Purification of Catalase by Modified Procedure and some Properties of Enzyme

Abstract

Catalase (EC 1.11.1.6) plays an important role in protecting organism from oxidative effect by breaking down H₂O₂ into H₂O and O₂ molecules. In this study, a modified procedure for catalase from bovine liver and its properties were reported. Bovine liver catalase was purified to electrophoretic homogeneity as a single protein band around 60 kDa on SDS-PAGE by acetone fractionation, followed by ion-exchange chromatography on DEAE-Sepharose and CM-Sepharose columns. The specific activity of the purified catalase was 79,277 units per mg of protein (U/mg) with 1.87% recovery and purification fold was roughly 60 times. The catalase was a homo-tetramer with a molecular mass of about 240.987 kDa as determined by native gel electrophoresis. The purified enzyme showed the highest activity at pH 7, 37°C, and remained active over a broad range of pH from 5 to 10 and range of temperature from 4°C to 40°C. Its activity was inactivated by incubating in 60°C for 30 min. The activity of the enzyme was induced by Ca²⁺ and inhibited by Na⁺, Ni²⁺, Cu²⁺, Zn²⁺, Fe²⁺, Fe³⁺ and NaN₃. Under the optimal conditions, K_m and K_cat/K_m values of the catalasewas found to be 23,69 mM and K_cat/K_m = 5.106 (M.s)^-1, respectively