. (2010). Alteration of the pH optimum of a family 11 xylanase, XynB6 of Dictyoglomus thermophilum. New Biotechnology, 27(6), 803–809.

    CAS  Article  Google Scholar 

  11. 11.

    Gibbs, M. D., Reeves, R. A., Hardiman, E. M., Choudhary, P. R., Daniel, R. M., & Bergquist, P. L. (2010). The activity of family 11 xylanases at alkaline pH. New Biotechnology, 27(6), 795–802.

    CAS  Article  Google Scholar 

  12. 12.

    Shibuya, H., Kaneko, S., & Hayashi, K. (2005). A single amino acid substitution enhances the catalytic activity of family 11 xylanase at alkaline pH. Bioscience, Biotechnology, and Biochemistry, 69(8), 1492–1497.

    CAS  Article  Google Scholar 

  13. 13.

    Xu, X., Liu, M. Q., Huo, W. K., & Dai, X. J. (2016). Obtaining a mutant of Bacillus amyloliquefaciens xylanase A with improved catalytic activity by directed evolution. Enzyme and Microbial Technology, 86, 59–66.

    CAS  Article  Google Scholar 

  14. 14.

    Zheng, H., Liu, Y., Sun, M., Han, Y., Wang, J., Sun, J., & Lu, F. (2014). Improvement of alkali stability and thermostability of Paenibacillus campinasensis Family-11 xylanase by directed evolution and site-directed mutagenesis. Journal of Industrial Microbiology & Biotechnology, 41(1), 153–162.

    Article  Google Scholar 

  15. 15.

    Eijsink, V. G., Bjork, A., Gaseidnes, S., Sirevag, R., Synstad, B., van den Burg, B., & Vriend, G. (2004). Rational engineering of enzyme stability. Journal of Biotechnology, 113(1–3), 105–120.

    CAS  Article  Google Scholar 

  16. 16.

    Lehmann, M., & Wyss, M. (2001). Engineering proteins for thermostability: the use of sequence alignments versus rational design and directed evolution. Current Opinion in Biotechnology, 12(4), 371–375.

    CAS  Article  Google Scholar 

  17. 17.

    Bommarius, A. S., Broering, J. M., Chaparro-Riggers, J. F., & Polizzi, K. M. (2006). High-throughput screening for enhanced protein stability. Current Opinion in Biotechnology, 17(6), 606–610.

    CAS  Article  Google Scholar 

  18. 18.

    Soskine, M., & Tawfik, D. S. (2010). Mutational effects and the evolution of new protein functions. Nature Reviews. Genetics, 11(8), 572–582.

    CAS  Article  Google Scholar 

  19. 19.

    DePristo, M. A., Weinreich, D. M., & Hartl, D. L. (2005). Missense meanderings in sequence space: a biophysical view of protein evolution. Nature Reviews. Genetics, 6(9), 678–687.

    CAS  Article  Google Scholar 

  20. 20.

    Pal, C., Papp, B., & Lercher, M. J. (2006). An integrated view of protein evolution. Nature Reviews. Genetics, 7(5), 337–348.

    CAS  Article  Google Scholar 

  21. 21.

    Denisenko, Y. A., Gusakov, A. V., Rozhkova, A. M., Osipov, D. O., Zorov, I. N., Matys, V. Y., Uporov, I. V., & Sinitsyn, A. P. (2017). Site-directed mutagenesis of GH10 xylanase A from Penicillium canescens for determining factors affecting the enzyme thermostability. International Journal of Biological Macromolecules, 104(Pt A), 665–671.

    CAS  Article  Google Scholar 

  22. 22.

    Bloom, J. D., & Arnold, F. H. (2009). In the light of directed evolution: pathways of adaptive protein evolution. Proceedings of the National Academy of Sciences of the United States of America, 106(Suppl 1), 9995–10000.

    CAS  Article  Google Scholar 

  23. 23.

    Bloom, J. D., Labthavikul, S. T., Otey, C. R., & Arnold, F. H. (2006). Protein stability promotes evolvability. Proceedings of the National Academy of Sciences of the United States of America, 103(15), 5869–5874.

    CAS  Article  Google Scholar 

  24. 24.

    Shah, V., Pierre, B., Kirtadze, T., Shin, S., & Kim, J. R. (2017). Stabilization of Bacillus circulans xylanase by combinatorial insertional fusion to a thermophilic host protein. Protein Engineering, Design & Selection, 30(4), 281–290.

    Google Scholar 

  25. 25.

    Kim, C. S., Pierre, B., Ostermeier, M., Looger, L. L., & Kim, J. R. (2009). Enzyme stabilization by domain insertion into a thermophilic protein. Protein Engineering, Design & Selection, 22(10), 615–623.

    Article  Google Scholar 

  26. 26.

    Pierre, B., Xiong, T., Hayles, L., Guntaka, V. R., & Kim, J. R. (2011). Stability of a guest protein depends on stability of a host protein in insertional fusion. Biotechnology and Bioengineering, 108(5), 1011–1020.

    CAS  Article  Google Scholar 

  27. 27.

    Pierre, B., Labonte, J. W., Xiong, T., Aoraha, E., Williams, A., Shah, V., Chau, E., Helal, K. Y., Gray, J. J., & Kim, J. R. (2015). Molecular determinants for protein stabilization by insertional fusion to a thermophilic host protein. Chembiochem, 16(16), 2392–2402.

    CAS  Article  Google Scholar 

  28. 28.

    Evdokimov, A. G., Anderson, D. E., Routzahn, K. M., & Waugh, D. S. (2001). Structural basis for oligosaccharide recognition by Pyrococcus furiosus maltodextrin-binding protein. Journal of Molecular Biology, 305(4), 891–904.

    CAS  Article  Google Scholar 

  29. 29.

    Joshi, M. D., Sidhu, G., Nielsen, J. E., Brayer, G. D., Withers, S. G., & McIntosh, L. P. (2001). Dissecting the electrostatic interactions and pH-dependent activity of a family 11 glycosidase. Biochemistry, 40(34), 10115–10139.

    CAS  Article  Google Scholar 

  30. 30.

    Pokhrel, S., Joo, J. C., Kim, Y. H., & Yoo, Y. J. (2012). Rational design of a Bacillus circulans xylanase by introducing charged residue to shift the pH optimum. Process Biochemistry, 47(12), 2487–2493.

    CAS  Article  Google Scholar 

  31. 31.

    Yang, J. H., Park, J. Y., Kim, S. H., & Yoo, Y. J. (2008). Shifting pH optimum of Bacillus circulans xylanase based on molecular modeling. Journal of Biotechnology, 133(3), 294–300.

    CAS  Article  Google Scholar 

  32. 32.

    Kim, S. H., Pokhrel, S., & Yoo, Y. J. (2008). Mutation of non-conserved amino acids surrounding catalytic site to shift pH optimum of Bacillus circulans xylanase. Journal of Molecular Catalysis B: Enzymatic, 55(3), 130–136.

    CAS  Article  Google Scholar 

  33. 33.

    Hanson-Manful, P., & Patrick, W. M. (2013). Construction and analysis of randomized protein-encoding libraries using error-prone PCR. Methods in Molecular Biology, 996, 251–267.

    CAS  Article  Google Scholar 

  34. 34.

    Vanhercke, T., Ampe, C., Tirry, L., & Denolf, P. (2005). Reducing mutational bias in random protein libraries. Analytical Biochemistry, 339(1), 9–14.

    CAS  Article  Google Scholar 

  35. 35.

    Gerrard, J. A. (2013). Protein nanotechnology : protocols, instrumentation and applications, in Methods in molecular biology. New York: Humana.

    Google Scholar 

  36. 36.

    Reitinger, S., Yu, Y., Wicki, J., Ludwiczek, M., D’Angelo, I., Baturin, S., Okon, M., Strynadka, N. C., Lutz, S., Withers, S. G., & McIntosh, L. P. (2010). Circular permutation of Bacillus circulans xylanase: a kinetic and structural study. Biochemistry, 49(11), 2464–2474.

    CAS  Article  Google Scholar 

  37. 37.

    Gill, S. C., & von Hippel, P. H. (1989). Calculation of protein extinction coefficients from amino acid sequence data. Analytical Biochemistry, 182(2), 319–326.

    CAS  Article  Google Scholar 

  38. 38.

    Wetlaufer, D. B., Edsall, J. T., & Hollingworth, B. R. (1958). Ultraviolet difference spectra of tyrosine groups in proteins and amino acids. The Journal of Biological Chemistry, 233(6), 1421–1428.

    CAS  Google Scholar 

  39. 39.

    Lawson, S. L., Wakarchuk, W. W., & Withers, S. G. (1997). Positioning the acid/base catalyst in a glycosidase: studies with Bacillus circulans xylanase. Biochemistry, 36(8), 2257–2265.

    CAS  Article  Google Scholar 

  40. 40.

    Zale, S. E., & Klibanov, A. M. (1983). On the role of reversible denaturation (unfolding) in the irreversible thermal inactivation of enzymes. Biotechnology and Bioengineering, 25(9), 2221–2230.

    CAS  Article  Google Scholar 

  41. 41.

    Palackal, N., Brennan, Y., Callen, W. N., Dupree, P., Frey, G., Goubet, F., Hazlewood, G. P., Healey, S., Kang, Y. E., Kretz, K. A., Lee, E., Tan, X., Tomlinson, G. L., Verruto, J., Wong, V. W., Mathur, E. J., Short, J. M., Robertson, D. E., & Steer, B. A. (2004). An evolutionary route to xylanase process fitness. Protein Science, 13(2), 494–503.

    CAS  Article  Google Scholar 

  42. 42.

    Davoodi, J., Wakarchuk, W. W., Surewicz, W. K., & Carey, P. R. (1998). Scan-rate dependence in protein calorimetry: the reversible transitions of Bacillus circulans xylanase and a disulfide-bridge mutant. Protein Science, 7(7), 1538–1544.

    CAS  Article  Google Scholar 

  43. 43.

    Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., & Ferrin, T. E. (2004). UCSF Chimera—a visualization system for exploratory research and analysis. Journal of Computational Chemistry, 25(13), 1605–1612.

    CAS  Article  Google Scholar 

  44. 44.

    Cherry, J. R., Lamsa, M. H., Schneider, P., Vind, J., Svendsen, A., Jones, A., & Pedersen, A. H. (1999). Directed evolution of a fungal peroxidase. Nature Biotechnology, 17(4), 379–384.

    CAS  Article  Google Scholar 

  45. 45.

    Shafikhani, S., Siegel, R. A., Ferrari, E., & Schellenberger, V. (1997). Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimerization. BioTechniques, 23(2), 304–310.

    CAS  Google Scholar 

  46. 46.

    McIntosh, L. P., Hand, G., Johnson, P. E., Joshi, M. D., Korner, M., Plesniak, L. A., Ziser, L., Wakarchuk, W. W., & Withers, S. G. (1996). The pKa of the general acid/base carboxyl group of a glycosidase cycles during catalysis: a 13C-NMR study of bacillus circulans xylanase. Biochemistry, 35(31), 9958–9966.

    CAS  Article  Google Scholar 

  47. 47.

    Tynan-Connolly, B. M., & Nielsen, J. E. (2006). pKD: re-designing protein pKa values. Nucleic Acids Res, 34(Web Server issue), W48–W51.

    CAS  Article  Google Scholar 

  48. 48.

    Shirai, T., Suzuki, A., Yamane, T., Ashida, T., Kobayashi, T., Hitomi, J., & Ito, S. (1997). High-resolution crystal structure of M-protease: phylogeny aided analysis of the high-alkaline adaptation mechanism. Protein Engineering, 10(6), 627–634.

    CAS  Article  Google Scholar 

  49. 49.

    Masui, A., Fujiwara, N., & Imanaka, T. (1994). Stabilization and rational design of serine protease AprM under highly alkaline and high-temperature conditions. Applied and Environmental Microbiology, 60(10), 3579–3584.

    CAS  Google Scholar 

  50. 50.

    Turunen, O., Vuorio, M., Fenel, F., & Leisola, M. (2002). Engineering of multiple arginines into the Ser/Thr surface of Trichoderma reesei endo-1,4-beta-xylanase II increases the thermotolerance and shifts the pH optimum towards alkaline pH. Protein Engineering, 15(2), 141–145.

    CAS  Article  Google Scholar 

  51. 51.

    Umemoto, H., Ihsanawatir, Inami, M., Yatsunami, R., Fukui, T., Kumasaka, T., Tanaka, N., & Nakamura, S. (2009). Improvement of alkaliphily of Bacillus alkaline xylanase by introducing amino acid substitutions both on catalytic cleft and protein surface. Bioscience, Biotechnology, and Biochemistry, 73(4), 965–967.

    CAS  Article  Google Scholar 

  52. 52.

    Gitlin, I., Carbeck, J. D., & Whitesides, G. M. (2006). Why are proteins charged? Networks of charge-charge interactions in proteins measured by charge ladders and capillary electrophoresis. Angewandte Chemie (International Ed. in English), 45(19), 3022–3060.

    CAS  Article  Google Scholar 

  53. 53.

    Irfan, M., Guler, H. I., Ozer, A., Sapmaz, M. T., Belduz, A. O., Hasan, F., & Shah, A. A. (2016). C-Terminal proline-rich sequence broadens the optimal temperature and pH ranges of recombinant xylanase from Geobacillus thermodenitrificans C5. Enzyme and Microbial Technology, 91, 34–41.

    CAS  Article  Google Scholar 

  54. 54.

    Zhou, C. Y., Li, T. B., Wang, Y. T., Zhu, X. S., & Kang, J. (2016). Exploration of a N-terminal disulfide bridge to improve the thermostability of a GH11 xylanase from Aspergillus niger. The Journal of General and Applied Microbiology, 62(2), 83–89.

    CAS  Article  Google Scholar 

  55. 55.

    Joo, J. C., Pohkrel, S., Pack, S. P., & Yoo, Y. J. (2010). Thermostabilization of Bacillus circulans xylanase via computational design of a flexible surface cavity. Journal of Biotechnology, 146(1–2), 31–39.

    CAS  Article  Google Scholar 

  56. 56.

    Davoodi, J., Wakarchuk, W. W., Carey, P. R., & Surewicz, W. K. (2007). Mechanism of stabilization of Bacillus circulans xylanase upon the introduction of disulfide bonds. Biophysical Chemistry, 125(2–3), 453–461.

    CAS  Article  Google Scholar 

  57. 57.

    Shah, V., Pierre, B., & Kim, J. R. (2013). Facile construction of a random protein domain insertion library using an engineered transposon. Analytical Biochemistry, 432(2), 97–102.

    CAS  Article  Google Scholar 

  58. 58.

    Shah, V., & Kim, J. R. (2016). Transposon for protein engineering. Mobile Genetics Elements, 6(6), e1239601.

    Article  Google Scholar 

  59. 59.

    Romero, P. A., & Arnold, F. H. (2009). Exploring protein fitness landscapes by directed evolution. Nature Reviews. Molecular Cell Biology, 10(12), 866–876.

    CAS  Article  Google Scholar 

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This work was supported by the National Science Foundation under Grant No. CBET-1134247.

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Correspondence to Jin Ryoun Kim.

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Shah, V., Charlton, T. & Kim, J.R. Laboratory Evolution of Bacillus circulans Xylanase Inserted into Pyrococcus furiosus Maltodextrin-Binding Protein for Increased Xylanase Activity and Thermal Stability Toward Alkaline pH. Appl Biochem Biotechnol 184, 1232–1246 (2018).

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  • Protein engineering
  • Thermal stabilization
  • Insertional fusion
  • Bacillus circulans xylanase
  • Maltodextrin-binding protein from Pyrococcus furiosus