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

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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.

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Acknowledgements

We thank André Justino for technical assistance.

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.

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Author notes

  1. Luana Parras Meleiro and Sibeli Carli contributed equally to this work.

Authors and Affiliations

  1. Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil

    Luana Parras Meleiro, Sibeli Carli, Marcela da Silva Torricillas, Ana Lucia Ribeiro Latorre Zimbardi, Richard John Ward & Rosa Prazeres Melo Furriel

  2. Instituto Federal de São Paulo, São José dos Campos, São Paulo, Brazil

    Raquel Fonseca-Maldonado

  3. Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil

    João Atílio Jorge

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  1. Luana Parras Meleiro
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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.

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Meleiro, L.P., Carli, S., Fonseca-Maldonado, R. et al. Overexpression of a Cellobiose-Glucose-Halotolerant Endoglucanase from Scytalidium thermophilum . Appl Biochem Biotechnol 185, 316–333 (2018). https://doi.org/10.1007/s12010-017-2660-8

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