Production and characterization of hemicellulase activities from Trichoderma harzianum strain T4

Xylan and mannan are the major constituent groups of hemicellulose in the cell wall of higher plants. The mesophilic fungus Trichoderma harzianum strain T4 produces extracellular xylanase and mannanase activities when grown in the presence of oat (Avena sativa)-spelt xylan and wheat bran as the carbon sources respectively. After the growth procedure, the crude extracts were submitted to ultrafiltration in an Amicon system fitted with a 10 kDa-cut-off membrane. Mannanase activity was only detected in the concentrated sample, whereas xylanase was also found in the permeate after ultrafiltration. Xylanase from the concentrated sample showed highest activity at 40 degrees C and pH 5.0. Mannanase activity was optimal at 65 degrees C and pH 2.6. Xylanase was stable in the temperature range 40-70 degrees C, presenting full stability for at least 48 h. Xylanase retained 100% of its original activity after incubation for 48 h at 70 degrees C. Xylanase was also stable at pH 5.0 and 6.0 for 48 h. However, mannanase activity was markedly less stable. The enzyme lost 50% of its activity at 55 degrees C after 45 min, whereas at 60 degrees C its half-life was 20 min. The Michaelis-Menten constant K(m) and V(max) for mannanase and xylanase activities were also calculated. Xylanase had more affinity for soluble xylan, with K(m) and V(max) values of 1.61 mg/ml and 10.03 units/ml respectively. The K(m) and V(max) values for crude mannanase were 6.0 mg/ml and 20.1 units/ml respectively. Xylanase and mannanase were activated by dithiothreitol, L-cysteine and L-tryptophan. Xylanase was partially purified by gel-filtration (Sephadex G-50) and hydrophobic-interaction (Phenyl-Sepharose) chromatographies. The partially purified enzyme was stable over the pH range 5-7 and temperature range of 40-60 degrees C. It was more active on soluble oat-spelt xylan and was activated by dithiothreitol, L-cysteine and L-tryptophan.