Identification, Fermentation And Characterization Of A Marine Bacterium Y-116 Producing Chitosanase

Application prospect of chitosanase has been considered in the field of medicine、food、cosmetics、functional materials、textile industry and feed, has a great potential in industrial application. A strain Y-116 of bacteria producing chitosanase was isolated from Yellow Sea water（stored by lab）. After purification culture, the strain was identified by physiological, biochemical and genetic characterization as Bacillus sp. In order to improve the yield of the chitosanase we optimized the fermentation conditions of marine Y-116. then the protease was preliminary purified and characterized,to study the enzymatic properties and stability, with a view to lay the foundation for future research, development and application.The paper chooses chitosanase as the research object, describes the producing chitosanase strain screening method, chitosanase fermentation optimization of culture conditions was discussed, measures to improve the yield of enzyme, and study the chitosanase separation and purification method, as well as the properties of the enzyme.Using the clarity circle method and shake fermention,a marine bacterium Y-116 producing chitosanase was screened. According to morphology and biochemistry characteristic result, and 16 S r DNA sequencing analyses,Y-116 is identified as a Bacillus sp. The strain is gram-positive bacteria;The spore which is at the middle is ellipse or columnar; The bacterial colony is round, protuberance and milk white or light yellow on the agar culture-medium. The surface of the bacterial colony is smooth.The culture medium and culture conditions of strain Y-116 were optimized in order to improve the yield of chitosanase by one-single factor and response surface methodology. The single factor was used to screen these eight factors: the nitrogen source, carbon source, initial p H, Mg²⁺, Na Cl, Yeast extract powder, inoculation, fermentation temperature and fermentation time. The best result carried out of each single factor was as follows: lactose 35 g/L, bean cake powder 30 g/L, initial p H 5.0, Mg²⁺ 2 mmol/L, Na Cl 5 g/L, Yeast extract powder5 g/L, inoculation 5%, the culture temperature at 30℃ and the culture time at 96 h. Secondly, those main variables were valuated through the Plackett-Burman design method. Results showed bean cake powder, initial p H and Mg²⁺ that were the significantly affecting variables. Then, the central composite design and the analysis by Design-Expert 8.05 software were adopted to determine the optimal level of the three main factors. The most suitable variables were identified as follows: bean cake powder 39.44 g/L, p H 5.94, Mg²⁺ 2.05 mmol/L. Comparison with that before optimization, the productivity of chitosanase increased 5 times under the optimum conditions.Using ammonium sulfate precipitation and ethanol precipitation separate the enzyme,then enzymatic and stability were studied.The result showed:The enzyme had temperature and p H optima of 55 ℃ and 5.5, of which condition the stability of the chitosanase is good. Li+、Ca²⁺、Al3+can inhibit the enzyme activity, Co²⁺、Zn²⁺、Fe²⁺ 、Cu²⁺have stronger inhibitory effect on the chitosanase among these metal ions, Mg²⁺、Mn²⁺、Ba²⁺can promote the enzyme activity.