Pectin methylesterase (PME) from orange peel (Valencia cv.) was extracted and purified using cation exchange and gel filtration chromatography. The purified orange peel PME contained a single band of 32000 Da with an isoelectric point higher than 9. The effect of High Pressure (HP) processing on the activity of PME in Tris-HCl buffer solution (pH 7.5) was studied, irreversible HP-induced conformotional changes were investigated by far and near – UV Circular dichroism (CD). The relative residual activity of PME decreased with the increase of pressure and treatment duration. HP inactivation of purified orange peel PME can be described by a first-order kinetic model. The inactivation rate constant k values were calculated as 0.018, 0.037, 0.085 and 0.169 min~(-1) at 200, 400, 500 and 700 MPa and 40°C respectively. As far as the CD analysis of PME samples is concerned, a characteristic for β-structure (β-sheets and turns) minimum at ～218 nm was observed. The secondary structure of PME was changed following HP treatment. Spectra of the thermal and high pressure treated samples showed different profiles in comparison to the native PME as the intensity of the minimum at the CD spectra decreased with the increase of pressure and treatment duration. It was observed that the conformational change of the secondary structure was accompanied by a corresponding loss of enzyme activity. The obtained results explain the inactivation of this enzyme after processing at certain conditions where irreversible conformational change was induced.
展开▼
