Author | Souza, Flavio Henrique Moreira | |
Author | Meleiro, Luana Parras | |
Author | Machado, Carla Botelho | |
Author | Zimbardi, Ana Lucia Ribeiro Latorre | |
Author | Maldonado, Raquel Fonseca | |
Author | Souza, Tatiana de Arruda Campos Brasil de | |
Author | Masui, Douglas Chodi | |
Author | Murakami, Mario Tyago | |
Author | Jorge, João Atilio | |
Author | Ward, Richard John | |
Author | Furriel, Rosa dos Prazeres Melo | |
Access date | 2014-11-11T14:48:59Z | |
Available date | 2015-12-01T06:30:05Z | |
Document date | 2014 | |
Citation | SOUZA, Flavio Henrique Moreira et al. Gene cloning, expression and biochemical characterization of a glucose- and xylose-stimulated β-glucosidase from Humicola insolens RP86. Journal of Molecular Catalysis B: Enzymatic, v. 106, p. 1-10, Aug. 2014. | pt_BR |
ISSN | 1381-1177 | |
URI | https://www.arca.fiocruz.br/handle/icict/8787 | |
Language | eng | pt_BR |
Rights | restricted access | pt_BR |
Title | Gene cloning, expression and biochemical characterization of a glucose- and xylose-stimulated β-glucosidase from Humicola insolens RP86 | pt_BR |
Type | Article | pt_BR |
DOI | 10.1016/j.molcatb.2014.04.007 | pt_BR |
Abstract | Efficient cellulose saccharification is crucial for the cost-effective production of ethanol from lignocellulosic biomass. Most β-glucosidases are inhibited by glucose, and high enzyme loads are needed to attain acceptable hydrolysis yields, increasing the process costs. Therefore, glucose-stimulated β-glucosidases with high catalytic efficiency for cellobiose hydrolysis are highly interesting, particularly those produced at low cost using convenient expression systems. The bglhi gene from Humicola insolens RP86, which encodes a glucose- and xylose-stimulated β-glucosidase (Bglhi), was cloned and expressed in E. coli. The recombinant Bglhi was expressed in soluble form and dynamic light scattering showed that the protein was monomeric (56.1 kDa). The enzyme showed broad substrate specificity, with optima of pH and temperature of 5.0–7.0 and 60 °C, and was stable from pH 4.5 to 8.0, and for 1 h at 50 °C in water. The recombinant Bglhi was highly stimulated by glucose and xylose. About 2-fold stimulation of pNP-glucosidase activity occurred at 50 mmol L−1 glucose or 100 mmol L−1 xylose. In addition, stimulation was observed over 50–370 mmol L−1 glucose or 100–1000 mmol L−1 xylose. Xylose maximally stimulated (2.3-fold) the cellobiase activity of the enzyme at 80 mmol L−1 concentration. The Vmax for cellobiose hydrolysis (183.4 ± 12.0 U mg−1) was 5-fold higher than that for pNP-Glc (36.4 ± 1.4 U mg−1), but a lower KM for pNP-Glc (0.20 ± 0.01 mmol L−1), compared to cellobiose (0.38 ± 0.01 mmol L−1), was estimated. More importantly, the recombinant Bglhi presented the highest catalytic efficiency for cellobiose hydrolysis (kcat/KM = 453.0 ± 29.6 s−1 mmol−1 L) among all glucose-stimulated and glucose-tolerant β-glucosidases known to date. Together, these properties indicate that the recombinant Bglhi is an attractive candidate to include in enzymatic cocktails for application in industrial processes for the saccharification of lignocellulosic materials. | pt_BR |
Affilliation | University of São Paulo. Faculty of Philosophy, Sciences and Languages of Ribeirão Preto. Department of Chemistry. Ribeirão Preto, SP, Brazil. | pt_BR |
Affilliation | University of São Paulo. Faculty of Philosophy, Sciences and Languages of Ribeirão Preto. Department of Chemistry. Ribeirão Preto, SP, Brazil. | pt_BR |
Affilliation | University of São Paulo. Faculty of Philosophy, Sciences and Languages of Ribeirão Preto. Department of Chemistry. Ribeirão Preto, SP, Brazil / National Center for Research in Energy and Materials. Brazilian Biosciences National Laboratory. Campinas, SP, Brazil. | pt_BR |
Affilliation | University of São Paulo. Faculty of Philosophy, Sciences and Languages of Ribeirão Preto. Department of Chemistry. Ribeirão Preto, SP, Brazil. | pt_BR |
Affilliation | University of São Paulo. Faculty of Medicine of Ribeirão Preto. Department of Biochemistry. Ribeirão Preto, SP, Brazil. | pt_BR |
Affilliation | Oswaldo Cruz Foundation. Carlos Chagas Institute. Curitiba, PR, Brazil. | pt_BR |
Affilliation | University of São Paulo. Faculty of Philosophy, Sciences and Languages of Ribeirão Preto. Department of Biology. Ribeirão Preto, SP, Brazil. | pt_BR |
Affilliation | National Center for Research in Energy and Materials. Brazilian Biosciences National Laboratory. Campinas, SP, Brazil | pt_BR |
Affilliation | University of São Paulo. Faculty of Philosophy, Sciences and Languages of Ribeirão Preto. Department of Biology. Ribeirão Preto, SP, Brazil. | pt_BR |
Affilliation | University of São Paulo. Faculty of Philosophy, Sciences and Languages of Ribeirão Preto. Department of Chemistry. Ribeirão Preto, SP, Brazil / National Center for Research in Energy and Materials. Brazilian Biosciences National Laboratory. Campinas, SP, Brazil. | pt_BR |
Affilliation | University of São Paulo. Faculty of Philosophy, Sciences and Languages of Ribeirão Preto. Department of Chemistry. Ribeirão Preto, SP, Brazil. | pt_BR |
Subject | Cellobiase | En |
Subject | Humicola insolens | pt_BR |
Subject | Cellulose saccharification | pt_BR |
Subject | Cellulosic ethanol | pt_BR |
Subject | Recombinant β-glucosidase | pt_BR |
Subject | Product stimulation | pt_BR |
Subject | Glucose stimulation | pt_BR |