Purification, characterization, immobilization and kinetic studies of catalase from a novel source Sechium edule

A catalase from a novel source Sechium edule (squash) has been purified to homogeneity using ammonium sulfate fractional precipitation, dialysis, and anion exchange chromatography on Diethylaminoethyl (DEAE) cellulose in sodium acetate buffer pH 6.0. The purity of the enzyme was analyzed by SDS-PAGE. The molecular weight of the enzyme using the SDS-PAGE method was found to be 55 kDa with specific activity 6.35 U/mg. The molecular weight of the enzyme was further confirmed by the INTACT mass spectrometric technique and found to be 52.5 kDa. The Reinheitzahl (Rz) value of the enzyme was 1.5. The MALDI-TOF analysis of the purified enzyme showed that it contains 529 amino acid residues. The K-m, V-max,V- optimum pH, and optimum temperature of the free enzyme using H2O2 as the substrate were found to be 0.03 mM, 200 mu mol/min, 7.6, and 24 degrees C, respectively. The purified enzyme was immobilized on chitosan beads which was prepared by extracting the chitosan from Cornu aspersum (garden snail) having highest degree of deacetylation%. The kinetic characteristics, K-m, V-max,V- optimum pH, and optimum temperature of the immobilized catalase were found to be 0.065 mM, 250 mu mol/min, 7.6, and 41 degrees C, respectively. The immobilized catalase is more thermostable in comparison to free catalase.