Quantification of Chitinase Activity in Fusarium oxysporum

Carmen Ruiz-Roldan, M. Roncero
2016 Bio-protocol  
Fungal morphogenetic development requires modification and plasticity of the cell wall, which implies synthesis and remodelling of its components, including chitin and glucan. Thus chitinase and glucanase activities are crucial for cell-wall biogenesis and cell division. Quantification of chitinase activity might be useful to identify structural defects that could negatively influence growth and morphogenesis of some filamentous fungi like Fusarium oxysporum, which produces both intracellular
more » ... d secreted chitinases. The chitinolytic enzymes are categorized based on their enzymatic action on chitin substrates. Endochitinases are defined as the enzymes catalyzing the random cleavage at internal points in the chitin chain. Exochitinases catalyze the progressive release of acetylchitobiose or Nacetylglucosamine from the non-reducing end of chitin, and thus, are referred to as chitobiosidase and β-N-acetylglucosaminidase, respectively. Here we describe a simple method to easily purify chitinases in order to compare both endo-and exo-chitinase activity of different F. oxysporum strains. The protocol can be adapted to any fungal species. Materials and Reagents 1. Monodur nylon filters 15 µm diameter (Filtravibracion S.L., catalog number: Nylon-15) 2. Sterile plastic funnels (80 mm diameter) (Tecnylab, catalog number: 45000150) 3. Eppendorf tubes 4. Sterile spatula (Instrumentación Científica, S.L., catalog number: 2004NE8044) 5. Microtiter plates (Thermo Fisher Scientific, catalog number: 2205) 6. Dialysis membranes, cut-off 12 kDa (Sigma-Aldrich, catalog number: D9527-100FT) 7. Fusarium oxysporum f.sp. lycopersici microconida suspensions from wild type (J. Tello, Universidad de Almeria, Spain), the Δcon7-1 and the cΔcon7-1 mutant strains (Ruiz-Roldan et al., 2015) 8. Glycerol (Merck, catalog number: 104092) 9. Potato dextrose broth medium (PDB) (Scharlau, catalog number: 01483) 10. Chitin (Sigma-Aldrich, catalog number: C6137) 11. Bio-Rad protein assay Dye reagent concentrate (Bio-Rad Laboratories, catalog number: 500-0006) 12. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number
doi:10.21769/bioprotoc.1882 fatcat:e2sdrwkvrjeyvee4ndoucrmjtu