层状氢氧化氨基苯甲酸锌固定化辣根过氧化物酶及其催化性质

Immobilization of Horseradish Peroxidase on Layered Zinc Aminobenzoate Hydroxide and Its Catalysis

利用层状氢氧化氨基苯甲酸锌对辣根过氧化物酶(HRP)进行固定化实验,通过紫外分析探讨了固定化效果与HRP质量浓度、体系p H的关系。利用Lineweaver-Burk双倒数做图法测定了固定化HRP的动力学常数,考察了固定化HRP的储存稳定性和重复利用性。实验结果表明,在体系p H=8.0、HRP质量浓度为1.0 mg/m L的条件下,层状氢氧化氨基苯甲酸锌对HRP的饱和固载量为100 mg/g,最大比活力为60.27 U/mg。固定化HRP的米氏常数(Km)、催化常数(Kcat)、酶活性(X)分别为3.23 mmol/L,42.5 s-1,57.5 U/mg。与游离HRP相比,固定化HRP的Km提高了44.2%,Kcat下降了74.0%,X值下降了74.4%。储存40 d后,固定化HRP的活性为游离HRP的5.35倍。循环利用8次后,固定化HRP的活性保留率仍为79.6%。

The immobilization of horseradish peroxidase（HRP） on layered zinc aminobenzoate hydroxide support was conducted. The effects of the HRP mass concentration and p H on the immobilization were investigated by means of UV analysis. The kinetics constants of the HRP immobilization were determined by Lineweaver-Burk double-reciprocal plot method. The storage stability and reusability of the immobilized HRP were studied. The experiment results showed that the immobilized HRP on layered zinc aminobenzoate hydroxide with a maximum immobilization capacity of 100 mg/g and specific activity of 60.27 U/mg could be prepared under the optimal conditions of p H 8.0 and HRP mass concentration 1.0 mg/m L. The michaelis constant（Km）,catalytic constant（Kcat） and enzyme activity（X） of the immobilized HRP were 3.23 mmol/L,42.5 s-1 and 57.5 U/mg,respectively. Compared with free enzyme,Km of the immobilized enzyme increased by 44.2%,while its Kcat and X decreased by 74.0% and 74.4%,respectively. The activity of the immobilized enzyme was 5.35 times that of free enzyme after they were stored 40 d,and still reached 79.6% of its original activity after being reused eight times.