| Summary: | 碩士 === 朝陽科技大學 === 應用化學系碩士班 === 96 === Verticillium lecanii (Zimm.) Viegas, an entompathgenic fungus, is a potential microbial biocontrol agent for aphid and Bemisia argentifolii. It is also the major chitinase production organism. In this study, a 5-L stirred-tank bioreactor was employed to produced the chitinase from Verticillium lecanii. Purification was achieved by means of 80% saturation of ammonium sulphate precipitation, DEAE-Sepharose CL-6B ion-exchange chromatography and subsequent extraction from a Sephadex G-100 gel filtration. Through these steps, the enzyme was purified 10.5-fold with a recovery of 10.35%. The total activity of the enzyme was counted to be 2.83 U. The molecular weight of chitinase was estimated to be 39 kDa as judged by SDS-PAGE and active stain. The pI of 4 was found as determined from 2DE. The magnetic nanoparticles were prepared by co-precipitating Fe2+ and Fe3+ ions supplemented with Triton X-100 surfactant. The minimal particle diameter of 2.74 μm was obtained. Almost 83% chitinase was immobilized while using 200 mg magnetic particles and 2% EDC was added at pH 5.0 for 30 mins. Over 50% immobilized efficiency was retained after the magnetic nanoparticles had been reused for 4 times. The immobilized enzyme showed a better thermal stability, the 60% relative activity still preserved at 70°C. No much difference was found in the optimal pH (pH 5) for both free and immobilized enzymes. The shelf-life for free enzyme was about 5 weeks, whereas 3 months for immobilized enzyme was achieved at 4°C.
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