Preparation Of High Performance Chitosan Based Vector Mediated Gene Delivery

Penicillium sp.ZD-Z1 screened out from the soil was applied to submerged fermentation to produce chitosanase. After fermented for 65-70 hours, fermentation liquid was managed by follow steps: ultrafiltration membrane with MWCo 2kDa and 10kDa , precipitation with 95% cold ethanol (3:1 V/V) and freezes centrifugal at the condition of 5℃ and 6000r/min. At last, two chitosanases ChA and ChB were isolated by IEC method. Degradabilities of two enzymes on chitosan were further studied, as the results of viscosity change and paper chromatogram shows, ChA was an Endo-chitosanase and ChB was an exo-chitosanase. The kinetic parameters were 5.64g/L and 2.47g/L respectively. Two chitosanses had good stability at the conserve condition of 4℃ and HAc-NaAc buffer with pH 4.4.ChA was used to prepare low molecular weight chitosan in the following experiment, using MHS equation η =1.02 × 10^-4M_w^1.27 to control the change of molecular weight. The optimal conditions of preparation of LMWC were as follows: concentration of chitosan 4%, ChA dosage 0.01U/mL, temperature and pH was 30°C and 5.0.Endo-Chitosanase ChA was immobilized on DEAE cellulose with glutaraldehyde by crossing-linking reaction. In this part, immobilization condition and the characterization of immobilized enzyme were discussed. The optimal condition determined as follows: 0.1g DEAE cellulose was treated with 5ml 5% solution of glutaraldehyde, then 0.3mg ChA with specific activity 32.8U/mg was immobilized on the carrier for 6 hours at 4 ℃. The optimal temperature and pH was 65 ℃ and 4.0. The immobilized enzyme showed good operation stability, its enzyme activity loss was less than 10% after 10 times reaction. The enzyme also showed good storage stability. Finally. The degradability of enzyme on 1% chitosan solution was tested in a reactor (Φ14.5cm×8.5cm) . As the result showed, when flow velocity was at 5mL/min, chitosan could be degraded effectively.Anion exchange resin ZGA454 screened out from four resins was used to desalt lactic acid ion, content of which in chitosan solution was comparatively high. After treated with ZGA454 resin , conductance of the solution decreased 87.7% and pH increased by 7.2 from 5.2. Solid chitosan product was prepared by spray dryness, conditions used in dryness were as follows: feed-in velocity 15%, temperature at entrance 155℃ or 160℃, spray airflow 50cm. The molecular weight of dryness product S1,S2 were 50377. 1062 by HPLC. Patentiometric Titration was applied todetermined the DDA of dryness product, as the result showed, DDA of product prepared by ChA was 84.85% and product separated from fermentation liquid was 76.82%.