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

Author(s)

Miotto, Lis Schwartz ^[1] ; de Rezende, Camila Alves ^[2] ; Bernardes, Amanda ^[1] ; Serpa, Viviane Isabel ^[1] ; Tsang, Adrian ^[3] ; Polikarpov, Igor ^[1]

Affiliation

^[1] Univ Sao Paulo, Inst Fis Sao Carlos, Grp Biotecnol Mol, BR-05508 Sao Paulo - Brazil

^[2] Univ Estadual Campinas, Inst Quim, Campinas, SP - Brazil

^[3] Concordia Univ, Ctr Struct & Funct Genom, Montreal, PQ - Canada

• Document type: Journal article
• Source: PLoS One; v. 9, n. 9 SEP 24 2014.
• Web of Science Citations: 12

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-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 degrees C on beta-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 beta-glucan as a substrate, the K-m was 3.2 +/- 0.5 mg mL(-1) and the V-max was 0.41 +/- 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. (AU)

• FAPESP's process: 09/52840-7 - Center of Biological and Industrial Processes for Biofuels - CeProBIO
• Grantee: Igor Polikarpov
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Thematic Grants
• FAPESP's process: 08/56255-9 - Structure and function of enzymes and auxiliary proteins from Trichoderma, active in cell-wall hydrolysis
• Grantee: Igor Polikarpov
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Thematic Grants
• FAPESP's process: 12/22802-9 - Functional and Structural Studies of Carbohydrate Binding Modules of Glycosil Hydrolases
• Grantee: Amanda Bernardes Muniz
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 10/52362-5 - Targeted analysis of microbial lignocellulolytic secretomes: a new approach to enzyme discovery
• Grantee: Igor Polikarpov
• Support Opportunities: Regular Research Grants
• FAPESP's process: 09/08233-9 - Molecular, structural and functional studies of the Cel12A from Gloeophyllum trabeum, an endo-1,4-²-glucanase from the family 12 of glycosyde hidrolases
• Grantee: Lis Schwartz Miotto
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)