Study On Optimal Fermentation Conditions Of A Chitosanase-producing Bacterium, Purification And Characerization Of The Chitosanase

Chitosanase which catalyze the hydrolysis of glycosidic bonds of chitosan, could hydrolyze chitosan into functional chitooligosaccharides and glusosamine. Our research focuses on finding new source of chitosanase from microorganisms, and providing technical support on enzymic preparation of chitooligosaccharides.A strain with chitosanase activity was isolated from soil samples. The optimal compositions of the medium for P003 strain producing chitosanase are as follows(%, w/v): powder chitosan 1.0, glucose 0.1, (NH₄)₂SO₄ 2.0, yeast extract 0.3, K₂HPO₄0.07, KH₂PO₄0.03, NaCl 0.5, MgSO₄·7H₂O 0.05, the initial pH5.0. The optimal cultivation conditions for P003 producing chitosanase are 500mL flask containing 150mL medium, 2.0% (v/v) amount of inoculation, 32℃ incubating temperature and shaking cultivation at 150rpm for 96 hours. The chitosanase activity of fermented broth can reach 108umol/mL·min when P003 is incubated under aforementioned conditions.The chitosanase of the fermented broth is extracted by ammonium sulfate precipitation (70% saturation) , and purified by Q-Sepharose Fast Flow colum and Sephadex G-75 colum, SDS-PAGE shows a single band for the purified chitosanase and the molecular weight is estimated to be 30.5kD. The optimal temperature for activity for hydrolysis of the chitosanase is 45℃ and the optimal pH is 5.8, respectively; The chitosanase activity is stable in the pH range of 5.0-11.0 and the temperature under 45℃; Mn²⁺ can enhance the enzyme activity significantly, whereas Hg²⁺, Cd²⁺, Fe³⁺, Cu²⁺and Ag⁺ can inhibit the enzyme activity; EDTA, GluNH₂·HCl and SDS inhibit the enzyme activity also, whereas P-mercaptoethanol has no effect on it; the enzyme show activity for colloidal chitosan, chitosan and colloidal chitin, and the hydrolysis with higher...