The Study Of Isolation And Characterization Of Peroxidase From Horseradish Roots Using Ultrafiltration

Horseradish peroxidase（HRP, E.C. 1.11.1.7） which catalyzes the oxidation of a large variety of substrate by hydrogen peroxide is one of the most important heme-containing enzyme for clinical diagnoses. Apart from the usage of medical treatment, HRP has been applied in decolorization of waste and treatment of wastewater containing phenolic compounds. However, the conventional techniques used for HRP isolation suffer from long process time and low product purity and difficulty to scale up. These drawbacks restrict the application of horseradish perxidase.In the present work, a two-stage ultrafiltration process was used for the separation and purification of HRP from horseradish roots and suitable operating parameters were selected by parameter scanning ultrafiltration. In addition, we also constructed the large-scale ultrafiltration technology for the isolation of HRP from horseradish roots by using a Centramate System from Pall Corporation. After the two-stage ultrafiltration process, HRP was purified 238-fold with a specific activity of 642.6 U/mg protein and a yield of 86%.The analysis results showed that the purified HRP had a molecular weight of 40 kDa and an isoelectric point of 8.3-8.6. The optimal p H for enzyme activity was found to be 4.5, and at this pH the enzyme exhibited maximum activity at 52 oC. Among the cations used, Cu²⁺ and Ca²⁺ were found to have a stimulating effect on the enzyme activity, whereas K⁺, Na⁺, Mg²⁺, Ba²⁺, Co²⁺, Mn²⁺, Zn²⁺ and Sr²⁺ were found to be strong inhibitors.