Characterization of a chitinase and an endo-β-1,3-glucanase from Trichoderma harzianum Rifai T24 involved in control of the phytopathogen Sclerotium rolfsii

Abstract.

Of 24 Trichoderma isolates, T. harzianum Rifai (T24) showed a potential for control of the phytopathogenic basidiomycete Sclerotium rolfsii. When T24 was grown on different carbon sources, growth inhibition of S. rolfsii by the T24 culture filtrate correlated with the activity of extracellular chitinase and β-1,3-glucanase. The 43-kilodalton (kDa) chitinase and the 74-kDa β-1,3-glucanase were purified from the T24 culture filtrate in two and three steps, respectively, using ammonium sulphate precipitation followed by hydrophobic interaction chromatography (phenyl-Sepharose) and gel filtration (β-1,3-glucanase). K _m and K _cat were 3.8 g l^–1 and 0.71 s^–1 for the chitinase (chitin) and 1.1 g l^–1 and 52 s^–1 for the β-1,3-glucanase (laminarin). The chitinase showed higher activity on chitin than on less-acetylated substrate analogues (chitosan), while the β-1,3-glucanase was specific for β-1,3-linkages in polysaccharides. Both enzymes were stable at 30°C, while at 60°C the chitinase and the β-1,3-glucanase were rapidly inactivated, showing half-lives of 15 and 20 min, respectively. The enzymes inhibited growth of S. rolfsii in an additive manner showing a promising ED₅₀ (50% effective dose) value of 2.7 µg/ml.