Screening Of Chitosanase-Producing Strain And Research On Immobilization Of Chitosanase

Four strains of producing chitosanase were isolated from marine sediments of Fujian Coastal. hrough the activity detection the strain which named M-2possessed the highest chitosanolytic activity among the isolated strains. The optimum fermenting conditions for the strain of M-2were determined as followings:chitosan1.0%,(NH4)2SO41.87%, pH6.5,4%Vaccination quantity,70mL liquid quantity in250mL triangular flask and shake cultivating for72h at speed of150r/min at30℃. The activity of chitosanase from strain of M-2in fermentation liquor could reach18U/mL under the best incubation condition. According to the morphologica observation and the sequencing analysis of26SrDNA, the strain of M-2belonged to the genus of Penicillium sp. It was named Penicilium sp. M-2.The purified enzyme displayed a significant band of protein by the SDS-PAGE analysis. The molecular masses of chitosanase from Penicillium sp.M-2was about40kD. The study on the enzyme properties of chitosanase from Penicillium sp.M-2showed the optimum pH was5.0. and the optimum temperature was55℃. When the ionic concentration was1x10-3mol/L, metal ions Na+, K+, Ca2+, Mg2+could accelerate the chitosanase activity, which was increased by4%,1%,2%,2%and metal ions Cu, Hg2+and Ag+could inhibite the activity, which was decreased by60%,65%and69%. There were different degradation of chitosanase to the different substrates and to the different degrees of deacetylation of substrates of chitosan. When water-solute chitosan as substrate the Kinetic parameters (Km) for enzymatic reaction was1.293g/L.Three different molecular weight of Chito-oligosacchasides were prepared by simulation of enzymatic hydrolysis and were applied to preliminary bacteriostatic experiment. Result showed that Chito-oligosacchaside with the concentration of4%and1215+20.0molecular weight had the best inhibition effect on Helminthosporium turcicum Pass and CoChliobolus sativus with inhibition ratio of79.3%and7%.The purified chitosanase was immobilized by chitosan which modified by Nano SiO2as immobilized carrier. Characterization of carrier was verified with Scanning Electron Microscopy (SEM), Infrared spectrum and X-ray diffraction. Immobilized chitosanase showed that the optimum temperature was60℃. The optimal pH for immobilized enzyme was7.0. The Km’for immobilized enzyme was10.64g/L. After the chitosanase immobilized, temperature, pH, and Km were increased. The immobilized chitosanase had strong operating stability, it could possess with70%of the original activity after ten times reused. The immobilized enzyme had a good low-temperature storage stability, under the conditions of4℃, the half-life of immobilized enzyme was about28days.