The Study On Purification And Activity Of Bioactive Substances With The Inhibition Of Chitinase

Chitinases play an important role in the growth and development of a variety of fungi and insects. Moreover, chitinases are closely related to mammalian asthma, atherosclerosis and other diseases according to recent studies. Taking chitinase as a target,the development of new bio-pesticide, antifungal agents and related drugs caused widespread attention at home and abroad.Based on the established strain library with chitinase inhibiting activity, Bombyx mori ehitinase was used as the target to screen strains able to inhibit chitinase by transparent ring plate screening and DNS colorimetric screening method. The strain A0101 and B2-9 with high inhibitory specificity were obtained. The purification and characterization of the chitinase inhibitors produced by A0101 and B2-9 were researched.According to the growth curve, the logarithmic phase of the actinomycete A0101 was at 2-3d, and the optimal time when the strain produced chitinase inhibitors was about at 6d. According to activity tracking, the separation and purification of fermentation products of strain A0101 were subjected to alcohol precipitation, dialysis, silica gel column chromatography and sephadexG-15 gel column chromatography, and the pure bioactive material was obtained. This bioactive material exhibited a single HPLC peak with a retention time of 4.1 min. Its inhibition rate on chitinase was evaluated by DNS colorimetric method to be 80.2%. Moreover, it was proved to be a multiple hydroxyl carbohydrate compound according to IR, H-NMR and Mass analyses. Under optimum fermentation conditions, a max inhibitor activity was observed after 6 days cultivation at 29℃,250r/min with 4% inoculum’s sizes containing soluble starch as a carbon source and KNO3 as a nitrogen source.The properties of the ehitinase inhibitor produced by strain A0101 are as follows:in the pH2-11 range, the temperature between 40-90℃, the chitinase inhibitor had high tolerance of temperature and pH. Cu2+ and Zn2+ can promote inhibitor activity, Mn2+ has blocked it and there were no effect by other ions basically. According to the antimicrobial spectrum experient:the inhibitor is a kind of broad spectrum bacteriostats. having certain inhibitory effect on yeast, fungus, Escherichia coli.The strain B2-9 was a gram-positive rod-shaped bacteria. According to its growth curve, the logarithmic phase was at 3-9h, and the optimal time when the strain produced chitinase inhibitors was about at 15-21h. The fermentation broth of strain B2-9 was separated by organic solvent extraction, DEAE -cellulose 52 column chromatography, cation exchange column chromatography, semi preparative Ci8 column chromatography, sephadexG-25 column chromatography. A compound with a purity of about 89.6% was detected at 5.9min by HPLC. The substance had high tolerance of temperature and pH.The active strain with chitinase inhibiting activity screened by this research and the research results of related properties provide the basis for further development and the creation of biological pesticides of chitinase inhibitors of microbial origin.