Extraction, Purification and Kinetics of Guaiacol Peroxidase from Leaves and Friuts of Black Blueberry (MORUS NIGRA)

Peroxidase (EC 1.11.1.7) is one of the key enzymes controlling plant growth, differentiation and development. Peroxidase from black mulberry (Morus Nigara) leaves and fruits was purified using ammonium sulfate salt precipitation and anion-exchange chromatography on DEAE-Sepharose column. The preparation gave an overall yield of 28.2%, 80-fold purification for leaves and of 12.4%, 97-fold purification for peroxidase of fruits. Specific activity of 15.3 and 31.1 U mg-1 protein for peroxidase of leaves and fruits were earned, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified enzymes were homogeneous and peroxidase of leaves have two isoenzymes with molecular weight of approximately 56 and 33 kDa, but peroxidase of fruits has a isoenzyme with molecular weight of approximately 55 kDa. Activity of peroxidase in leaves and fruits in the presence of guaiacol and H2O2 were optimum after incubation at 40 and 45 °C, respectively. Temperature stability profile showed that the purified Guaiacol peroxidase of leaves had maximum stability at 60 °C and retained 79% activity after incubation for 30 min. PGPF was 100% stable for 1 h at 45 °C and retained 45% of its initial activity after 4 h of preincubation at this temperature. The purified Guaiacol peroxidase of leaves seemed to have considerable thermostability, which can be favorable in industrial operations for traditional brewing and food processing. Kinetic parameters assesment for the purified Guaiacol peroxidase of leaves and purified Guaiacol peroxidase of friuts showed Vmax of 16.8 and 25.3 U mg-1, and Km of 6.8 and 5.6 mM, respectively. NaN3 and kojic acid were potent inhibitors of peroxidase and ZnSO4 showed an activatory effect on peroxidase in leaves and fruits of black mulberry