Characterization and antifungal activity of chitosanase produced by Pedobacter sp. PR-M6.

To investigate the temperature requirements of chitosanase activity, as well as the degradation patterns generated by enzyme-induced chitosan oligomer hydrolysis, Pedobacter sp. PR-M6 was inoculated onto 0.5% colloidal chitosan medium agar plates. Cell growth was higher at 30 °C than at 20 °C during the initial 2 days of incubation. The protein content rapidly increased on day 1 at both temperatures and then it slowly increased at 20 °C and slowly decreased at 30 °C during the following 5 days of incubation. In order to characterize the electrophoretic pattern, Pedobacter sp. PR-M6 was cultured in 1% powder chitosan medium at 20 °C and 30 °C for 5 days after incubation and analyzed by SDS-PAGE. Four bands were visible, corresponding to ct1 (25 kDa), ct2 (17 kDa), ct3 (15 kDa), and ct4 (14 kDa), at both 20 °C and 30 °C. The optimal conditions for the activity of chitosanase produced from Pedobacter sp. PR-M6 were 60 °C and 1.81 enzyme units/mg protein. Two major isozyme bands (ct3 and ct4) exhibited their strongest chitosanase activity at 50 °C in SDS-PAGE gel. The reaction products generated from (GlcN)-(GlcN) substrates at 60 °C after a 1 h incubation were investigated by thin-layer chromatography. Low-molecular weight chitosan and oligochitosan (LCOC) and soluble chitosan showed antifungal activity against A. brassicicola, B. cinerea, F. solani, and R. solani. LCOC exhibited higher antifungal activity than soluble chitosan. Moreover, LCOC treatments (500 ppm and 1000 ppm) inhibited conidia germination in A. brassicicola.