| Chitosanase is enzyme, which hydrolyze chitosan molecules, a deacetylated derivative of chitin, to give chitosan oligosaccharides. The chitosan oligosaccharides have diverse physiological functio- ns in certain conditions and were widely used in the fields of agricuture, industry and environment protection. As an elicitor, chitosan oligomers has an important roles in the interaction between path- ogenic fungi and its host. Chitosan oligomers could inhibit growth of many pathogenic fungi and induce the production of pathogenesis-related proteins such asβ-1,3-glucanase, chitinase and chitos- anase etc in plants.Chitosanase showed activity in both antifungal properties and induction of plant resisitance.The main objective of this study were: to screen and identify microorganism that can pro- duce chitosanase; study on the optimal conditions of chitosanase formation of chitosanase producing strains; to purify and characterize chitosanase from chitosanase producing strains and study of the b- iocontrol efficacy and mechnisms of chitosanase producing strains and chitosanase against the disea- se of Rhizoctonia damp-off of mungbean and cucumber.Fifteen strains capable of utilizing chitosan as a sole carbon source and producing chitosanase were screened by the method of brilliant clear circle from 100 strains of plant endophytic bacteria.A strain designated K7, with the most chitosansae activity among the 15 strains previously isolated was obtained by liquid culture.The chitosanase activity of K7 was 1.17μmol/mL. The strain K7 was ide- ntified as Bacillus subtilis,according to the morphological characters such as colony,cell shape, stai- ning reactions and other biological, physiological and biochemical characteristics.Fermentation process for chitosanase production from Bacillus subtilis K7 was optimized. The optimum cultural conditions were determined as followings: Bacillus subtilis K7 was cultivated by 100 mL LB liquid medium in a 250 mL flask and cultivated at 30℃, shaked with 200 rpm for 24 h. One milliliter of the precultured broth was inoculated into 100mL same liquid medium in a 250 mL flask and continued cultivated at 30℃, 200 rpm for 30 h. The results showed that under this conditi- ons, the chitosanase activity from K7 was improved from 1.17μmol/mL to 17.41μmol/mL. The stu- dy of flask culture showed that the chitosanase of K7 can be produced without chitosan, which sugg- ested that the production of chitosanase was constitutive.The chitosanase was purified from the centrifuged supernatant of fermentation broth of Bacillus subtilis K7.Crude enzyme was salted out by adding ammonium sulfate to make 80% saturation. Aft- er precipitation ,the emzyme solution was purified by Sephadex G-100 colum chromatography and the molecular weight was determined with SDS-PAGE. The results indicated the molecular weight of one component of chitosanase was 40~43KD. The enzymatic activities was studied and the results showed that the purified chitosanase could hydrolyze colloidal chitosan and carboxymethyl cellulose, however, .it could not hydrolyze colloidal chitin. The optimum conditions of chitosan (DD 90%) hydrolyzed by chitosanase from Bacillus subtilis K7 were pH 5.0, 51℃,and 15 min reaction time.Biological control against the plant disease of Rhizoctonia dump-off with K7 strains and the chit- osanase from it was studied, the results showed that both bacteria and chitosanase could inhibit the growth of the pathogenic Rhizoctonia solani in vitro and could decrease the incidence and severity of the disease under two crops of mungbean and cucumber.The results also showed that the control efficacy to the disease was related to the concentrations of the enzymes. According to the results pre- viously, it suggested that the strain K7 was a potential biocontrol strain against the disease of dump- off and the chitosanase play a key role in the model of action in the biocontrol strain.
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