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Applied Biochemistry and Biotechnology

, Volume 185, Issue 1, pp 316–333 | Cite as

Overexpression of a Cellobiose-Glucose-Halotolerant Endoglucanase from Scytalidium thermophilum

  • Luana Parras MeleiroEmail author
  • Sibeli Carli
  • Raquel Fonseca-Maldonado
  • Marcela da Silva Torricillas
  • Ana Lucia Ribeiro Latorre Zimbardi
  • Richard John Ward
  • João Atílio Jorge
  • Rosa Prazeres Melo Furriel
Article

Abstract

Enzyme reaction products and by-products from pretreatment steps can inhibit endoglucanases and are major factors limiting the efficiency of enzymatic lignocellulosic biomass hydrolysis. The gene encoding the endoglucanase from Scytalidium thermophilum (egst) was cloned and expressed as a soluble protein in Pichia pastoris GS115. The recombinant enzyme (Egst) was monomeric (66 kDa) and showed an estimated carbohydrate content of 53.3% (w/w). The optimum temperature and pH of catalysis were 60–70 °C and pH of 5.5, respectively. The enzyme was highly stable at pH 3.0–8.0 with a half-life in water of 100 min at 65 °C. The Egst presented good halotolerance, retaining 84.1 and 71.4% of the control activity in the presence of 0.5 and 2.0 mol L−1 NaCl, respectively. Hydrolysis of medium viscosity carboxymethylcellulose (CMC) by Egst was stimulated 1.77-, 1.84-, 1.64-, and 1.8-fold by dithiothreitol, β-mercaptoethanol, cysteine, and manganese at 10, 10, 10, and 5 mmol L−1 concentration, respectively. The enzyme hydrolyzed CMC with maximal velocity and an apparent affinity constant of 432.10 ± 16.76 and 10.5 ± 2.53 mg mL−1, respectively. Furthermore, the Egst was tolerant to reaction products and able to act on pretreated fractions sugarcane bagasse demonstrating excellent properties for application in the hydrolysis of lignocellulosic biomass.

Keywords

Endoglucanase GH5 Halotolerance Pichia pastoris Scytalidium thermophilum 

Notes

Acknowledgements

We thank André Justino for technical assistance.

Authors’ Contributions

Luana P. Meleiro, Sibeli Carli and Rosa P. M. Furriel conceived and designed the experiments; Luana P. Meleiro, Sibeli Carli, Raquel F. Maldonado, Marcela S. Torricillas and Ana L. R. L. Zimbardi performed the experiments; Raquel F. Maldonado and Richard J. Ward conducted the cloning and expression experiments; Luana P. Meleiro, Sibeli Carli and Marcela S. Torricillas analyzed the data; Richard J. Ward, Rosa P. M. Furriel and João A. Jorge contributed reagents, materials and analysis tools; Luana P. Meleiro and Sibeli Carli wrote the paper.

Funding

This investigation was supported by research grants from CNPq (Conselho deDesenvolvimento Científico e Tecnológico), FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). Luana P. Meleiro and Raquel F. Maldonado received post-doctoral scholarship from FAPESP; Marcela S. Torricillas received a scientific initiation scholarship from CNPq; Sibeli Carli received a Ph.D. scholarship from CAPES; Richard J. Ward; João A. Jorge and Rosa P. M. Furriel received researcher stipends from CNPq.

Compliance with Ethical Standards

Competing Interests

The authors declare that they have no conflict of interest.

Supplementary material

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Fig. S1 (PDF 381 kb)
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Fig. S2 (PDF 400 kb)
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Fig. S3 (PDF 135 kb)
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Fig. S4 (PDF 146 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2017

Authors and Affiliations

  • Luana Parras Meleiro
    • 1
    Email author
  • Sibeli Carli
    • 1
  • Raquel Fonseca-Maldonado
    • 2
  • Marcela da Silva Torricillas
    • 1
  • Ana Lucia Ribeiro Latorre Zimbardi
    • 1
  • Richard John Ward
    • 1
  • João Atílio Jorge
    • 3
  • Rosa Prazeres Melo Furriel
    • 1
  1. 1.Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão PretoUniversidade de São PauloRibeirão PretoBrazil
  2. 2.Instituto Federal de São PauloSão José dos CamposBrazil
  3. 3.Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão PretoUniversidade de São PauloRibeirão PretoBrazil

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