Preparation Of Xanthine-Oxidase-Magenetic-Microspheres And Its Separation Of Uric-Acid-Lowering Peptides

In this paper,a method for the screening and determination of uric acid peptides in vitro was established.On the basis of this,magnetic chitosan microspheres were prepared for the immobilization of xanthine oxidase?XO?to construct the XO-magenetic microspheres,which was used to affinity adsorb the uric-acid-lowering peptides from protein hydrolyzates.The results provide a theoretical guidance and technical support for the enrichment of uric-acid-lowering peptides from protein hydrolyzates.The in vitro XO inhibitory activity of peptides was determined by double-enzyme-coupling method combined with high performance liquid chromatography?HPLC?.The optimal reaction conditions by double-enzyme-coupling method were as follows: 0.22 mM xanthine,0.52 u/mL XO,Tris-HCl pH 8.0,30 ?,20 min for catalytic reaction.Meanwhile,the optimal conditions for the determination of uric acid by HPLC were as follows: 95% 15 mM NH4H2PO4 and 5% methanol at pH 6.5,1 mL/min of flow rate at 25 ?,290 nm for detecting.The results of double-enzyme coupling assay combing with HPLC method showed that reduced glutathione?GSH?and oxidized glutathione?GSSG?had xanthine oxidase inhibitory activity and could be used as uric-acid-lowering peptides.Fe₃O₄ magnetic particles were prepared with chemical coprecipitation methodology,and formed into magnetic chitosan microspheresas by emulsion cross-linking method.Afterwards,the magnetic chitosan microspheres were activated with the surface modified with epoxy groups for the xanthine oxidase immobilization.Taking the epoxy density of microsphere surface as an activation marker,the optimal process conditions were 40%?v/v?of epichlorohydrin,0.60 g/L of NaBH₄ and 1.20 mol/L of NaOH.The results of microspheres for structural characterization showed that the Fe₃O₄ magnetic particles were successfully wrapped in chitosan and the surface of activated microspheres were surrounded by epoxy reactive groups.The medium diameter of Fe₃O₄ magnetic particles,the unactivated and activated magnetic chitosan microspheres respectively were 2.16 20.30 24.69 ?m.After the activation,xanthine oxidase was immobilized on the megnetic microspheres to construct the XO-magenetic microspheres.Taking the enzyme activity as indicators,the optimal immobilzation process was conducted at 30 ? and pH 8.0 for 1 h.The study of enzymatic properties of XO-magenetic-microspheres indicated that the optimum temperature and pH of the enzyme was at 48 ? and pH 8.5.Moreover,the enzyme possessed reusability and good thermal,pH and storage stability.The optimal affinity absorption for allopurinol by XO-magenetic-microspheres were summarized as follows: 3 mM allopurinol,2 h for absorption at 40 ?,pH 7.5,and the maximum adsorption amount of allopurinol was 67.19 ± 1.89 ?M/g.Meanwhile,the optimal affinity absorption for GSH by XO-magenetic-microspheres were as follows: 50 mg/mL GSH,2 h for absorption at 40 ?,pH 7.5,and the maximum adsorption amount of GSH was 1572 ± 49.36 mg/g.The effects of different eluents on the elution of GSH were studied by HPLC,consequently,NaCl+HCl solutions was used as the eluent of GSH.The adsorption of GSH via XO-magenetic-microspheres and magenetic microspheres was studied by HPLC.The results showed that the adsorption of XO-magenetic-microspheres for GSH was specific.