title: The characterization of the endoglucanase Cel12A from Gloeophyllum trabeum reveals an enzyme highly active on β-glucan.
abstract: The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-β-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on β-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50 °C on β-glucan. Under these conditions specific activity was 239.2 ± 9.1 U mg(-1) and the half-life of the enzyme was 84.6 ± 3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using β-glucan as a substrate, the Km was 3.2 ± 0.5 mg mL(-1) and the Vmax was 0.41 ± 0.02 µmol min(-1). Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process.
authors.affiliation: Grupo de Biotecnologia Molecular, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil.
authors.forename: Lis Schwartz
authors.initials: LS
authors.lastname: Miotto
authors.affiliation: Instituto de Química, Universidade Estadual de Campinas, Campinas, SP, Brazil.
authors.forename: Camila Alves
authors.initials: CA
authors.lastname: de Rezende
authors.affiliation: Grupo de Biotecnologia Molecular, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil.
authors.forename: Amanda
authors.initials: A
authors.lastname: Bernardes
authors.affiliation: Grupo de Biotecnologia Molecular, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil.
authors.forename: Viviane Isabel
authors.initials: VI
authors.lastname: Serpa
authors.affiliation: Centre for Structural and Functional Genomics, Concordia University, Montreal, QC, Canada.
authors.forename: Adrian
authors.initials: A
authors.lastname: Tsang
authors.affiliation: Grupo de Biotecnologia Molecular, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil.
authors.forename: Igor
authors.initials: I
authors.lastname: Polikarpov
mesh_terms: D001234:Aspergillus niger / Q000235:genetics; D001487:Basidiomycota / Q000201:enzymology* / Q000235:genetics; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods; D008855:Microscopy, Electron, Scanning / Q000379:methods; D011994:Recombinant Proteins / Q000302:isolation & purification; D013379:Substrate Specificity; D013696:Temperature; D047071:beta-Glucans / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D047071:beta-Glucans; D002480:Cellulase