title: Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme.
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.
authors.affiliation: Departamento de Física e Informática, Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Sãocarlense, 400, São Carlos, SP, 13566-590, Brazil.
authors.forename: Vanessa O A
authors.initials: VO
authors.lastname: Pellegrini
authors.affiliation: Departamento de Física e Informática, Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Sãocarlense, 400, São Carlos, SP, 13566-590, Brazil.
authors.forename: Viviane Isabel
authors.initials: VI
authors.lastname: Serpa
authors.affiliation: Departamento de Física e Informática, Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Sãocarlense, 400, São Carlos, SP, 13566-590, Brazil.
authors.forename: Andre S
authors.initials: AS
authors.lastname: Godoy
authors.affiliation: Departamento de Física e Informática, Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Sãocarlense, 400, São Carlos, SP, 13566-590, Brazil.
authors.forename: Cesar M
authors.initials: CM
authors.lastname: Camilo
authors.affiliation: Departamento de Física e Informática, Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Sãocarlense, 400, São Carlos, SP, 13566-590, Brazil.
authors.forename: Amanda
authors.initials: A
authors.lastname: Bernardes
authors.affiliation: Instituto de Química, Universidade de Campinas, Caixa Postal 6154, Campinas, SP, 13083-970, Brazil.
authors.forename: Camila A
authors.initials: CA
authors.lastname: Rezende
authors.affiliation: Escola de Química, Departamento de Engenharia Bioquímica, Universidade Federal do Rio de Janeiro, Bloco E, Sala 121, Ilha do Fundão, Rio de Janeiro, RJ, 21949-900, Brazil.
authors.forename: Nei Pereira
authors.initials: NP
authors.lastname: Junior
authors.affiliation: Laboratório Nacional de Ciência e Tecnologia do Bioetanol - CTBE, Centro Nacional de Pesquisa em Energia e Materiais - CNPEM, Rua Giuseppe Máximo Scolfaro, 10000, 13083-970, Campinas, SP, Brazil.
authors.forename: João Paulo L
authors.initials: JP
authors.lastname: Franco Cairo
authors.affiliation: Laboratório Nacional de Ciência e Tecnologia do Bioetanol - CTBE, Centro Nacional de Pesquisa em Energia e Materiais - CNPEM, Rua Giuseppe Máximo Scolfaro, 10000, 13083-970, Campinas, SP, Brazil.
authors.forename: Fabio M
authors.initials: FM
authors.lastname: Squina
authors.affiliation: Departamento de Física e Informática, Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Sãocarlense, 400, São Carlos, SP, 13566-590, Brazil. ipolikarpov@ifsc.usp.br.
authors.forename: Igor
authors.initials: I
authors.lastname: Polikarpov
mesh_terms: D050260:Carbohydrate Metabolism; D044602:Cellulases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D002482:Cellulose; D044602:Cellulases
keywords: Aspergillus niger; Cellulase; Endoglucanase; Second-generation ethanol; Trichoderma harzianum