Comparative Structural Changes and Inactivation Kinetics of Pectin Methylesterases from Different Orange Cultivars Processed by High Pressure

Pectin methylesterases (PMEs) from two different orange sources, Valencia and Navel cv., were extracted and purified using cation exchange, heparin chromatography, and finally, gel filtration chromatography, yielding a single peak corresponding to a protein of molecular weight 34 and 35 kDa for Valencia and Navel PMEs, respectively. Effects of high pressure (HP) and thermal processing for various treatment durations on the activity of PMEs in Tris-HCl buffer solution (pH 7.5) were explored. Higher levels of pressure, temperature, and treatment duration resulted in an analogous reduction of the PME residual activity. HP-induced inactivation of both purified PMEs was described by a first-order kinetic model. Kinetic parameters were estimated and a multiparameter equation was developed to predict the PME inactivation rate constant at any combination of pressure and temperature conditions for both enzymes. The PME from Valencia orange peel appeared to be more heat- and pressure-sensitive compared to Navel PME. HP-induced conformational changes of the PME molecules were also investigated using circular dichroism (CD) spectroscopy. A direct comparison of the CD results for treated and untreated proteins reveals that pressure treatment has negligible effects upon far-UV CD spectra, while significant irreversible changes are depicted in near UV for both PMEs. It is, thus, evidenced that exposure to HP may lead to a structurally molten globulelike state, where the PME maintains a secondary structure of untreated protein molecules, while a tertiary structure is substantially affected bearing subsequent impact on substrate-enzyme binding interaction, leading to reduction of enzyme activity.