The Characterization of the Endoglucanase Cel12A from Gloeophyllum trabeum Reveals an Enzyme Highly Active on beta -Glucan: e108393

• Published in: PloS one Vol. 9; no. 9
• Main Authors: Miotto, Lis Schwartz, Rezende, Camila Alvesde, Bernardes, Amanda, Serpa, Viviane Isabel, Tsang, Adrian, Polikarpov, Igor
• Format: Journal Article
• Language: English
• Published: 01.09.2014
• Subjects: Aspergillus niger; Basidiomycetes; Gloeophyllum trabeum
• DOI: 10.1371/journal.pone.0108393

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- beta -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 beta -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 degree C on beta -glucan. Under these conditions specific activity was 239.2 plus or minus 9.1 U mg-1 and the half-life of the enzyme was 84.6 plus or minus 3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using beta -glucan as a substrate, the Km was 3.2 plus or minus 0.5 mg mL-1 and the Vmax was 0.41 plus or minus 0.02 mu mol 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.