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Substituting Both the N-Terminal and “Cord” Regions of a Xylanase from Aspergillus oryzae to Improve Its Temperature Characteristics

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Abstract

To improve the temperature characteristics of AoXyn11A, a mesophilic glycoside hydrolase family (GHF) 11 xylanase from Aspergillus oryzae CICC40186, its N-terminal and “cord” regions were selected to be substituted by means of the computer-aided analysis and calculation. In brief, one mutant, named ATX11A41, possessing the lowest root-mean-square deviation (RMSD) value was designed based on the molecular dynamics (MD) simulation by substituting the N-terminal 41 amino acids of AoXyn11A with the corresponding 42 ones of pXYL11, a thermophilic GHF11 xylanase from Thermobifida fusca. On the basis of the primary structure alignment of pXYL11 with ATX11A41 (or AoXyn11A), another mutant, named ATX11A41/cord, was designed by substituting the cord region (93GTYNPGSGG101) of ATX11A41 with the corresponding one (93GTYRPTG99) of pXYL11. Both mutant-encoding genes, ATx11A41 and ATx11A41/cord, were constructed as designed theoretically by a megaprimer PCR technique and were expressed in Pichia pastoris GS115. The specific activities of recombinant (re) AoXyn11A, ATX11A41, and ATX11A41/cord were 2916.7, 2667.6, and 2457.0 U/mg, respectively. The analysis of temperature characteristics displayed that the temperature optimum (Topt) of reATX11A41 or reATX11A41/cord was 65 °C, which was 15 °C higher than that of reAoXyn11A. The thermal inactivation half-life (t1/2) values of reATX11A41 and reATX11A41/cord at 60 °C were 55 and 83 min, respectively, whereas that of reAoXyn11A was only 18 min at 50 °C. The melting temperature (Tm) values of reAoXyn11A, reATX11A41, and reATX11A41/cord were 54.2, 66.7, and 71.9 °C, respectively. In conclusion, the above findings indicated that the substitution of both the N-terminal and cord regions of a mesophilic AoXyn11A greatly contributed to its improved temperature characteristics.

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References

  1. Trevizano, L. M., Ventorim, R. Z., de Rezende, S. T., Junior, F. P. S., & Guimarães, V. M. (2012). Thermostability improvement of Orpinomyces sp. xylanase by directed evolution. Journal of Molecular Catalysis B: Enzymatic, 81, 12–18. https://doi.org/10.1016/j.molcatb.2012.04.021.

    Article  CAS  Google Scholar 

  2. Fu, X. Y., Zhao, W., Xiong, A. S., Tian, Y. S., & Peng, R. H. (2011). High expression of recombinant Streptomyces sp. S38 xylanase in Pichia pastoris by codon optimization and analysis of its biochemical properties. Molecular Biology Reports, 38(8), 4991–4997. https://doi.org/10.1007/s11033-010-0644-7.

    Article  CAS  PubMed  Google Scholar 

  3. Conejo-Saucedo, U., Cano-Camacho, H., Villa-Rivera, M. G., Lara-Marquez, A., López-Romero, E., & Zavala-Páramo, M. G. (2017). Protein homology modeling, docking, and phylogenetic analyses of an endo-1,4-β-xylanase GH11 of Colletotrichum lindemuthianum. Mycological Progress, 16(6), 577–591. https://doi.org/10.1007/s11557-017-1291-3.

    Article  Google Scholar 

  4. Paës, G., Berrin, J. G., & Beaugrand, J. (2012). GH11 xylanases: structure/function/properties relationships and applications. Biotechnology Advances, 30(3), 564–592. https://doi.org/10.1016/j.biotechadv.2011.10.003.

    Article  CAS  PubMed  Google Scholar 

  5. Zhang, H. M., Li, J. F., Wang, J. Q., Yang, Y. J., & Wu, M. C. (2014). Determinants for the improved thermostability of a mesophilic family 11 xylanase predicted by computational methods. Biotechnology for Biofuels, 7(1), 3. https://doi.org/10.1186/1754-6834-7-3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Robledo, A., Aguilar, C. N., Belmares-Cerda, R. E., Flores-Gallegos, A. C., Contreras-Esquivel, J. C., Montañez, J. C., & Mussatto, S. I. (2016). Production of thermostable xylanase by thermophilic fungal strains isolated from maize silage. CyTA-Journal of Food, 14(2), 302–308. https://doi.org/10.1080/19476337.2015.1105298.

    Article  CAS  Google Scholar 

  7. Kumar, S., Tsai, C. J., & Nussinov, R. (2000). Factors enhancing protein thermostability. Protein Engineering, 13(3), 179–191. https://doi.org/10.1093/protein/13.3.179.

    Article  CAS  PubMed  Google Scholar 

  8. Yu, H. R., & Huang, H. (2014). Engineering proteins for thermostability through rigidifying flexible sites. Biotechnology Advances, 32(2), 308–315. https://doi.org/10.1016/j.biotechadv.2013.10.012.

    Article  CAS  PubMed  Google Scholar 

  9. Wang, Q., & Xia, T. (2008). Importance of C-terminal region for thermostability of GH11 xylanase from Streptomyces lividans. Applied Biochemistry and Biotechnology, 144(3), 273–282. https://doi.org/10.1007/s12010-007-8016-z.

    Article  CAS  PubMed  Google Scholar 

  10. Yin, X., Li, J. F., Wang, J. Q., Tang, C. D., & Wu, M. C. (2013). Enhanced thermostability of a mesophilic xylanase by N-terminal replacement designed by molecular dynamics simulation. Journal of the Science of Food and Agriculture, 93(12), 3016–3023. https://doi.org/10.1002/jsfa.6134.

    Article  CAS  PubMed  Google Scholar 

  11. Dumon, C., Varvak, A., Wall, M. A., Flint, J. E., Lewis, R. J., Lakey, J. H., Morland, C., Luginbühl, P., Healey, S., Todaro, T., DeSantis, G., Sun, M., Parra-Gessert, L., Tan, X., Weiner, D. P., & Gilbert, H. J. (2008). Engineering hyperthermostability into a GH11 xylanase is mediated by subtle changes to protein structure. Journal of Biological Chemistry, 283(33), 22557–22564. https://doi.org/10.1074/jbc.M800936200.

    Article  CAS  PubMed  Google Scholar 

  12. Hakulinen, N., Turunen, O., Janis, J., Leisola, M., & Rouvinen, J. (2003). Three-dimensional structures of thermophilic β-1,4-xylanases from Chaetomium thermophilum and Nonomuraea flexuosa: comparison of twelve xylanases in relation to their thermal stability. European Journal of Biochemistry, 270(7), 1399–1412. https://doi.org/10.1046/j.1432-1033.2003.03496.x.

    Article  CAS  PubMed  Google Scholar 

  13. Li, J. F., Gao, S. J., Liu, X. T., Gong, Y. Y., Chen, Z. F., Wei, X. H., Zhang, H. M., & Wu, M. C. (2013). Modified pPIC9K vector-mediated expression of a family 11 xylanase gene, Aoxyn11A, from Aspergillus oryzae in Pichia pastoris. Annals of Microbiology, 63(3), 1109–1120. https://doi.org/10.1007/s13213-012-0568-7.

    Article  CAS  Google Scholar 

  14. Cheng, Y. F., Yang, C. H., & Liu, W. H. (2005). Cloning and expression of Thermobifida xylanase gene in the methylotrophic yeast Pichia pastoris. Enzyme Microbial Technology, 37(5), 541–546. https://doi.org/10.1016/j.enzmictec.2005.04.006.

    Article  CAS  Google Scholar 

  15. Reetz, M. T., & Carballeira, J. D. (2007). Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nature Protocols, 2(4), 891–903. https://doi.org/10.1038/nprot.2007.72.

    Article  CAS  PubMed  Google Scholar 

  16. Xie, Z. H., & Shi, X. J. (2009). Fast and almost 100% efficiency site-directed mutagenesis by the megaprimer PCR method. Progress in Biochemistry and Biophysics, 36(11), 1490–1494. https://doi.org/10.3724/SP.J.1206.2009.00139.

    Article  CAS  Google Scholar 

  17. Jang, M. K., Lee, S. W., Lee, D. G., Kim, N. Y., Yu, K. H., Jang, H. J., Kim, S., Kim, A., & Lee, S. H. (2010). Enhancement of the thermostability of a recombinant β-agarase, AgaB, from Zobellia galactanivorans by random mutagenesis. Biotechnology Letters, 32(7), 943–949. https://doi.org/10.1007/s10529-010-0237-5.

    Article  CAS  PubMed  Google Scholar 

  18. Dong, Y. H., Li, J. F., Hu, D., Yin, X., Wang, C. J., Tang, S. H., & Wu, M. C. (2016). Replacing a piece of loop-structure in the substrate-binding groove of Aspergillus usamii β-mannanase, AuMan5A, to improve its enzymatic properties by rational design. Applied Microbiology and Biotechnology, 100(9), 3989–3998. https://doi.org/10.1007/s00253-015-7224-7.

    Article  CAS  PubMed  Google Scholar 

  19. Le, Q. A., Joo, J. C., Yoo, Y. J., & Kim, Y. H. (2012). Development of thermostable Candida antarctica lipase B through novel in silico design of disulfide bridge. Biotechnology and Bioengineering, 109(4), 867–876. https://doi.org/10.1002/bit.24371.

    Article  CAS  PubMed  Google Scholar 

  20. Badieyan, S., Bevan, D. R., & Zhang, C. M. (2012). Study and design of stability in GH5 cellulases. Biotechnology and Bioengineering, 109(1), 31–44. https://doi.org/10.1002/bit.23280.

    Article  CAS  PubMed  Google Scholar 

  21. Radestock, S., & Gohlke, H. (2008). Exploiting the link between protein rigidity and thermostability for data-driven protein engineering. Engineering in Life Sciences, 8(5), 507–522. https://doi.org/10.1002/elsc.200800043.

    Article  CAS  Google Scholar 

  22. Niesen, F. H., Berglund, H., & Vedadi, M. (2007). The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nature Protocols, 2(9), 2212–2221. https://doi.org/10.1038/nprot.2007.321.

    Article  CAS  PubMed  Google Scholar 

  23. Zhang, S., Zhang, K., Chen, X. Z., Chu, X., Sun, F., & Dong, Z. Y. (2010). Five mutations in N-terminus confer thermostability on mesophilic xylanase. Biochemical and Biophysical Research Communications, 395(2), 200–206. https://doi.org/10.1016/j.bbrc.2010.03.159.

    Article  CAS  PubMed  Google Scholar 

  24. You, C., Huang, Q., Xue, H. P., Xu, Y., & Lu, H. (2010). Potential hydrophobic interaction between two cysteines in interior hydrophobic region improves thermostability of a family 11 xylanase from Neocallimastix patriciarum. Biotechnology and Bioengineering, 105(5), 861–870. https://doi.org/10.1002/bit.22623.

    Article  CAS  PubMed  Google Scholar 

  25. Tina, K. G., Bhadra, R., & Srinivasan, N. (2007). PIC: Protein Interactions Calculator. Nucleic Acids Research, 35(Web Server), W473–W476. https://doi.org/10.1093/nar/gkm423.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Georis, J., Esteves, F. D., Lamotte-Brasseur, J., Bougnet, V., Devreese, B., Giannotta, F., et al. (2000). An additional aromatic interaction improves the thermostability and thermophilicity of a mesophilic family 11 xylanase: structural basis and molecular study. Protein Science, 9, 466–475.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lee, J. H., Heo, S. Y., Lee, J. W., Yoon, K. H., Kim, Y. H., & Nam, S. W. (2009). Thermostability and xylan-hydrolyzing property of endoxylanase expressed in yeast Saccharomyces cerevisiae. Biotechnology and Bioprocess Engineering, 14(5), 639–644. https://doi.org/10.1007/s12257-009-0014-2.

    Article  CAS  Google Scholar 

  28. Anbarasan, S., Jänis, J., Paloheimo, M., Laitaoja, M., Vuolanto, M., Karimäki, J., et al. (2010). Effect of glycosylation and additional domains on the thermostability of a family 10 xylanase produced by Thermopolyspora flexuosa. Applied and Environmental Microbiology, 76(1), 356–360. https://doi.org/10.1128/AEM.00357-09.

    Article  CAS  PubMed  Google Scholar 

  29. Thompson, M. J., & Eisenberg, D. (1999). Transproteomic evidence of a loop-deletion mechanism for enhancing protein thermostability. Journal of Molecular Biology, 290(2), 595–604. https://doi.org/10.1006/jmbi.1999.2889.

    Article  CAS  PubMed  Google Scholar 

  30. Hawwa, R., Aikens, J., Turner, R. J., Santarsiero, B. D., & Mesecar, A. D. (2009). Structural basis for thermostability revealed through the identification and characterization of a highly thermostable phosphotriesterase-like lactonase from Geobacillus stearothermophilus. Archives of Biochemistry and Biophysics, 488(2), 109–120. https://doi.org/10.1016/j.abb.2009.06.005.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Xue, H. P., Zhou, J. G., You, C., Huang, Q., & Lu, H. (2012). Amino acid substitutions in the N-terminus, cord and α-helix domains improved the thermostability of a family 11 xylanase XynR8. Journal of Industrial Microbiology and Biotechnology, 39, 1279–1288.

    Article  CAS  PubMed  Google Scholar 

  32. Donald, J. E., Kulp, D. W., & DeGrado, W. F. (2011). Salt bridges: geometrically specific, designable interactions. Proteins, 79, 898–915.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This work was financially supported by the National Natural Science Foundation of China (21676117) and the Postgraduate Innovation Training Project of Jiangsu (KYLX16_0804). The authors are grateful to Prof. Xianzhang Wu (School of Biotechnology, Jiangnan University, Jiangsu, China) for providing the technical assistance.

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  1. Chuang Li and Jianfang Li contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, People’s Republic of China

    Chuang Li & Rui Wang

  2. State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, People’s Republic of China

    Jianfang Li & Xueqing Li

  3. Wuxi School of Medicine, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, People’s Republic of China

    Jinping Li, Chao Deng & Minchen Wu

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  1. Chuang Li
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Correspondence to Minchen Wu.

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Li, C., Li, J., Wang, R. et al. Substituting Both the N-Terminal and “Cord” Regions of a Xylanase from Aspergillus oryzae to Improve Its Temperature Characteristics. Appl Biochem Biotechnol 185, 1044–1059 (2018). https://doi.org/10.1007/s12010-017-2681-3

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