生物化学

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 and 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 N-acetylglucosamine 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.