Applied Microbiology and Biotechnology

, Volume 99, Issue 22, pp 9591–9604 | Cite as

Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme

  • Vanessa O. A. Pellegrini
  • Viviane Isabel Serpa
  • Andre S. Godoy
  • Cesar M. Camilo
  • Amanda Bernardes
  • Camila A. Rezende
  • Nei Pereira Junior
  • João Paulo L. Franco Cairo
  • Fabio M. Squina
  • Igor PolikarpovEmail author
Biotechnologically relevant enzymes and proteins


Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for β-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl β-d-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.


Trichoderma harzianum Second-generation ethanol Cellulase Endoglucanase Aspergillus niger 



We would like to acknowledge support of the Brazilian funding agencies Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) via grants #2008/56255-9, 2009/52840-7, 2009/05328-9, 2010/18773-8, 2011/20977-3, and 2011/05712-3; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) via grants #490022/2009-0, 301981/2011-6 and, 400045/2012–5; CAPES and Universidade de São Paulo via grants “Centro de Instrumentação para estudos avançados de materiais nanoestruturados e biossistemas” and “Núcleo de Apoio à Pesquisa em Bioenergia e Sustentabilidade (NAPBS).”

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

253_2015_6772_MOESM1_ESM.pdf (431 kb)
ESM 1 (PDF 431 kb)


  1. Badhan A, Wang Y, Gruninger R, Patton D, Powlowski J, Tsang A, McAllister T (2014) Formulation of enzyme blends to maximize the hydrolysis of alkaline peroxide pretreated alfalfa hay and barley straw by rumen enzymes and commercial cellulases. BMC Biotechnol 14:31CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bailey MJ, Siika-aho M, Valkeajärvi A, Penttilä ME (1993) Hydrolytic properties of two cellulases of Trichoderma reesei expressed in yeast. Biotechnol Appl Biochem 17:65–76PubMedGoogle Scholar
  3. Becker D, Braet C, Brumer H, Claeyssens M, Divne C, Fagerström BR, Harris M, Jones TA, Kleywegt GJ, Koivula A, Mahdi S, Piens K, Sinnott ML, Ståhlberg J, Teeri TT, Underwood M, Wohlfahrt G (2001) Engineering of a glycosidase family 7 cellobiohydrolase to more alkaline pH optimum: the pH behaviour of Trichoderma reesei Cel7A and its E223S/A224H/L225V/T226A/D262G mutant. Biochem J 356:19–30CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bhat MK (2000) Cellulases and related enzymes in biotechnology. Biotechnol Adv 18:355–383CrossRefPubMedGoogle Scholar
  5. Biely P, Vrsanská M, Claeyssens M (1991) The endo-1,4-β-glucanase I from Trichoderma reesei. Eur J Biochem 200:157–163CrossRefPubMedGoogle Scholar
  6. Bubner P, Plank H, Nidetzky B (2013) Visualizing cellulase activity. Biotechnol Bioeng 110:1529–1549CrossRefPubMedGoogle Scholar
  7. Buckeridge MS, Dos Santos WD, De Souza AP (2010) Routes for cellulosic ethanol in Brazil. In: LAB C (ed) Sugarcane bioethanol: R&D for productivity and sustainability. Edgard Blucher, Sao Paulo, Brazil, pp. 365–380Google Scholar
  8. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The carbohydrate-active enzymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 37:D233–D238CrossRefPubMedGoogle Scholar
  9. Chundawat SPS, Beckham GT, Himmel ME, Dale BE (2011) Deconstruction of lignocellulosic biomass to fuels and chemicals. Annu Rev Chem Biomol Eng 2:121–145CrossRefPubMedGoogle Scholar
  10. Colussi F, Garcia W, Rosseto FR, de Mello BL, de Oliveira NM, Polikarpov I (2012) Effect of pH and temperature on the global compactness, structure, and activity of cellobiohydrolase Cel7A from Trichoderma harzianum. Eur Biophys J 41:89–98CrossRefPubMedGoogle Scholar
  11. de Castro AM, de Albuquerque de Carvalho ML, Leite SG, Pereira Jr N (2010) Cellulases from Penicillium funiculosum: production, properties and application to cellulose hydrolysis. J Ind Microbiol Biotechnol 37:151–158CrossRefPubMedGoogle Scholar
  12. Dias MOS, Junqueira TL, Cavalett O, Cunha MP, Jesus CDF, Rossell CEV, Maciel Filho R, Bonomi A (2012) Integrated versus stand-alone second generation ethanol production from sugarcane bagasse and trash. Bioresour Technol 103(1):152–161CrossRefPubMedGoogle Scholar
  13. Ding SJ, Ge W, Buswell JA (2001) Endoglucanase I from the edible straw mushroom, Volvariella volvacea. Purification, characterization, cloning and expression. Eur J Biochem 268(22):5687–5695CrossRefPubMedGoogle Scholar
  14. Eriksson T, Stals I, Collén A, Tjerneld F, Claeyssens M, Stålbrand H, Brumer H (2004) Heterogeneity of homologously expressed Hypocrea jecorina (Trichoderma reesei) Cel7B catalytic module. Eur J Biochem 271:1266–1276CrossRefPubMedGoogle Scholar
  15. Ganner T, Bubner P, Eibinger M, Mayrhofer C, Plank H, Nidetzky B (2012) Dissecting and reconstructing synergism: in situ visualization of cooperativity among cellulases. J Biol Chem 287:43215–43222CrossRefPubMedPubMedCentralGoogle Scholar
  16. García R, Cremata JA, Quintero O, Montesino R, Benkestock K, Ståhlberg J (2001) Characterization of protein glycoforms with N-linked neutral and phosphorylated oligosaccharides: studies on the glycosylation of endoglucanase 1 (Cel7B) from Trichoderma reesei. Biotechnol Appl Biochem 33:141–152CrossRefPubMedGoogle Scholar
  17. Hasper AA, Dekkers E, van Mil M, van de Vondervoort PJ, de Graaff LH (2002) EglC, a new endoglucanase from Aspergillus niger with major activity towards xyloglucan. Appl Environ Microbiol 68:1556–1560CrossRefPubMedPubMedCentralGoogle Scholar
  18. Hartley JL, Temple GF, Brasch MA (2000) DNA cloning using in vitro site-specific recombination. Genome Res 10:1788–1795CrossRefPubMedPubMedCentralGoogle Scholar
  19. Hilden L, Valjamae P, Johansson G (2005) Surface character of pulp fibres studied using endoglucanases. J Biotechnol 118:386–397CrossRefPubMedGoogle Scholar
  20. Himmel ME, Ding SY, Johnson DK, Adney WS, Nimlos MR, Brady JW, Foust TD (2007) Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315:804–807CrossRefPubMedGoogle Scholar
  21. Horn SJ, Vaaje-Kolstad G, Westereng B, Eijsink VG (2012) Novel enzymes for the degradation of cellulose. Biotechnol Biofuels 5:45CrossRefPubMedPubMedCentralGoogle Scholar
  22. Kalsner I, Hintz W, Reid LS, Schachter H (1995) Insertion into Aspergillus nidulans of functional UDP-GlcNAc: α 3-D-mannoside β-1,2-N-acetylglucosaminyl-transferase I, the enzyme catalysing the first committed step from oligomannose to hybrid and complex N-glycans. Glycoconj J 12:360–370CrossRefPubMedGoogle Scholar
  23. Karlsson J, Siika-aho M, Tenkanen M, Tjerneld F (2002) Enzymatic properties of the low molecular mass endoglucanases Cel12A (EG III) and Cel45A (EG V) of Trichoderma reesei. J Biotechnol 99:63–78CrossRefPubMedGoogle Scholar
  24. Katzen F (2007) Gateway(®) recombinational cloning: a biological operating system. Expert Opin Drug Discov 2:571–589CrossRefPubMedGoogle Scholar
  25. Kim HM, Lee YG, Patel DH, Lee KH, Lee DS, Bae HJ (2012) Characteristics of bifunctional acidic endoglucanase (Cel5B) from Gloeophyllum trabeum. J Ind Microbiol Biotechnol 39:1081–1089CrossRefPubMedGoogle Scholar
  26. Kont R, Kurasin M, Teugjas H, Valjamae P (2013) Strong cellulase inhibitors from the hydrothermal pretreatment of wheat straw. Biotechnol Biofuels 6:135CrossRefPubMedPubMedCentralGoogle Scholar
  27. Lee I, Evans BR, Woodward J (2000) The mechanism of cellulase action on cotton fibers: evidence from atomic force microscopy. Ultramicroscopy 82:213–221CrossRefPubMedGoogle Scholar
  28. Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66:506–577CrossRefPubMedPubMedCentralGoogle Scholar
  29. Mikkelson A, Maaheimo H, Hakala TK (2013) Hydrolysis of konjac glucomannan by Trichoderma reesei mannanase and endoglucanases Cel7B and Cel5A for the production of glucomannooligosaccharides. Carbohydr Res 372:60–68CrossRefPubMedGoogle Scholar
  30. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  31. Miotto LS, de Rezende CA, Bernardes A, Serpa VI, Tsang A, Polikarpov I (2014) The characterization of the endoglucanase Cel12A from Gloeophyllum trabeum reveals an enzyme highly active on β-glucan. PLoS One 9:e108393CrossRefPubMedPubMedCentralGoogle Scholar
  32. Momeni MH, Payne CM, Hansson H, Mikkelsen NE, Svedberg J, Engström A, Sandgren M, Beckham MG, Ståhlberg J (2013) Structural, biochemical, and computational characterization of the glycoside hydrolase family 7 cellobiohydrolase of the tree-killing fungus Heterobasidion irregulare. J Biol Chem 8:5861–5872CrossRefGoogle Scholar
  33. Naran R, Pierce ML, Mort AJ (2007) Detection and identification of rhamnogalacturonan lyase activity in intercellular spaces of expanding cotton cotyledons. Plant J 50:95–107CrossRefPubMedGoogle Scholar
  34. Okada H, Tada K, Sekiya T, Yokoyama K, Takahashi A, Tohda H, Kumagai H, Morikawa Y (1998) Molecular characterization and heterologous expression of the gene encoding a low-molecular-mass endoglucanase from Trichoderma reesei QM9414. Appl Environ Microbiol 64:555–563PubMedPubMedCentralGoogle Scholar
  35. Payne CM, Resch MG, Chen L, Crowley MF, Himmel ME, Taylor LE, Sandgren M, Ståhlberg J, Stals I, Tan Z, Beckham GT (2013) Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose. Proc Natl Acad Sci U S A 110:14646–14651CrossRefPubMedPubMedCentralGoogle Scholar
  36. Penttilä M, Nevalainen H, Rättö M, Salminen E, Knowles J (1987) A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 61:155–164CrossRefPubMedGoogle Scholar
  37. Punt PJ, van Biezen N, Conesa A, Albers A, Mangnus J, van den Hondel C (2002) Filamentous fungi as cell factories for heterologous protein production. Trends Biotechnol 20:200–206CrossRefPubMedGoogle Scholar
  38. Saha BC (2004) Production, purification and properties of endoglucanase from a newly isolated strain of Mucor circinelloides. Process Biochem 39:1871CrossRefGoogle Scholar
  39. Sanchez OJ, Cardona CA (2008) Trends in biotechnological production of fuel ethanol from different feedstocks. Bioresour Technol 99:5270–5295CrossRefPubMedGoogle Scholar
  40. Sandgren M, Ståhlberg J, Mitchinson C (2005) Structural and biochemical studies of GH family 12 cellulases: improved thermal stability, and ligand complexes. Prog Biophys Mol Biol 89:246–291CrossRefPubMedGoogle Scholar
  41. Schuster A, Schmoll M (2010) Biology and biotechnology of Trichoderma. Appl Microbiol Biotechnol 87:787–799CrossRefPubMedPubMedCentralGoogle Scholar
  42. Schwarz W (2001) The cellulosome and cellulose degradation by anaerobic bacteria. Appl Microbiol Biotechnol 56:634–649CrossRefPubMedGoogle Scholar
  43. Serpa VI, Polikarpov I (2011) Enzymes in bioenergy. In: Buckeridge MS, Goldman GH (eds) Routes to cellulosic ethanol. Springer, New York, London, pp. 97–115CrossRefGoogle Scholar
  44. Shumiao Z, Huang J, Zhang C, Deng L, Hu N, Liang Y (2010) High-level expression of an Aspergillus niger endo-β-1,4-glucanase in Pichia pastoris through gene codon optimization and synthesis. J Microbiol Biotechnol 20:467–473PubMedGoogle Scholar
  45. Storms R, Zheng Y, Li H, Sillaots S, Martinez-Perez A, Tsang A (2005) Plasmid vectors for protein production, gene expression and molecular manipulations in Aspergillus niger. Plasmid 53:191–204CrossRefPubMedGoogle Scholar
  46. Sun Y, Cheng J (2002) Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol 83(1):1–11CrossRefPubMedGoogle Scholar
  47. Suurnäkki A, Tenkanen M, Siika-Aho M, Niku-Paavola ML, Viikari L, Buchert J (2000) Trichoderma reesei cellulases and their core domains in the hydrolysis and modification of chemical pulp. Cellulose 7:189–209CrossRefGoogle Scholar
  48. Teeri TT (1997) Crystalline cellulose degradation: new insight into the function of cellobiohydrolases. Trends Biotechnol 15:160–167CrossRefGoogle Scholar
  49. Varnai A, Huikko L, Pere J, Siika-Aho M, Viikari L (2011) Synergistic action of xylanase and mannanase improves the total hydrolysis of softwood. Bioresour Technol 102:9096–9104CrossRefPubMedGoogle Scholar
  50. Vlasenko E, Schülein M, Cherry J, Xu F (2010) Substrate specificity of family 5, 6, 7, 9, 12, and 45 endoglucanases. Bioresour Technol 101:2405–2411CrossRefPubMedGoogle Scholar
  51. Voutilainen SP, Boer H, Alapuranen M, Janis J, Vehmaanpera J, Koivula A (2009) Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B. Appl Microbiol Biotechnol 83:261–272CrossRefPubMedGoogle Scholar
  52. Wang J, Quirk A, Lipkowski J, Dutcher JR, Hill C, Mark A, Clarke AJ (2012) Real-time observation of the swelling and hydrolysis of a single crystalline cellulose fiber catalyzed by cellulase 7B from Trichoderma reesei. Langmuir 28:9664–9672CrossRefPubMedGoogle Scholar
  53. Wood TM (1988) Preparation of crystalline, amorphous, and dyed cellulase substrates. Methods Enzymol 160:19–25CrossRefGoogle Scholar
  54. Xiang L, Li A, Tian C, Zhou Y, Zhang G, Ma Y (2014) Identification and characterization of a new acid-stable endoglucanase from a metagenomic library. Protein Expr Purif 102:20–26CrossRefPubMedGoogle Scholar
  55. Yelton MM, Hamer JE, Timberlake WE (1984) Transformation of Aspergillus nidulans by using a trpC plasmid. Proc Natl Acad Sci U S A 81:1470–1474CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Vanessa O. A. Pellegrini
    • 1
  • Viviane Isabel Serpa
    • 1
  • Andre S. Godoy
    • 1
  • Cesar M. Camilo
    • 1
  • Amanda Bernardes
    • 1
  • Camila A. Rezende
    • 2
  • Nei Pereira Junior
    • 3
  • João Paulo L. Franco Cairo
    • 4
  • Fabio M. Squina
    • 4
  • Igor Polikarpov
    • 1
    Email author
  1. 1.Departamento de Física e Informática, Instituto de Física de São CarlosUniversidade de São PauloSão CarlosBrazil
  2. 2.Instituto de QuímicaUniversidade de CampinasCampinasBrazil
  3. 3.Escola de Química, Departamento de Engenharia BioquímicaUniversidade Federal do Rio de JaneiroRio de JaneiroBrazil
  4. 4.Laboratório Nacional de Ciência e Tecnologia do Bioetanol - CTBECentro Nacional de Pesquisa em Energia e Materiais - CNPEMCampinasBrazil

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