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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 Polikarpov
Biotechnologically relevant enzymes and proteins

Abstract

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.

Keywords

Trichoderma harzianum Second-generation ethanol Cellulase Endoglucanase Aspergillus niger 

Notes

Acknowledgments

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)

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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
  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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