title: The functional properties of a xyloglucanase (GH12) of Aspergillus terreus expressed in Aspergillus nidulans may increase performance of biomass degradation.
abstract: Filamentous fungi are attractive hosts for heterologous protein expression due to their capacity to secrete large amounts of enzymes into the extracellular medium. Xyloglucanases, which specifically hydrolyze xyloglucan, have been recently applied in lignocellulosic biomass degradation and conversion in many other industrial processes. In this context, this work aimed to clone, express, and determine the functional properties of a recombinant xyloglucanase (AtXEG12) from Aspergillus terreus, and also its solid-state (SSF) and submerged (SmF) fermentation in bioreactors. The purified AtXEG12 showed optimum pH and temperature of 5.5 and 65 °C, respectively, demonstrating to be 90 % stable after 24 h of incubation at 50 °C. AtXEG12 activity increased in the presence of 2-mercaptoethanol (65 %) and Zn+2(45 %), while Cu+2and Ag+ions drastically decreased its activity. A substrate assay showed, for the first time for this enzyme's family, xylanase activity. The enzyme exhibited high specificity for tamarind xyloglucan (KM1.2 mg mL-1) and Vmaxof 17.4 μmol min-1mg-1of protein. The capillary zone electrophoresis analysis revealed that AtXEG12 is an endo-xyloglucanase. The heterologous xyloglucanase secretion was greater than the production by wild-type A. terreus cultivated in SmF. On the other hand, AtXEG12 activity reached by SSF was sevenfold higher than values achieved by SmF, showing that the expression of recombinant enzymes can be significantly improved by cultivation under SSF.
authors.affiliation: Departamento de Bioquímica e Imunologia, FMRP, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Gabriela Leal
authors.initials: GL
authors.lastname: Vitcosque
authors.affiliation: Departamento de Bioquímica e Imunologia, FMRP, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.|Chemical Biochemical Environmental Engineering Department, University of Maryland, Baltimore County, MD, USA.
authors.forename: Liliane Fraga Costa
authors.initials: LF
authors.lastname: Ribeiro
authors.affiliation: Departamento de Bioquímica e Imunologia, FMRP, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.|Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, Ribeirão Preto, SP, 14040-901, Brazil.
authors.forename: Rosymar Coutinho
authors.initials: RC
authors.lastname: de Lucas
authors.affiliation: Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, Ribeirão Preto, SP, 14040-901, Brazil.|Instituto de Ciências da Saúde, Agrárias e Humanas do Centro Universitário do Planalto de Araxà (UNIARAXÀ), Araxà, MG, Brazil.
authors.forename: Tony Marcio
authors.initials: TM
authors.lastname: da Silva
authors.affiliation: Departamento de Bioquímica e Imunologia, FMRP, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.|Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Lucas Ferreira
authors.initials: LF
authors.lastname: Ribeiro
authors.affiliation: Departamento de Bioquímica e Biologia Tecidual, Instituto de Biologia, Universidade de Campinas, Campinas, SP, Brazil.
authors.forename: André Ricardo
authors.initials: AR
authors.lastname: de Lima Damasio
authors.affiliation: Empresa Brasileira de Pesquisa Agropecuária, EMBRAPA Instrumentação, São Carlos, SP, Brazil.
authors.forename: Cristiane Sanchez
authors.initials: CS
authors.lastname: Farinas
authors.affiliation: Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, Ribeirão Preto, SP, 14040-901, Brazil.
authors.forename: Aline Zorzetto Lopes
authors.initials: AZ
authors.lastname: Gonçalves
authors.affiliation: Departamento de Biotecnologia, Escola de Engenharia de Lorena, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Fernando
authors.initials: F
authors.lastname: Segato
authors.affiliation: Departamento de Botanica, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Marcos Silveira
authors.initials: MS
authors.lastname: Buckeridge
authors.affiliation: Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, Ribeirão Preto, SP, 14040-901, Brazil.
authors.forename: João Atilio
authors.initials: JA
authors.lastname: Jorge
authors.affiliation: Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, Ribeirão Preto, SP, 14040-901, Brazil. polizeli@ffclrp.usp.br.
authors.forename: Maria de Lourdes T M
authors.initials: ML
authors.lastname: Polizeli
mesh_terms: D001230:Aspergillus / Q000201:enzymology*; D019149:Bioreactors / Q000382:microbiology; D003001:Cloning, Molecular; D020536:Enzyme Activators / Q000032:analysis; D004791:Enzyme Inhibitors / Q000032:analysis; D004795:Enzyme Stability; D005285:Fermentation; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008031:Lignin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D029861:Tamarindus / Q000737:chemistry; D013696:Temperature
chemical_list: D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; C036909:lignocellulose; D008031:Lignin; D006026:Glycoside Hydrolases; C072818:xyloglucan endo(1-4)-beta-D-glucanase
keywords: Aspergillus terreus; Recombinant enzymes; SSF; SmF; Xyloglucanases