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Santos CA, Zanphorlin LM, Crucello A, et al. Crystal structure and biochemical characterization of the recombinant ThBgl, a GH1 β-glucosidase overexpressed in Trichoderma harzianum under biomass degradation conditions. Biotechnol Biofuels. 2016;9:71doi: 10.1186/s13068-016-0487-0.
Santos, C. A., Zanphorlin, L. M., Crucello, A., Tonoli, C. C. C., Ruller, R., Horta, M. A. C., Murakami, M. T., & de Souza, A. P. (2016). Crystal structure and biochemical characterization of the recombinant ThBgl, a GH1 β-glucosidase overexpressed in Trichoderma harzianum under biomass degradation conditions. Biotechnology for biofuels, 971. https://doi.org/10.1186/s13068-016-0487-0
Santos, Clelton A, et al. "Crystal structure and biochemical characterization of the recombinant ThBgl, a GH1 β-glucosidase overexpressed in Trichoderma harzianum under biomass degradation conditions." Biotechnology for biofuels vol. 9 (2016): 71. doi: https://doi.org/10.1186/s13068-016-0487-0
Santos CA, Zanphorlin LM, Crucello A, Tonoli CCC, Ruller R, Horta MAC, Murakami MT, de Souza AP. Crystal structure and biochemical characterization of the recombinant ThBgl, a GH1 β-glucosidase overexpressed in Trichoderma harzianum under biomass degradation conditions. Biotechnol Biofuels. 2016 Mar 22;9:71. doi: 10.1186/s13068-016-0487-0. eCollection 2016. PMID: 27006690; PMCID: PMC4802607.
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Biotechnol Biofuels. 2016 Mar 22;9:71. doi: 10.1186/s13068-016-0487-0. eCollection 2016.

Crystal structure and biochemical characterization of the recombinant ThBgl, a GH1 β-glucosidase overexpressed in Trichoderma harzianum under biomass degradation conditions.

Biotechnology for biofuels

Clelton A Santos, Letícia M Zanphorlin, Aline Crucello, Celisa C C Tonoli, Roberto Ruller, Maria A C Horta, Mario T Murakami, Anete Pereira de Souza

Affiliations

  1. Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, SP Brazil.
  2. Laboratório Nacional de Ciência e Tecnologia do Bioetanol, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP Brazil.
  3. Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP Brazil.
  4. Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP Brazil.

PMID: 27006690 PMCID: PMC4802607 DOI: 10.1186/s13068-016-0487-0

Abstract

BACKGROUND: The conversion of biomass-derived sugars via enzymatic hydrolysis for biofuel production is a challenge. Therefore, the search for microorganisms and key enzymes that increase the efficiency of the saccharification of cellulosic substrates remains an important and high-priority area of study. Trichoderma harzianum is an important fungus known for producing high levels of cellulolytic enzymes that can be used for cellulosic ethanol production. In this context, β-glucosidases, which act synergistically with cellobiohydrolases and endo-β-1,4-glucanases in the saccharification process, are potential biocatalysts for the conversion of plant biomass to free glucose residues.

RESULTS: In the present study, we used RNA-Seq and genomic data to identify the major β-glucosidase expressed by T. harzianum under biomass degradation conditions. We mapped and quantified the expression of all of the β-glucosidases from glycoside hydrolase families 1 and 3, and we identified the enzyme with the highest expression under these conditions. The target gene was cloned and heterologously expressed in Escherichia coli, and the recombinant protein (rThBgl) was purified with high yields. rThBgl was characterized using a comprehensive set of biochemical, spectroscopic, and hydrodynamic techniques. Finally, we determined the crystallographic structure of the recombinant protein at a resolution of 2.6 Å.

CONCLUSIONS: Using a rational approach, we investigated the biochemical characteristics and determined the three-dimensional protein structure of a β-glucosidase that is highly expressed by T. harzianum under biomass degradation conditions. The methodology described in this manuscript will be useful for the bio-prospection of key enzymes, including cellulases and other accessory enzymes, for the development and/or improvement of enzymatic cocktails designed to produce ethanol from plant biomass.

Keywords: Biomass degradation; Enzyme prospection; Overexpression; Trichoderma harzianum; β-Glucosidase

References

  1. Biotechnol Appl Biochem. 1999 Aug;30 ( Pt 1):81-7 - PubMed
  2. Nat Rev Genet. 2009 Jan;10(1):57-63 - PubMed
  3. Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501 - PubMed
  4. J Biochem. 1999 Apr;125(4):728-36 - PubMed
  5. Mol Biotechnol. 2013 Mar;53(3):289-99 - PubMed
  6. Biotechnol Bioeng. 1990 Jul;36(3):275-87 - PubMed
  7. Science. 2007 Feb 9;315(5813):804-7 - PubMed
  8. Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):133-44 - PubMed
  9. J Bacteriol. 1969 Dec;100(3):1355-63 - PubMed
  10. Nat Methods. 2008 Jul;5(7):621-8 - PubMed
  11. Bioresour Technol. 2013 Jan;127:500-7 - PubMed
  12. Appl Biochem Biotechnol. 2010 Mar;160(5):1407-14 - PubMed
  13. Appl Environ Microbiol. 2002 Sep;68(9):4546-53 - PubMed
  14. PLoS One. 2014 Feb 18;9(2):e88689 - PubMed
  15. Microb Cell Fact. 2012 Feb 17;11:25 - PubMed
  16. Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W615-9 - PubMed
  17. N Biotechnol. 2015 Jan 25;32(1):13-20 - PubMed
  18. Eur J Biochem. 1987 Jun 1;165(2):333-41 - PubMed
  19. Can J Microbiol. 1977 Feb;23(2):139-47 - PubMed
  20. Front Microbiol. 2014 Apr 11;5:142 - PubMed
  21. Biotechnol Bioeng. 1998 Sep 5;59(5):621-34 - PubMed
  22. Enzyme Microb Technol. 2011 Jun 10;49(1):94-9 - PubMed
  23. Biomed Res Int. 2013;2013:203735 - PubMed
  24. Crit Rev Biotechnol. 2010 Dec;30(4):302-9 - PubMed
  25. Biotechnol Biofuels. 2013 Sep 09;6(1):127 - PubMed
  26. Curr Opin Chem Biol. 2008 Oct;12(5):539-55 - PubMed
  27. Enzyme Microb Technol. 2012 Dec 10;51(6-7):319-24 - PubMed
  28. Acta Crystallogr D Biol Crystallogr. 2014 Jun;70(Pt 6):1631-9 - PubMed
  29. Genome Announc. 2015 Jun 11;3(3):null - PubMed
  30. Bioresour Technol. 2010 Jul;101(13):4862-74 - PubMed
  31. Microbiologyopen. 2013 Aug;2(4):595-609 - PubMed
  32. Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):213-21 - PubMed
  33. Appl Biochem Biotechnol. 2004 Spring;113-116:1115-26 - PubMed
  34. Cell Mol Life Sci. 2010 Oct;67(20):3389-405 - PubMed
  35. Protein Expr Purif. 2004 Dec;38(2):248-57 - PubMed
  36. Curr Opin Chem Biol. 2006 Apr;10(2):141-6 - PubMed
  37. FEBS Lett. 2007 Apr 3;581(7):1514-20 - PubMed
  38. BMC Genomics. 2013 Aug 09;14:541 - PubMed
  39. Biosci Biotechnol Biochem. 2001 Sep;65(9):2028-32 - PubMed
  40. Microb Cell Fact. 2012 May 20;11:63 - PubMed
  41. Adv Biochem Eng Biotechnol. 2007;108:95-120 - PubMed
  42. Biol Proced Online. 2009 May 15;11:32-51 - PubMed
  43. Int J Biol Macromol. 2016 Jan;82:375-80 - PubMed
  44. Nucleic Acids Res. 2009 Jan;37(Database issue):D233-8 - PubMed
  45. Biotechnol Biofuels. 2014 Jan 28;7(1):14 - PubMed
  46. Biotechnol Biofuels. 2013 Jul 24;6(1):105 - PubMed
  47. Science. 2007 Feb 9;315(5813):801-4 - PubMed
  48. J Struct Biol. 2011 Jan;173(1):46-56 - PubMed
  49. Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):125-32 - PubMed
  50. Microbiol Mol Biol Rev. 2002 Sep;66(3):506-77, table of contents - PubMed
  51. Biotechnol Lett. 2011 Dec;33(12):2475-9 - PubMed
  52. Biotechnol Biofuels. 2010 Feb 11;3(1):3 - PubMed
  53. Nat Biotechnol. 2008 May;26(5):553-60 - PubMed
  54. Front Microbiol. 2014 Apr 17;5:172 - PubMed

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