The characterization of the endoglucanase Cel12A from Gleophyllum trabeum reveals an enzyme highly active on β-glucan
Monday, April 28, 2014
Exhibit/Poster Hall, lower level (Hilton Clearwater Beach)
Lis Schwartz Miotto1, Camila A. Rezende2, Amamda Bernardes1, Viviane Isabel Serpa1, Adrian Tsang3 and Igor Polikarpov1, (1)Molecular Biology, University of Sao Paulo, Sao Carlos, Brazil, (2)Institute of Chemistry, University of Campinas, Campinas, Brazil, (3)Centre for Structural and Functional Genomics, Concordia University, Canada
Endoglucanases are important enzymes applied to the conversion of biomass aiming for the 2nd generation biofuel production. The extracellular Cel12A is one of the few endo-1,4-β-glucanase produced by Gloeophyllum trabeum, a basidiomycete fungus which causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. 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 Cel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A shows 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 are, respectively, 4.5 and 50℃ on β-glucan. Under these conditions specific activity is 239.2 ± 9.1 U mg-1 and the half-life of the enzyme is 84.6 ± 3.5 hours. Thermofluor studies revealed that the enzyme is most thermal stable at pH 3. Using β-glucan as a substrate, the Km is 3.2 ± 0.5 mg mL-1 and the Vmax is 0.41 ± 0.02 µmol min-1. Analysis of the effects of Cel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process.