PURIFICATION AND CHARACTERIZATION OF PECTIN METHYLESTERASE ISOENZYMES FROM ORANGE PEEL

Pectin methylesterase (PME) isoenzymes from Citrus sinensis (Balady orange) peel have been purified and characterized. The purification procedure included CM-cellulose, DEAE-celiuIose and Sephacryl S-200 columns. PME1, PME2 and PME4 were purified to homogeneity. The molecular weights of PME1, PME2 and PME4 were determined using Sephacryl S-200 column and estimated to be 23,000, 47,400 and 79,500 Da, respectively. PME1, PME2 and PME4 had isoelectric points of 5.4, 5.9 and 5.6, and pH optima of 6.0, 6.0 and 5.5, respectively. Maximum PME activity was reached at 50 mM NaCI for PME1 and PME4, and 25 mM NaCl for PME2. Km values of PME1, PME2 and PME4 using pectin with degree of esterification (DE) of 8% were 0.55, 1.0 and 3.33 mg pectin/ml, respectively. The affinity of PMEs was increased with the increasing of the DE of pectin. The time-dependent heat-inactivation curves for the three isoforms exhibited exponential behaviour and showed that PME1 was more heat resistant than PME2 and PME4. Effect of pH on enzyme stability, kinetic properties (Vmax and Kcat) of isoenzymes, and effect of different metal cations on enzyme activity were investigated. A study of the inhibitory effect of ferrous sulfate (FeSO4) on the PME activity in freshly prepared Balady orange juice revealed that 1 mM FeSO4 was the best concentration that maintained the homogeneity and stability of the juice.