Purification And Immobilization Of Chitosanase From Penicillium Sp.ZDZ1

Chitosanase was isolated and purified from the supernatant of fermentation broth ofPenicillium sp. The purification procedure involved ammonium sulfate fraction, gel filtration chromatography on Sephadex G-100 column. The optimal temperature and pH were 50癈 and 5.0 respectively. Metal ions like Mg^and Ca2+ could speed the enzymatic reaction, but heavy metal ions like Zn2+, Ni2^ and Cu2+ inhibited the enzyme strongly. The chitosanase showed substrate specificity. Kinetic parameters Km was 2.60Ig/L.Chitosanase was immobilized on chitin with glutaraldehyde by cross-link reaction, the immobilization conditions and characterization of the immobilized enzyme were studied. The optimal conditions for immobilization were as follows: O.lg chitin was treated with 5mL 5% solution of glutaraldehyde, then 2mg chitosanase was immobilized on the carrier, the optimal reaction time was 8 hr. Optimal temperature and pH for the immobilized enzyme was 60 癈 and 4.0 respectively. Kinetic parameter Km was 27.01g/L. The immobilized enzyme showed good temperature stability, and its operation and storage stability were also very good.Chitooligosaccharides were prepared by enzymatic hydrolysis with the chitosanase, the optimal conditions for hydrolysis was determined. At the same time, hydrolysis was performed with a substrate concentration of 3% (w/v). then the components of the hydrolysis product were isolated by ion-exchange chromatography, and the Chitooligosaccharides with DP of 2-9 were obtained. The average molecular weight of hydrolysis product with different hydrolysis time were estimated as 1746, 3295, 3482 respectively by vapor pressure osmometer (VPO).