Optimization of Chitinase Production by Bacillus sp. B26 and Its Antifungal Activity

This study was carried out on the screening of Bacillus species isolated from marine samples for the production of chitinase and identification of the selected strain. Then fermentation parameters influencing chitinase production by the selected strain were optimized and tested its antifungal activity. Out of 83 Bacillus isolates, 14 bacterial isolates found producing chitinase characterized by clear inhibition around bacterial colonies on the colloidal chitin agar plate. Based on the chitinolytic index, five isolates were selected and tested for their antagonistic activity against pathogenic fungi. The results showed that the Bacillus sp. B26 had an antagonistic effect on Alternaria alternata TISTR 3435, Fusarium solani TISTR 3436, Mucor ruber TISTR 3006, and Penicillium chrysogenum TISTR 3554 with the percentage inhibition of 74.51, 69.49, 30.95, and 46.51, respectively. Five isolates were then tested for their ability to produce chitinase in the colloidal chitin broth medium. It was found that the Bacillus sp. B26 produced chitinase showing inhibiting activity against Fusarium solani TISTR 3436 (44.5%) and Penicillium chrysogenum TISTR 3554 (34.76%). Finally, Bacillus sp. B26 was selected for the characterization and optimization process of enzyme production. Bacillus sp. B26 showed positive results for urease production, catalase test, starch hydrolysis, and casein hydrolysis but negative results with citrate utilization, oxidase test, and Voges-Proskauer reaction. It produced acids by fermentation of sugar such as arabinose, lactose, maltose, glucose, sucrose, galactose, mannitol, and fructose. Bacillus sp. B26 could grow in medium containing 0-5 % NaCl with optimum at 2% NaCl, pH 7-8, at 37°C. Based on the 16S rRNA gene sequence, Bacillus sp. B26 had sequence similarity (99%) with Bacillus paramycoides. The composition of an optimized medium for the production of chitinase by Bacillus sp. B26 was found to be 2% of squid pen powder, 0.5% of sodium nitrate as the best additional nitrogen source and 2% of NaCl in the basal medium containing 0.05% of yeast extract, 0.1% of (NH4)2SO4, 0.03% of MgSO4•7H2O and 0.136% of KH2PO4 with initial medium pH 7. Under the optimized conditions, Bacillus sp. B26 gave a maximum chitinase activity of 6.52 U/mL/h, which was achieved after 72 h of incubation. The yield of 2.83-fold was observed as compared to the unoptimized basal medium with squid pen powder as a substrate. Antifungal activity of the obtained crude chitinase was tested against several fungal strains. It was found that the crude chitinase proved to be more effective in inhibiting the mycelial growth of F. solani TISTR 3436 (83.39%) and P. chrysogenum TISTR 3554 (80.12%).