Purification and characterization of a β-glucanase produced by Trichoderma harzianum showing biocontrol potential

Abstract

A β-1,3-glucanase was produced by Trichoderma harzianum in cultures containing chitin as the sole substrate. Two proteins showing β-1,3-glucanase activity were purified to apparent homogeneity by hydrophobic chromatography. The molecular masses of these proteins were 29 and 36 kDa. The 36 kDa protein was further characterized. It was active on a broad pH range, and maximal activity was detected at pH 5.0. The optimum temperature of the 36 kDa β-1,3-glucanase was 50°C, but the purified enzyme was very sensitive to temperature. It lost about 60% or more of the activity after incubation for 30 min at 45, 50 and 60°C. The apparent KM and Vmax for hydrolysis of laminarin at pH 5.0 and 37°C, were 0.099 mg of reducing sugar/mL and 0.3 mg of reducing sugar/min.mL, respectively. The enzyme was insensitive to organic compound and metal ions, except for the ferric ion which inhibited about 100% of the original activity at the concentration of 1 mM. In contrast to other hydrolytic enzymes (a chitinase and a protease) produced by the same T. harzianum isolate (1051), the β-1,3-glucanase showed no effect on the cell wall of the phytopathogenic fungus Crinipellis perniciosa.