Study On Isolation Of Marine Chitinase-Producing Strains, Purification And Characterization Of The Chitinase

Chitin is the most abundant polysaccharides in marine and the second large renewable resources on the earth. Chitinase are glycosyl hydrolases, which can catalyze the hydrolyzation of chitin. The degradation products of chitin including chitooligosaccharides and N-acetyl D-glucosamines have many kinds of bioactivities, such as the antifungal activity, antitumor activity and enhancing organic immunity activity. They also have important application values on food, medical, cosmetics and prevention of plant pests. Using chitinase to prepare chitooligosaccharides has caused widely concerning in recent years. It was showed that many chitinases isolated from land microorganisms have poor activity. Since the ocean amassed many chitins, there may be many microbes which could use it. In vies of the marine microorganisms possess outstanding indurations for alkali, salt and cold, it is significant to extract chitinase from marine microorganisms.Samples from the North Sea were collected, and 52 strains which could produce chitinase were selected by using transparent ring method. The chitinase activities of 10 strains from the selected strains were measured by DNS method. The Y1 strain isolated from shell surface showed a high enzyme activity. The 16S rDNA of Y1 was cloned and sequenced.The result showed that Y1 is serratia marcescens.The culture condition of Y1 was studied. The results showed that the optimized protocol for strain Y1 to produce chitinase involves 0.8% colloid chitin, 0.5%peptone, 1.5% sodium chloride, 100 ml liquid in 250 ml triangle bottled, initial pH 6.0, the initial inoculation volume is 8%, the cultural time is 24 hours, the culture temperature is 30℃and the shaking cultivation is 150 rpms.The chitinase was purified from the fermented broth using an ammonium sulfate precipitate, ion-exchange chromatography and gel filtering chromatography method. The purified chitinase was examined by SDS-PAGE and a 60 KDa strip was obtained. The enzymatic properties of the chitinase were investigated. The results showed that the most suitable reaction temperature of the chitinase is 45℃, the stable temperature is under the 35℃, the most suitable pH is 8.5, Ba 2+ has apparent accelerate the enzyme activity and the most suitable colloid chitin concentration is 0.5%. In the optimized condition, the enzyme activity could reach 6.28 U/mg. The hydrolysis products were analyzed by TLC,and the results showed that the main product is NAG₄ .