Studies On The Preparation And Adsorption Of The Tetrodotoxin-Specific Affinity Microspheres

A new method of synthesis microsphere was prepared by the reaction of 25% glutaraldehyde with chitosan in acetic acid solution.The effect factors were discussed in detail. The concentration of chitosan and connector are important factors for the quality of synthetic chitosan microspheres. The results showed that the reaction was carried out at 40℃⁶0℃, and also proved that the 3.0% of chitosan in acetic acid solution and the higher connector volume are appropriate conditions for the output of synthetic chitosan microspheres . The synthetic chitosan microspheres are insoluble in many kinds of solvents and are stability for heat, diluted acid, diluted base and organic solvent. The product has a great improvement performance of easy regeneration.The crude extraction of TTX was obtained by the aceticacidethanol solution from the ovary of Fugu oblongus,then defatted with chloroform. The crude extract of TTX was absorbed by activated charcoal and futher purified by the affinity adsorption chitosan microspheres.The adsorption capability of TTX was measured with the mouse bioassay. The adsorptive capacity of the affinity adsorption chitosan microspheres reached about 10μgTTX/g(wet),the recovery rate was about 51% and 80%,and the protein concentration was lowered about 3 and 4 times ,compared with the activated charcoal .The purification efficiency and the recovery rate of the aflEinity adsorption chitosan microspheres are higher than of the activated charcoal.The novel chitosan microspheres are also used to adsorpt the Cu²⁺in solution. The Cu²⁺concentration and the adsorption time, pH, tempreture, dosage of chitosan microspheres on the adsorption effects were investigated.The results showed that the chitosan microspheres had higher adsorption properties for Cu²⁺,the adsorption capacity reached 178mg/g, and can be reused several times after simple regeneration.The lysozyme purification from egg white was carried out and compared with the three adsorbents: chitosan microspheres , D724 and D155 resinas on a column.The most of proteins are removed from extraction solution by heat-denaturati on isoelectric point precipitation. Regulating the pH 3.5 with HAC ,then the lysozyme fliud was further desalted. The collected activated fraction was detected by SDS-PAGE. The experimental results showed that the chitosan microspheres absorbtion is more rapid and effective and the proteins were almost separated. The lysozyme activity was 7793U/mg protein,its purity was raised 36.2 folds by using bioactivity assay. Comparing with the D724 and D155 resinas ion exchange adsorption, this chitosan microspheres can reduce denatured albumin and decrease impurity of residue egg white that can be reused in some way ,so the lysozyme production cost will be lowered.