Abstract
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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References
Collins, T., Gerday, C., & Feller, G. (2005). Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiology Reviews, 29(1), 3–23.
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.
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.
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.
Bajpai, P. (1999). Application of enzymes in the pulp and paper industry. Biotechnology Progress, 15(2), 147–157.
Kulkarni, N., Shendye, A., & Rao, M. (1999). Molecular and biotechnological aspects of xylanases. FEMS Microbiology Reviews, 23(4), 411–456.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Soskine, M., & Tawfik, D. S. (2010). Mutational effects and the evolution of new protein functions. Nature Reviews. Genetics, 11(8), 572–582.
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.
Pal, C., Papp, B., & Lercher, M. J. (2006). An integrated view of protein evolution. Nature Reviews. Genetics, 7(5), 337–348.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Vanhercke, T., Ampe, C., Tirry, L., & Denolf, P. (2005). Reducing mutational bias in random protein libraries. Analytical Biochemistry, 339(1), 9–14.
Gerrard, J. A. (2013). Protein nanotechnology : protocols, instrumentation and applications, in Methods in molecular biology. New York: Humana.
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.
Gill, S. C., & von Hippel, P. H. (1989). Calculation of protein extinction coefficients from amino acid sequence data. Analytical Biochemistry, 182(2), 319–326.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Tynan-Connolly, B. M., & Nielsen, J. E. (2006). pKD: re-designing protein pKa values. Nucleic Acids Res, 34(Web Server issue), W48–W51.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Shah, V., & Kim, J. R. (2016). Transposon for protein engineering. Mobile Genetics Elements, 6(6), e1239601.
Romero, P. A., & Arnold, F. H. (2009). Exploring protein fitness landscapes by directed evolution. Nature Reviews. Molecular Cell Biology, 10(12), 866–876.
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This work was supported by the National Science Foundation under Grant No. CBET-1134247.
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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). https://doi.org/10.1007/s12010-017-2619-9
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DOI: https://doi.org/10.1007/s12010-017-2619-9