Department of Agricultural Chemistry, Institute of Environmentally-Friendly Agriculture (IEFA), College of Agricultural and Life Sciences, Chonnam National University, Gwangju, 61186, Republic of Korea.
Department of Agricultural Chemistry, Institute of Environmentally-Friendly Agriculture (IEFA), College of Agricultural and Life Sciences, Chonnam National University, Gwangju, 61186, Republic of Korea.
Microb Pathog. 2019 Apr;129:277-283. doi: 10.1016/j.micpath.2019.02.026. Epub 2019 Feb 27.
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.
为了研究壳聚糖酶活性的温度要求，以及酶诱导壳聚糖低聚物水解产生的降解模式，将 Pedobacter sp. PR-M6 接种到 0.5%胶体壳聚糖培养基琼脂平板上。在培养的最初 2 天，30°C 时细胞生长高于 20°C。在这两种温度下，第 1 天的蛋白质含量迅速增加，然后在 20°C 下缓慢增加，在接下来的 5 天培养期间在 30°C 下缓慢下降。为了表征电泳模式，将 Pedobacter sp. PR-M6 在 20°C 和 30°C 下培养 1%粉末壳聚糖培养基 5 天，然后进行 SDS-PAGE 分析。在 20°C 和 30°C 下，都可以看到 4 条带，对应于 ct1（25 kDa）、ct2（17 kDa）、ct3（15 kDa）和 ct4（14 kDa）。壳聚糖酶活性的最佳条件是 Pedobacter sp. PR-M6 产生的 60°C 和 1.81 酶单位/mg 蛋白。在 SDS-PAGE 凝胶中，两种主要同工酶带（ct3 和 ct4）在 50°C 时表现出最强的壳聚糖酶活性。在 60°C 孵育 1 小时后，用薄层层析法研究（GlcN）-（GlcN）底物产生的反应产物。低分子量壳聚糖和寡壳聚糖（LCOC）和可溶性壳聚糖对 A. brassicicola、B. cinerea、F. solani 和 R. solani 具有抗真菌活性。LCOC 表现出比可溶性壳聚糖更高的抗真菌活性。此外，LCOC 处理（500 ppm 和 1000 ppm）抑制了 A. brassicicola 分生孢子的萌发。
