Understanding the role of fungal pectin methylesterase in fruit texture engineering

Pectin methylesterase (PME) catalyzes the demethoxylation of pectin, a major polysaccharide in plant cell walls. Demethoxylated pectin can be cross-linked by divalent ions and is less susceptible to chemical depolymerization by β-elimination. PME is therefore applied for improvement of textural properties of plant based foods. The application of PME in food industry has generally been developed empirically. However, the increasing insight in the cell wall architecture, the pectin structure, its relation to physico-chemical properties and the structure and action of enzymes (including PME), will enable the design of enzyme preparations that tailor-make pectins with specific functionalities. In this context, the objective of the present work was to study the effect of relevant processing parameters (temperature, high pressure and pH) on the structural and functional stability and the kinetics and the mode of action of recombinant Aspergillus aculeatus PME, purified from a commercial preparation currently applied in fruit processing. The aim was to generate a scientific basis that allows a more reasoned application of the enzyme and the development of products with new functionalities[I1] . As a case study, the effect of pretreatments involving infusion of the enzyme preparation on the texture of thermally and high pressure processed strawberries was investigated. High pressure was selected as a processing parameter, next to the more conventional parameters pH and temperature, because high pressure processing is gaining interest as a food preservation technique due to the better balance between food safety and food quality, compared to conventional processes, obtained for various food products. The main structural component of recombinant A. aculeatus PME, as revealed by FTIR studies, was β-helix, which supports the idea that the fungal enzyme adopts a parallel β-helix three-dimensional architecture, similar to the crystalline structures proposed for bacterial and plant PME and other pectinolytic enzymes. The ordered structural components of recombinant A. aculeatus PME started to unfold at temperatures exceeding 50°C. This irreversible protein denaturation was reflected in a loss of catalytic activity in the same temperature range. The thermal inactivation kinetics of recombinant A. aculeatus PME could be described be a first-order model. Recombinant A. aculeatus PME was a very pressure stable enzyme. No loss of activity was observed after prolonged treatments up to 700 MPa at moderate temperatures. Pressures higher than 1 GPa had to be applied to induce any conformational changes of the protein. Moreover, pressure protected recombinant A. aculeatus PME against thermal denaturation. At elevated pressures higher temperatures were required to unfold and inactivate the enzyme. Temperature, pressure and pH strongly affected the rate of pectin demethoxylation catalyzed by recombinant A. aculeatus PME. The enzyme was active in acidic conditions, with highest activity observed between pH 4.0-4.5 (depending on temperature). At atmospheric pressure recombinant A. aculeatus PME was optimally active at 40-45°C. Increasing the pressure shifted the optimal temperature to higher values, due to the protective effect of pressure against thermal inactivation. Highest activity was detected when temperatures of 50-55°C were combined with pressures of 200-300 MPa. At all elevated pressures studied (100-500 MPa) the activity was higher compared to atmospheric pressure. The mode of action of PME was investigated by enzymatic fingerprinting of the demethoxylated pectins using endo-polygalacturonase from Kluyveromyces fragilis, with subsequent analysis with HPAEC and HPSEC. A multiple-chain mechanism was proposed to describe the action of recombinant A. aculeatus PME, although some multiple-attack mechanism on the intrachain level, yielding limited blocks of demethoxylated units, can be suspected. The mode of action of the fungal PME (with acidic pI and acidic pH optimum) clearly differed from the mode of action of PME purified from Lycopersicon esculentum (tomato) (with an alkaline pI and neutral to alkaline pH optimum), which was suggested to act according to a multiple-attack mechanism producing large blocks of demethoxylated galacturonic acids residues. Neither for recombinant A. aculeatus PME, nor for tomato PME an effect of pH (pH 4.5 vs pH 8.0) on the mode of action was detected with the enzymatic fingerprinting method applied. Furthermore, increasing temperature (50°C vs 22.0°C) and/or pressure (300 MPa vs 0.1 MPa) did not have an influence on the mode of pectin demethoxylation by recombinant A. aculeatus PME. In a final part, it was demonstrated that recombinant A. aculeatus PME can be applied as a tool to improve the textural properties of processed fruit with a delicate structure. It was shown that vacuum infusion of the fungal PME together with Ca2+-ions can significantly increase the hardness of strawberries. The difference in hardness was maintained after thermal and high pressure treatments of the fruit.status: publishe