Purification and Characterization of a Salt-Dependent Pectin Methylesterase from Carica papaya Fruit Mesocarp-Exocarp...

Pectin methylesterase (PME) is a ubiquitous cell wall enzyme, which de-esterifies and modifies pectins for food applications. Functional properties of pectin rely on molecular weight and degree of esterification, and thus de-esterification by PME influences the pectin functionality. The main aim of the study is to purify and biochemically characterize PME from the outer mesocarp-exocarp tissue of unripe Carica papaya L. fruit. The ion-exchange and gel-permeation chromatography purified enzyme exhibited a specific activity of 2363.1 ± 92.8 units/mg protein, with a fold purification of 10.6, and final recovery of 9.0%. The PME showed a low apparent mass of 27 kDa by SDS-PAGE. The optimal activity of purified PME was found at pH 7.0, and at 60 °C. The enzyme is fairly stable at 60 °C for 10 min, retaining 60% activity. The optimum activity was found with 0.25 mol/L monovalent salts indicating that this PME is salt-dependent. The K_m of PME was 0.22 mg/mL, and the V_max value was 1289.15 ± 15.9 units/mg. The increase in the calcium sensitivity of the PME-treated pectin indicated a blockwise mode of action. The PME significantly differs from other known plant PMEs in their biochemical properties. Manual inspection and MASCOT searching of generated tryptic peptides confirmed no homology to known papaya PME sequences. The preliminary results indicate that the papaya PME can be potentially utilized to modify pectin functionality at elevated temperature. However, further investigation is required to understand the usefulness of this enzyme for the modification of pectins for various food applications.

In this work, a small, 27 kDa papaya PME was purified by ion-exchange and gel-permeation chromatography and biochemically characterized. The papaya PME significantly differs from other known plant PMEs in their biochemical properties. The preliminary results like fair thermostability coupled with high temperature optimum indicate that the papaya PME can be potentially utilized to modify pectin functionality at high temperature. Modification of pectin functionality at elevated temperatures is advantageous since it evades the detrimental action of other pectinolytic enzymes.