Preparation And Application Of Immobilized Metal Affinity Carrier

Twelve kinds of agar powders were used as matrices to prepare the immobilized metal affinity supports: Agar powders were rinsed through pH4.0, 0.04 M sodium acetic acid buffer and pH 9.0, 0.05 M sodium phosphate buffer. The rinsed agar was activated by epichlorohydrin and modified with imidodiacetic acid, then chelated with metal ion. Using the poly-his-tagged GL-7-ACA acylase expressed in Escherichia coli as a model enzyme, the one which exhibiting the highest immobilized enzyme activity was selected to be used in the following experiment. For studying poly-his-tagged GL-7-ACA acylase adsorption capacity on IMAC, factors such as chelator surface density, chelating metal ion, sodium chloride concentration were investigated. The optimal conditions were found at 15℃, 21 μmol Co2+ per milliliter with 0.1M, pH 8.0 sodium phosphate buffer. Basing upon the condition determined above, the support was used to prepare immobilized GL-7-ACA acylase and immobilized PGA, with the immobilized enzyme activities of 100U/g and 557.57U/g, respectively. When the rate of adsorption of GL-7-ACA acylase on prepared support was compared with those of on commercial ones, it is correspond to TALON metal resin and FP-IDA, and 1.5 folds to EC-IDA. The immobilized activity of prepared support for PGA is 1.47 folds to that of Eupergit C, and 2.2 folds to those of Sepabeads Ec-Ep and Amberzyme oxirane resin. The optimal condition of column adsorption and purification of D-amino acid oxidase by FP-IDA was studied. When the enzyme activity was about 400U/ml, the volume of crude enzyme solution penetrating through the support was 1BV. The enzyme was purified 3.2-fold with a yield of 20% using IMAC, and the natural protein, especially catalase were washed by 10 mM Tris-Cl buffer with 0.5 M sodium chloride, 5% glycerol, 50mM imidazole.