四氧化三铁吸附包埋固定辣根过氧化物酶及其应用

Preparation and Application of Horseradish Peroxidase Immobilized in Fe_3O_4-Asorption-Gelatin-Entrapping Method

建立了Fe_3O_4吸附包埋固定辣根过氧化物酶,并与明胶、开孔明胶、海藻酸钠3种不同的固定化方法载体进行了比较,发现该固定化方法具有较高的固定效率。对固定化酶与自由酶的稳定性进行了比较,发现Fe3O4吸附包埋固定化HRP的稳定性高于其他固定化HRP和自由酶。此外,测定了不同戊二醛浓度、交联时间、给酶量、Fe3O4使用量及明胶浓度对辣根过氧化物酶固定效果的影响。结果表明,固定化反应的最佳条件如下最佳给酶量与Fe3O4用量比例约为95uHRP/gFe3O4,最佳Fe3O4用量与凝胶比例为1.0gFe3O4/10mL10%～20%,最适戊二醛浓度和交联时间分别为0.5%和30min。在此条件下制备的辣根过氧化物酶活性约为1.1u/g,酶活固定率约为15%。该固定化酶重复应用于PCP催化去除反应,可获得稳定的PCP去除率。

Fe3O4-asorption-gelatin-entrapping method for immobilization of horseradish peroxidase was established. Immobilization efficiency of this carrier was compared with that of sodium alginate, gelatin and open pore gelatin cube. It was found that higher efficiency could be achieved by Fe3O4-asorption-gelatin-entrapping method. The stabilities of different immobilized enzymes and free HRP were also compared, and the stability of immobilized HRP with Fe3O4-gelatin as carrier was considerably higher than the stabilities of other enzymes. Effects of concentration of glutaraldehyde and gelatin, crosslinking time, dosage of HRP and Fe3O4 on the activity of immobilized HRP were studied. The results of optimal conditions of immobilization were as follows： the optimal＇ratio of dosage of HRP and Fe3O4 was about 95 u HRP/g Fe3O4, the optimal ratio of Fe3O4 and gelatin was 1.0 g Fe3O4/10 mL 10% - 20% gelatin, the optimal glutaraldehyde concentration was 0.5 % and the optimal crosslinking time was 30 min. On this condition, the activity of immobilized HRP was about 1.1 u/g and the recovery of enzyme activity was around 15% . Repetitive use of immobilized HRP for the elimination of PCP was examined and stable efficiency of PCP degradation was achieved.