Study On Isolation And Fermentation Conditions Of A Chitinase-producing Strain HD001, Purification And Characerization Of The Chitinase

Chitin, aβ-1,4-linked biopolymer of N-acetylucosamine, is the second main natural resources and the component of fungal cell walls and peritrophic membrane of insects. Decomposition product of chitin has various application. Chitinase can catalyse the reaction of hydrolyzing chitin into N-acetylglucosamine(GlcNac) and (GlcNac)n. Chitinase widely exists in animals, plants and fungi. Therefore, chitinase is important in a wide variety of biological and biotechnological processes, agricultural and industrial production, natural resources utilization, biological control, environmental protection, food and medical exploitation. Study on chitinase from microbe is important in finding new sources of chitinase.With the purposes of screening and selecting an effectively chitinase-producing srtain,a strain HD001 with activity of chitinase is isolated from marine soil samples, which can be induced high enzyme activity of chitinase by chitin.The optimal compositions of the medium for strain HD001 to produce chitinase are as follows (%, w/v): (NH4)2SO4 2.0, NaCl 0.5, K2HPO40.07, KH2PO40.03, MgSO4·7H2O 0.05, glucose 0.1, yeast extract 0.3, powder chitin 1.5. The optimal cultivation conditions for HD001 producing chitinase are 500mL flask containing 150mL medium, 1.0%(v/v) amount of inoculation, 30℃incubating temperature and shaking cultivation at 160r/min for 120h in the liquid medium with initial pH 6.0. The maximal chitinase activity of fermented liquid can reach 1.7U/mL when strain HD001 is incubated under aforementioned conditions. The activity of the chitinase in this experiment is higher than that of others reported.The fermented liquid of strain HD001 is extracted by ammonium sulfate precipitation (30%⁹0% saturation) after procedure of centrifugal effect. Then the crude chitinase is purified by ion-exchange chromatography (DEAE-cellulose column) and gel filtration (Sephacryl S-100 column). SDS-PAGE shows a single band for the purified chitinase and the molecular weight is estimated to be about 85.7 kDa.The optimal temperature for hydrolysis of chitinase is 50℃and the optimal pH is 5.6, respectively. The chitinase activity is stable in the pH range of 5.5⁷.0 and the temperature under 60℃. It remains activity if it is put at 25℃for 24 hour. The enzyme activity is slightly activated by Mg²⁺, whereas Zn²⁺, Fe3+, Cu²⁺, Fe²⁺, Mn²⁺ can inhibit the enzyme activity, especially Mn²⁺. The kinetic parameter Km was 1.931mg/mL and Vmax was 2.381μmol/min·mL.