Purification, characterization and synergistic activity of β-1,3-glucanase and antibiotic extract from an antagonistic Bacillus subtilis NSRS 89-24 against rice blast and sheath blight

Antifungal compounds in the culture filtrate from Bacillus subtilis NSRS 89-24 that inhibited the growth of Pyricularia grisea and Rhizoctonia solani were mainly heat stable as the filter sterilized culture filtrate showed higher activity than an autoclaved one. The heat stable and labile components were due to an antibiotic and a β-1,3-glucanase, respectively. This β-1,3-glucanase was purified and characterized. Glucanase activity in the culture medium of B. subtilis NSRS 89-24 was inducible in the presence of 0.3% chitin, reaching a maximum on day 5. After purification, activity was associated with a protein of molecular mass of approximately 95.5 kDa by both gel filtration and native PAGE. Two major bands of Mr 64.6 and 32.4 kDa were revealed by SDS–PAGE. The enzyme had a Km of 0.9 mg/ml, and Vmax of 0.11 U, the optimal pH was 6.5–9.5 and was stable up to 50 °C. Both the pure enzyme and the antibiotic extract from the culture filtrate of the B. subtilis separately inhibited R. solani and P. grisea with MIC values of 12.5 and 6.25 mU/ml and 3.13 and 1.56 μg/ml, respectively. The glucanase enzyme in combination with the antibiotic showed a strong synergistic inhibitory effect on the hyphal growth of both fungi.