Isolation, Purification and Characterization of Peroxidase from Cucumber

Electrophoresis-purity peroxidase was extracted from fresh cucumber and purified through homogenization, extraction, ammonium sulfate precipitation, CM-Sepharose and Superdex-200 chromatography. The specific activity, recovery and purification fold of the peroxidase were 64 177.67 U/mg, 9.58% and 61.93, respectively. Molecular mass of this purified enzyme was 40.21 kD as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE), while native gel filtration confirmed a monomer form of 41.15 kD. Besides, the peroxidase, whose optimum temperature and pH were 60 ℃ and 6, respectively, was comparatively stable in the temperature range of 25-45 ℃ and pH range of 5-9. The purified peroxidase showed Km value of 53.79 mmol/L under definite conditions. In addition, its activity was scarcely affected by potassium thiocyanate(KSCN). The peroxidase was found to be activated by urea, K+, Mn2+, Ca2+, Mg2+, Ba2+and Cu2+ by 12%, 27%, 13%, 28%, 39%, 99% and 248% at 50 mmol/L, respectively. However, the peroxidase activity was significantly inhibited by SDS, ascorbic acid and oxalic acid. Moreover, Zn2+, methanol, ethanol and isopropanol caused partial inhibitory effects on the peroxidase.