Display options
Cite
Ribeiro LF, Tullman J, Nicholes N, et al. A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination. Biotechnol Biofuels. 2016;9:119doi: 10.1186/s13068-016-0529-7.
Ribeiro, L. F., Tullman, J., Nicholes, N., Silva, S. R., Vieira, D. S., Ostermeier, M., & Ward, R. J. (2016). A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination. Biotechnology for biofuels, 9119. https://doi.org/10.1186/s13068-016-0529-7
Ribeiro, Lucas Ferreira, et al. "A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination." Biotechnology for biofuels vol. 9 (2016): 119. doi: https://doi.org/10.1186/s13068-016-0529-7
Ribeiro LF, Tullman J, Nicholes N, Silva SR, Vieira DS, Ostermeier M, Ward RJ. A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination. Biotechnol Biofuels. 2016 Jun 06;9:119. doi: 10.1186/s13068-016-0529-7. eCollection 2016. PMID: 27274356; PMCID: PMC4896006.
Copy Download .nbib Format: NLM
Share it on

Biotechnol Biofuels. 2016 Jun 06;9:119. doi: 10.1186/s13068-016-0529-7. eCollection 2016.

A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination.

Biotechnology for biofuels

Lucas Ferreira Ribeiro, Jennifer Tullman, Nathan Nicholes, Sérgio Ruschi Bergamachi Silva, Davi Serradella Vieira, Marc Ostermeier, Richard John Ward

Affiliations

  1. Johns Hopkins University, Baltimore, MD USA ; Departamento de Bioquímica e Imunologia, FMRP-Universidade de São Paulo-USP, Ribeirão Preto, SP Brazil.
  2. Johns Hopkins University, Baltimore, MD USA ; Institute for Bioscience and Biotechnology Research, Rockville, MD USA.
  3. Johns Hopkins University, Baltimore, MD USA.
  4. Universidade Federal do Rio Grande do Norte, Natal, Brazil.
  5. Laboratório Nacional de Ciência e Tecnologia do Bioetanol-CTBE, Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP Brazil ; Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, Ribeirão Preto, SP 14040-901 Brazil.

PMID: 27274356 PMCID: PMC4896006 DOI: 10.1186/s13068-016-0529-7

Abstract

BACKGROUND: Saccharification of lignocellulosic material by xylanases and other glycoside hydrolases is generally conducted at high concentrations of the final reaction products, which frequently inhibit the enzymes used in the saccharification process. Using a random nonhomologous recombination strategy, we have fused the GH11 xylanase from Bacillus subtilis (XynA) with the xylose binding protein from Escherichia coli (XBP) to produce an enzyme that is allosterically stimulated by xylose.

RESULTS: The pT7T3GFP_XBP plasmid containing the XBP coding sequence was randomly linearized with DNase I, and ligated with the XynA coding sequence to create a random XynA-XBP insertion library, which was used to transform E. coli strain JW3538-1 lacking the XBP gene. Screening for active XBP was based on the expression of GFP from the pT7T3GFP_XBP plasmid under the control of a xylose inducible promoter. In the presence of xylose, cells harboring a functional XBP domain in the fusion protein (XBP+) showed increased GFP fluorescence and were selected using FACS. The XBP+ cells were further screened for xylanase activity by halo formation around xylanase producing colonies (XynA+) on LB-agar-xylan media after staining with Congo red. The xylanase activity ratio with xylose/without xylose in supernatants from the XBP+/XynA+ clones was measured against remazol brilliant blue xylan. A clone showing an activity ratio higher than 1.3 was selected where the XynA was inserted after the asparagine 271 in the XBP, and this chimera was denominated as XynA-XBP271. The XynA-XBP271 was more stable than XynA at 55 °C, and in the presence of xylose the catalytic efficiency was ~3-fold greater than the parental xylanase. Molecular dynamics simulations predicted the formation of an extended protein-protein interface with coupled movements between the XynA and XBP domains. In the XynA-XBP271 with xylose bound to the XBP domain, the mobility of a β-loop in the XynA domain results in an increased access to the active site, and may explain the observed allosteric activation.

CONCLUSIONS: The approach presented here provides an important advance for the engineering enzymes that are stimulated by the final product.

Keywords: Allosteric regulation; Directed evolution; Enzyme engineering; Molecular dynamics simulation; Nonhomologous recombination; Xylanase

References

  1. Protein Eng Des Sel. 2009 Oct;22(10):615-23 - PubMed
  2. Protein Eng Des Sel. 2013 Jan;26(1):15-23 - PubMed
  3. Science. 2003 Sep 26;301(5641):1904-8 - PubMed
  4. Biotechnol Biofuels. 2013 Jul 24;6(1):104 - PubMed
  5. Bioinformatics. 2013 Apr 1;29(7):845-54 - PubMed
  6. J Comput Chem. 2004 Oct;25(13):1656-76 - PubMed
  7. Proteins. 1995 Nov;23(3):318-26 - PubMed
  8. J Biol Chem. 1982 Mar 25;257(6):2926-31 - PubMed
  9. J Chem Phys. 2007 Jan 7;126(1):014101 - PubMed
  10. Structure. 2009 Jan 14;17(1):66-78 - PubMed
  11. Biochem Mol Biol Educ. 2005 Nov;33(6):399-403 - PubMed
  12. FEBS Lett. 2005 Nov 21;579(28):6505-10 - PubMed
  13. Biotechnol Biofuels. 2012 Jul 26;5(1):52 - PubMed
  14. Nucleic Acids Res. 2008 Aug;36(13):e78 - PubMed
  15. Biochim Biophys Acta. 2013 Aug;1834(8):1492-500 - PubMed
  16. Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11591-6 - PubMed
  17. J Biol Chem. 2011 Dec 16;286(50):43026-38 - PubMed
  18. J Mol Biol. 2002 Apr 19;318(1):161-77 - PubMed
  19. Chem Biol. 2004 Nov;11(11):1483-7 - PubMed
  20. Nucleic Acids Res. 2014 Jan;42(Database issue):D490-5 - PubMed
  21. Biochim Biophys Acta. 2009 Oct;1790(10):1301-6 - PubMed
  22. Appl Environ Microbiol. 2010 Jan;76(1):338-46 - PubMed
  23. Biotechnol Adv. 2010 May-Jun;28(3):407-25 - PubMed
  24. FEMS Microbiol Rev. 2005 Jan;29(1):3-23 - PubMed
  25. Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14116-21 - PubMed
  26. J Biotechnol. 2013 Dec;168(4):440-5 - PubMed
  27. Protein Expr Purif. 2005 Jul;42(1):122-30 - PubMed
  28. Biotechnol Bioeng. 2016 Apr;113(4):852-8 - PubMed
  29. J Comput Chem. 2005 Dec;26(16):1701-18 - PubMed
  30. Biotechnol Biofuels. 2013 Jan 28;6(1):16 - PubMed
  31. Appl Environ Microbiol. 2014 Feb;80(3):917-27 - PubMed
  32. FEBS Lett. 2010 Nov 5;584(21):4435-41 - PubMed
  33. Nat Chem Biol. 2008 Aug;4(8):474-82 - PubMed
  34. Biotechnol Biofuels. 2015 Aug 15;8:118 - PubMed
  35. Appl Biochem Biotechnol. 2008 Mar;144(3):273-82 - PubMed
  36. J Mol Biol. 2004 Feb 6;336(1):263-73 - PubMed
  37. Curr Opin Microbiol. 2003 Jun;6(3):219-28 - PubMed
  38. Curr Opin Struct Biol. 1998 Oct;8(5):631-9 - PubMed
  39. Curr Opin Struct Biol. 2000 Apr;10(2):153-9 - PubMed
  40. Proc Natl Acad Sci U S A. 2005 Aug 9;102(32):11224-9 - PubMed
  41. Chembiochem. 2015 Nov 2;16(16):2392-402 - PubMed

Publication Types

Grant support