Purification Of Sweet Potato Peroxidase And Its Characteristics Of Catalyzing Luminol Chemiluminescence

Peroxidases are a group of oxidoreductases that catalyse a variety of reactions in the presence of peroxides such as hydrogen peroxide. They are found widely in plants, animals and microorganisms. Some applications of peroxidases include enzyme immunoassays, peroxidase biosensors, diagnostic kits, organic synthesis and treatment of waste waters.In this research, purication and characteristics of catalyzing luminol chemiluminescence of sweet potato peroxidase from sweet potato (Ipomoea batatas) peel were studied. The results showed that:Sweet potato peroxidase was purified to electrophoretic homogeneity by combined consecutive treatments consisting of aqueous two-phase extraction, gel filtration on Sepharose CL-6B, hydrophobic interaction chromatography on Phenyl Sepharose 6 fast flow and affinity chromatography on Con A-Sepharose 4B. The specific activity of purified peroxidase was 6930 U/mg, the purification fold was 165, and the recovery rate was 17%. The enzyme had been demonstrated by SDS-PAGE as a single band, and its molecular weight was 34000. The RZ value of this enzyme was about 2.0.The optimal activity of sweet potato peroxidase was at pH5.5 and at 70℃, respectively. The enzyme was stable at pH2.2-10.0. The activity of this enzyme still retained 60% when keeped in 60℃for 1 h. The results showed that sweet potato peroxidase was able to retain its catalytic activity under wide range of pH and temperature. Furthermore, sweet potato peroxidase was very stable at high temperature and extreme pH. The enzyme activity was seriously inhibited by Fe2+ and Fe3+, yet enhanced by Ca2+ and Mg2+. PEG and SDS had no obvious inhibition on the enzyme activity. Organic solvents such as ethanol, methanol and acetone could inhibit the activity of this enzyme and the order according to the inhibitory effect from strong to weak was ethanol, acetone and methanol.The pH, Tris buffer, luminol and hydrogen peroxide concentrations were optimized for maximum sensitivity using the sweet potato peroxidase, and we determined favourable conditions for sweet potato peroxidase catalysis (60 mmol/L Tris-HCl buffer, pH9.0, containing 4 mmol/L hydrogen peroxide and 5 mmol/L luminol). The sweet potato peroxidase detection limit in luminol was 5 fmol/L. As in the case of other anionic peroxidases, the catalytic efficiency of sweet potato peroxidases did not depend on the presence of enhancers in the reaction medium. The long-term chemiluminescence signal produced during sweet potato peroxidase-dependent luminol peroxidation was a characteristic feature of the sweet potato peroxidase. High sensitivity in combination with the long-term chemiluminescence signal and high stability suggests that sweet potato peroxidase will be a promising tool in chemiluminescence enzyme immunoassay.