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

Abstract

Peroxidase (EC 1.11.1.7) is one of the key enzymes controlling plant growth, differentiation and development. Peroxidase from black blueberry (Morus Nigara) leaves and friuts was purified using ammonium sulfate salt precipitation and Anion-exchange chromatography on DEAE-sepharose. 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 isoenzyme 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 friuts in presence of guaiacol and H2O2 were optimum after incubation at 40 and 45°C, respectively. Temperature stability profile showed that PGPL had maximum stability at 60°C and retained 79% activity after incubation for 30 minutes. PGPF was 100% stable for 1 h at 45°C and retained 45% activity after 4 h of preincubation. PGPL seemed to have considerable thermostability, which can be favorable in industrial operations for traditional brewing and food processing. Vmax of PGPL and PGPF were 16.8 and 25.3 unit/mg protein, so showed km of 6.8 and 5.6 mM, respectively. NaN3 and kojic acid were potent inhibitors of peroxidase and ZnSO4 showed that has an activatory effect on peroxidase in Leaves and fruits fo blueberry.