BioEnergy Research

, Volume 9, Issue 3, pp 931–941 | Cite as

Triggering the Expression of Cellulolytic Genes Using a Recombinant Endoxylanase from Trichoderma harzianum IOC-3844

  • Wesley Cardoso Generoso
  • Wilson Malagó-Jr
  • Nei Pereira-Jr
  • Flávio Henrique-SilvaEmail author


Xylanases are used in several biotechnological processes, primarily for biopulping and biobleaching in the paper industry and as accessory enzymes for bioethanol production. In this study, a recombinant family 10 endoxylanase from Trichoderma harzianum strain IOC-3844 was expressed and characterized biochemically. Concomitantly, the effects of different culture conditions on the regulation of xylanases and cellulolytic genes in T. harzianum were investigated. The recombinant protein was expressed in two major forms by Pichia pastoris: one form with a molecular mass of 35 kDa (non-glycosylated) and another form with a molecular mass of 60 kDa (glycosylated). Both forms showed optimal xylanolytic activity at 40 °C and pH 6.5. Glycosylation resulted in a twofold higher catalytic efficiency for the recombinant enzyme. In transcriptional studies, in contrast to the findings reported for Trichoderma reesei xyn3, the xyn3 gene from T. harzianum IOC-3844 was up-regulated in the presence of xylan. Moreover, stronger and faster expression of the analyzed genes (xyn2, xyn3, and egl3) was observed during cultivation with cellulose and xylan together. We employed the recombinant xyn3 during the cultivation of T. harzianum in steam-exploded bagasse to release the remaining xylan bound to the cellulose. Interestingly, the addition of Xyn3 accelerated the expression of xylanases and other genes of the cellulolytic system (egl1, egl3, cbh2, and swo1). Our results provide a potential strategy for triggering the expression of xylanases and cellulases in T. harzianum IOC-3844. The addition of Xyn3 can improve the expression of lignocellulolytic genes considerably when the fungus is cultivated with sugarcane bagasse.


Xylanase Trichoderma harzianum Second generation ethanol Lignocellulolytic enzymes Cellulases 



The authors thank the Brazilian fostering agencies National Council for Scientific and Technological Development (CNPq) and São Paulo State Foundation for Science and Technology (FAPESP) for financial support. The authors also thank Professor Dr. Roberto de Campos Giordano from the Laboratory for Development and Automation of Bioprocesses (UFSCar, Brazil) for providing the steam-exploded sugarcane bagasse.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12155_2016_9748_MOESM1_ESM.pdf (703 kb)
ESM 1 (PDF 703 kb)


  1. 1.
    Wyman CE (2007) What is (and is not) vital to advancing cellulosic ethanol. Trends Biotechnol 25(4):153–157. doi: 10.1016/j.tibtech.2007.02.009 CrossRefPubMedGoogle Scholar
  2. 2.
    Soccol CR, Vandenberghe LP, Medeiros AB, Karp SG, Buckeridge M, Ramos LP, Pitarelo AP, Ferreira-Leitao V, Gottschalk LM, Ferrara MA, da Silva Bon EP, de Moraes LM, Araujo Jde A, Torres FA (2010) Bioethanol from lignocelluloses: status and perspectives in Brazil. Bioresour Technol 101(13):4820–4825. doi: 10.1016/j.biortech.2009.11.067 CrossRefPubMedGoogle Scholar
  3. 3.
    Alvira P, Tomas-Pejo E, Jose Negro M, Ballesteros AM (2011) Strategies of xylanase supplementation for an efficient saccharification and cofermentation process from pretreated wheat straw. Biotechnol Prog. doi: 10.1002/btpr.623 PubMedGoogle Scholar
  4. 4.
    Pereira Jr. N, Couto MAPG, Anna LMMS (2008) Biomass of lignocellulosic composition for fuel ethanol production within the context of biorefinery, vol 2. Series on Biotechnology, 1 edn. Biblioteca Nacional, Rio de JaneiroGoogle Scholar
  5. 5.
    Banerjee G, Scott-Craig JS, Walton JD (2010) Improving enzymes for biomass conversion: a basic research perspective. Bioenergy Res 3:82–92. doi: 10.1007/s12155-009-9067-5 CrossRefGoogle Scholar
  6. 6.
    Beg QK, Kapoor M, Mahajan L, Hoondal GS (2001) Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol 56:326–38. doi: 10.1007/s002530100704 CrossRefPubMedGoogle Scholar
  7. 7.
    Biely P, Vrsanska M, Tenkanen M, Kluepfel D (1997) Endo-beta-1,4-xylanase families: differences in catalytic properties. J Biotechnol 57(1–3):151–166CrossRefPubMedGoogle Scholar
  8. 8.
    Zhang JH, 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. Bioresour Technol 102:9090–9095. doi: 10.1016/j.biortech.2011.06.085 CrossRefPubMedGoogle Scholar
  9. 9.
    Polizeli MLTM, Rizzatti ACS, Monti R, Terenzi HF, Jorge JA, Amorim DS (2005) Xylanases from fungi: properties and industrial applications. Appl Microbiol Biotechnol 67:577–591. doi: 10.1007/s00253-005-1904-7 CrossRefPubMedGoogle Scholar
  10. 10.
    Xu J, Nogawa M, Okada H, Morikawa Y (2000) Regulation of xyn3 gene expression in Trichoderma reesei PC-3-7. Appl Microbiol Biotechnol 54(3):370–375. doi: 10.1007/s002530000410 CrossRefPubMedGoogle Scholar
  11. 11.
    Mach RL, Zeilinger S (2003) Regulation of gene expression in industrial fungi: Trichoderma. Appl Microbiol Biotechnol 60(5):515–522. doi: 10.1007/s00253-002-1162-x CrossRefPubMedGoogle Scholar
  12. 12.
    de Castro AM, Pedro KC, da Cruz JC, Ferreira MC, Leite SG, Pereira N Jr (2010) Trichoderma harzianum IOC-4038: a promising strain for the production of a cellulolytic complex with significant beta-glucosidase activity from sugarcane bagasse cellulignin. Appl Biochem Biotechnol 162(7):2111–2122. doi: 10.1007/s12010-010-8986-0 CrossRefPubMedGoogle Scholar
  13. 13.
    de Castro AM, Ferreira MC, da Cruz JC, Pedro KC, Carvalho DF, Leite SG, Pereira N (2010) High-yield endoglucanase production by Trichoderma harzianum IOC-3844 cultivated in pretreated sugarcane mill byproduct. Enzyme Res 2010:854526. doi: 10.4061/2010/854526 PubMedPubMedCentralGoogle Scholar
  14. 14.
    Mandels M, Reese ET (1957) Induction of cellulase in Trichoderma viride as influenced by carbon sources and metals. J Bacteriol 73(2):269–278PubMedPubMedCentralGoogle Scholar
  15. 15.
    Guo Y, Ribeiro JMC, Anderson JM, Bour S (2009) dCAS: a desktop application for cDNA sequence annotation. Bioinformatics 25(9):1195–1196. doi: 10.1093/bioinformatics/btp129 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795. doi: 10.1016/j.jmb.2004.05.028 CrossRefPubMedGoogle Scholar
  17. 17.
    Julenius K, Molgaard A, Gupta R, Brunak S (2005) Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites. Glycobiology 15:153–164. doi: 10.1093/glycob/cwh151 CrossRefPubMedGoogle Scholar
  18. 18.
    Cregg JM (2007) Pichia protocols. Methods in molecular biology, vol 389, 2nd edn. Humana Press, TotowaGoogle Scholar
  19. 19.
    Morales P, Madarro A, Perez-Gonzalez JA, Sendra JM, Pinaga F, Flors A (1993) Purification and characterization of alkaline xylanases from Bacillus polymyxa. Appl Environ Microbiol 59(5):1376–1382PubMedPubMedCentralGoogle Scholar
  20. 20.
    Generoso WC, Malago-Jr W, Pereira N Jr, Henrique-Silva F (2012) Recombinant expression and characterization of an endoglucanase III (cel12a) from Trichoderma harzianum (Hypocreaceae) in the yeast Pichia pastoris. Genet Mol Res 11(2):1544–1557. doi: 10.4238/2012.May.21.11 CrossRefPubMedGoogle Scholar
  21. 21.
    Bailey MJ, Biely P, Poutanen K (1992) Interlaboratory testing of methods for assay of xylanase activity. J Biotechnol 23(3):257–270. doi: 10.1016/0168-1656(92)90074-J CrossRefGoogle Scholar
  22. 22.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 −ΔΔct method. Methods 25:402–408. doi: 10.1006/meth.2001.1262 CrossRefPubMedGoogle Scholar
  23. 23.
    van de Vyver WFJ, Dawson KA, Casey NH, Tricarico JM (2004) Effect of glycosylation on the stability of fungal xylanase exposed to proteases or rumen fluid in vitro. Anim Feed Sci Technol 116:259–269. doi: 10.1016/j.anifeedsci.2004.07.012 CrossRefGoogle Scholar
  24. 24.
    Xu J, Takakuwa N, Nogawa M, Okada H, Morikawa Y (1998) A third xylanase from Trichoderma reesei PC-3-7. Appl Microbiol Biotechnol 49:718–724. doi: 10.1007/s002530051237 CrossRefGoogle Scholar
  25. 25.
    Moraïs S, Barak Y, Caspi J, Hadar Y, Lamed R, Shoham Y, Wilson DB, Bayer EA (2010) Contribution of a xylan-binding module to the degradation of a complex cellulosic substrate by designer cellulosomes. Appl Environ Microbiol 76:3787–96. doi: 10.1128/AEM.00266-10 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Carle-Urioste JC, Escobar-Vera J, El-Gogary S, Henrique-Silva F, Torigoi E, Crivellaro O, Herrera-Estrella A, El-Dorry H (1997) Cellulase induction in Trichoderma reesei by cellulose requires its own basal expression. J Biol Chem 272:10169–10174. doi: 10.1074/jbc.272.15.10169 CrossRefPubMedGoogle Scholar
  27. 27.
    Dodd D, Cann IK (2009) Enzymatic deconstruction of xylan for biofuel production. Glob Change Biol Bioenergy 1(1):2–17. doi: 10.1111/j.1757-1707.2009.01004.x CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Subramaniyan S, Prema P (2002) Biotechnology of microbial xylanases: enzymology, molecular biology, and application. Crit Rev Biotechnol 22:33–64. doi: 10.1080/07388550290789450 CrossRefPubMedGoogle Scholar
  29. 29.
    Looser V, Bruhlmann B, Bumbak F, Stenger C, Costa M, Camattari A, Fotiadis D, Kovar K (2015) Cultivation strategies to enhance productivity of Pichia pastoris: a review. Biotechnol Adv. doi: 10.1016/j.biotechadv.2015.05.008 PubMedGoogle Scholar
  30. 30.
    Zheng Y, Pan Z, Zhang R (2009) Overview of biomass pretreatment for cellulosic ethanol production. Int J Agric Biol Eng 2:51–68. doi: 10.3965/j.issn.1934-6344.2009.03.051-068 Google Scholar
  31. 31.
    Fernández-Espinar M, Piñaga F, Graaff L, Visser J, Ramón D, Vallés S (1994) Purification, characterization and regulation of the synthesis of an Aspergillus nidulans acidic xylanase. Appl Microbiol Biotechnol 42(4):555–562. doi: 10.1007/bf00173920 CrossRefGoogle Scholar
  32. 32.
    Salles BC, Cunha RB, Fontes W, Sousa MV, Filho EX (2000) Purification and characterization of a new xylanase from Acrophialophora nainiana. J Biotechnol 81:199–204. doi: 10.1016/S0168-1656(00)00280-7 CrossRefPubMedGoogle Scholar
  33. 33.
    Silveira FQD, de Sousa MV, Ricart CAO, Milagres AMF, de Medeiros CL, Filho EXF (1999) A new xylanase from a Trichoderma harzianum strain. J Ind Microbiol Biotechnol 23(1):682–685CrossRefGoogle Scholar
  34. 34.
    Herold S, Bischof R, Metz B, Seiboth B, Kubicek CP (2013) Xylanase gene transcription in Trichoderma reesei is triggered by different inducers representing different hemicellulosic pentose polymers. Eukaryot Cell 12:390–398. doi: 10.1128/EC.00182-12 CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Canilha L, Chandel AK, Milessi TSD, Antunes FAF, Freitas WLD, Felipe MDA, da Silva SS (2012) Bioconversion of sugarcane biomass into ethanol: an overview about composition, pretreatment methods, detoxification of hydrolysates, enzymatic saccharification, and ethanol fermentation. J Biomed Biotechnol. doi: 10.1155/2012/989572 PubMedPubMedCentralGoogle Scholar
  36. 36.
    Kimura T, Suzuki H, Furuhashi H, Aburatani T, Morimoto K, Sakka K, Ohmiya K (2002) Molecular cloning, characterization, and expression analysis of the xynF3 gene from Aspergillus oryzae. Biosci Biotechnol Biochem 66(2):285–292. doi: 10.1271/bbb.66.285 CrossRefPubMedGoogle Scholar
  37. 37.
    Rahman Z, Shida Y, Furukawa T, Suzuki Y, Okada H, Ogasawara W, Morikawa Y (2009) Evaluation and characterization of Trichoderma reesei cellulase and xylanase promoters. Appl Microbiol Biotechnol 82(5):899–908. doi: 10.1007/s00253-008-1841-3 CrossRefPubMedGoogle Scholar
  38. 38.
    Nakazawa H, Kawai T, Ida N, Shida Y, Kobayashi Y, Okada H, Tani S, J-i S, Kawaguchi T, Morikawa Y, Ogasawara W (2012) Construction of a recombinant Trichoderma reesei strain expressing Aspergillus aculeatus β-glucosidase 1 for efficient biomass conversion. Biotechnol Bioeng 109(1):92–99. doi: 10.1002/bit.23296 CrossRefPubMedGoogle Scholar
  39. 39.
    Furukawa T, Shida Y, Kitagami N, Mori K, Kato M, Kobayashi T, Okada H, Ogasawara W, Morikawa Y (2009) Identification of specific binding sites for XYR1, a transcriptional activator of cellulolytic and xylanolytic genes in Trichoderma reesei. Fungal Genet Biol 46(8):564–574. doi: 10.1016/j.fgb.2009.04.001 CrossRefPubMedGoogle Scholar
  40. 40.
    Stricker AR, Grosstessner-Hain K, Wurleitner E, Mach RL (2006) Xyr1 (xylanase regulator 1) regulates both the hydrolytic enzyme system and D-xylose metabolism in Hypocrea jecorina. Eukaryotic Cell 5(12):2128–2137. doi: 10.1128/Ec.00211-06 CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Furukawa T, Shida Y, Kitagami N, Ota Y, Adachi M, Nakagawa S, Shimada R, Kato M, Kobayashi T, Okada H, Ogasawara W, Morikawa Y (2008) Identification of the cis-acting elements involved in regulation of xylanase III gene expression in Trichoderma reesei PC-3-7. Fungal Genet Biol 45:1094–1102. doi: 10.1016/j.fgb.2008.03.006 CrossRefPubMedGoogle Scholar
  42. 42.
    Delabona PS, Farinas CS, da Silva MR, Azzoni SF, Pradella JGC (2012) Use of a new Trichoderma harzianum strain isolated from the Amazon rainforest with pretreated sugar cane bagasse for on-site cellulase production. Bioresour Technol 107:517–521. doi: 10.1016/j.biortech.2011.12.048 CrossRefGoogle Scholar
  43. 43.
    Qing Q, Wyman CE (2011) Supplementation with xylanase and β-xylosidase to reduce xylo-oligomer and xylan inhibition of enzymatic hydrolysis of cellulose and pretreated corn stover. Biotechnol Biofuels 4:18. doi: 10.1186/1754-6834-4-18 CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Martín Medina CO, Marcet M, Thomsen AB (2008) Comparison of wet oxidation and steam explosion as pretreatment methods for bioethanol production from sugarcane bagasse. Bio Res 3:670–683Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Wesley Cardoso Generoso
    • 1
  • Wilson Malagó-Jr
    • 1
  • Nei Pereira-Jr
    • 2
  • Flávio Henrique-Silva
    • 1
    Email author
  1. 1.Department of Genetics and EvolutionFederal University of São CarlosSão PauloBrazil
  2. 2.Department of Biochemical EngineeringFederal University of Rio de JaneiroRio de JaneiroBrazil

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