Preparation Of Metal Chelated Affinity Ligand And Purification Of Soybean Hull Peroxidase Using Aqueous Two-phase Affinity Partitioning

The recent progresses and key problems in aqueous two-phase partitioning (ATPS) were reviewed. Especially, the forming, characteristics and utilization of metal immobilized affinity partition, the preparation of affinity ligand, and the purification of peroxidase were disccused.The synthesis process of iminodiacetic acid-polyethylene glycol (IDA-PEG) with varying content of IDA was investigated. The process was optimized under BFi concentration, NaOH concentration, reaction time, and IDA coupling conditions. It was found that the amount of copper( II) on the immobilized metal ion affinity ligand was related to the molecular ratio of epichlorohydrin to PEG in the synthesis process. Under optimal conditions, three affinity ligands of PEG-IDA-Cu( II )-A (0.24 molCu2+/mol PEG)^ PEG-IDA-Cu( II )-B (0.51 molCu2+/molPEG^ PEG-IDA-Cu( II) -C(0.75mol Cu2+/mol PEG) were prepared respectively. The optimized conditions of the reaction of epichlorohydrin to PEG were 0.50% BF3; 2.5% NaOH (40% wt), 20h reaction time at room temperature, and the optimized condition of the reaction of IDA to epichlorohydrin-PEG were in the buffer of 2mol/L Na2COs, mol ratio of IDA to PEG at 20 and 24h reaction time at 65 癈.The liquid-liquid equilibria for polyethylene glycol (PEG) + (NH4) 2864 +water and hydorxypropyl starch (PES)+ PEG+water, which the PEG was substituted by metal chelated affinity ligand partially, were measured at 20 癈 by the formaldehyde titration method or the optical rotation-vacuum-drying method. The phase diagrams of these systems were similar to those without IDA-PEG. The effect of the immobilized metal ion affinity ligand on the tie lines of the PEG/(NH4) 2864 system showed that the slope of the tie line increases firstly and then decreases with increasing PEG-IDA-Cu( II), but the slope was larger than that of PEG-IDA- Cu(II)/ (NH4) 2864 system.The affinity partition behavior of soybean hull peroxidase (SHP) in ATPS was studied. In the systems of PEG4000/Na 2SO4 and PEG4000/(NH4) 2SO4, the enzyme was enriched little by little in the top phase with the PEG substituted by PEG-IDA-Cu( II). In PEG/PES systems, the partition coefficients of the enzyme were higher comparatively. The pH had little effect on the partition. With the decrease of the molecular weight of PEG and the increase of the molecular weight of PES, the partition coefficient of SHP was enhanced. With the increase of the concentration of PEG in the system, SHP trends to enrich in the top phase. When the ratio of PEG substituted by PEG-IDA-Cu( II) was over 10%, the partition coefficient of SHP reached maximum value. Acceding to the yields and purification factor, the optimizedpartition conditions were found as PEG2000 10%, PES200 14%, 10% PEG substituted by PEG-IDA-Cu( II). In this system, the partition coefficient was 27, the recovery was 96%, and the purification factor was 2.8.