Thomas Duvetter

Pectin methylesterase and its proteinaceous inhibitor: a review.

Pectin methylesterase (PME) catalyses the demethoxylation of pectin, a major plant cell wall polysaccharide. Through modification of the number and distribution of methyl-esters on the pectin backbone, PME affects the susceptibility of pectin towards subsequent (non-) enzymatic conversion reactions (e.g., pectin depolymerisation) and gel formation, and, hence, its functionality in both plant cell wall and pectin-containing food products. The enzyme plays a key role in vegetative and reproductive plant development in addition to plant-pathogen interactions. In addition, PME action can impact favourably or deleteriously on the structural quality of plant-derived food products. Consequently, PME and also the proteinaceous PME inhibitor (PMEI) found in several plant species and specifically inhibiting plant PMEs are highly relevant for plant biologists as well as for food technologists and are intensively studied in both fields. This review paper provides a structured, comprehensive overview of the knowledge accumulated over the years with regard to PME and PMEI. Attention is paid to both well-established and novel data concerning (i) their occurrence, polymorphism and physicochemical properties, (ii) primary and three-dimensional protein structures, (iii) catalytic and inhibitory activities, (iv) physiological roles in vivo and (v) relevance of (endogenous and exogenous) enzyme and inhibitor in the (food) industry. Remaining research challenges are indicated.

Strawberry Pectin Methylesterase (PME): Purification, Characterization, Thermal and High-Pressure Inactivation

Pectin methylesterase (PME) was extracted from strawberries ( Fragaria ananassa, cv Elsanta) and purified by affinity chromatography on a CNBr‐Sepharose 4B‐PME‐inhibitor column. A single protein and PME activity peak was obtained. A biochemical characterization in terms of molecular mass, pI, and kinetic parameters of strawberry PME was performed. In a second step, the thermal and high‐pressure stability of the enzyme was studied. Isothermal and combined isothermal‐isobaric inactivation of purified strawberry PME could be described by a fractional‐conversion model. Purified strawberry PME is much more stable toward high‐pressure treatments in comparison to those from oranges and bananas.

Effect of Temperature and High Pressure on the Activity and Mode of Action of Fungal Pectin Methyl Esterase

Pectin was de‐esterified with purified recombinant Aspergillus aculeatus pectin methyl esterase (PME) during isothermal‐isobaric treatments. By measuring the release of methanol as a function of treatment time, the rate of enzymatic pectin conversion was determined. Elevated temperature and pressure were found to stimulate PME activity. The highest rate of PME‐catalyzed pectin de‐esterification was obtained when combining pressures in the range 200–300 MPa with temperatures in the range 50–55 °C. The mode of pectin de‐esterification was investigated by characterizing the pectin reaction products by enzymatic fingerprinting. No significant effect of increasing pressure (300 MPa) and/or temperature (50 °C) on the mode of pectin conversion was detected.

The in situ observation of the temperature and pressure stability of recombinant Aspergillus aculeatus pectin methylesterase with Fourier transform IR spectroscopy reveals an unusual pressure stability of β-helices

The stability of recombinant Aspergillus aculeatus PME (pectin methylesterase), an enzyme with high beta-helix content, was studied as a function of pressure and temperature. The conformational stability was monitored using FTIR (Fourier transform IR) spectroscopy whereas the functional enzyme stability was monitored by inactivation studies. Protein unfolding followed by amorphous aggregation, which makes the process irreversible, was observed at temperatures above 50 degrees C. This could be correlated to the irreversible enzyme inactivation observed at that temperature. Hydrostatic pressure greater than 1 GPa was necessary to induce changes in the enzymes secondary structure. No enzyme inactivation was observed at up to 700 MPa. Pressure increased PME stability towards thermal denaturation. At 200 MPa, temperatures above 60 degrees C were necessary to cause significant PME unfolding and loss of activity. These results may be relevant for an understanding of the extreme stability of amyloid fibrils for which beta-helices have been proposed as a structural element.

A pectin-methylesterase-inhibitor-based molecular probe for in situ detection of plant pectin methylesterase activity.

In the quest of obtaining a molecular probe for in situ detection of pectin methylesterase (PME), the PME inhibitor (PMEI) was biotinylated and the biotinylated PMEI (bPMEI) was extensively characterized. Reaction conditions for single labeling of the purified PMEI with retention of its inhibitory capacity were identified. High-performance size-exclusion chromatography (HPSEC) analysis revealed that the bPMEI retained its ability to form a complex with plant PME and that it gained the capacity to strongly bind an avidin species. By means of dot-blot binding assays, the ability of the probe to recognize native and high-temperature or high-pressure denatured plant PMEs, coated on an absorptive surface, was investigated and compared to the binding characteristics of recently reported anti-PME monoclonal antibodies. Contrary to the antibodies, bPMEI only detected active PME molecules. Subsequently, both types of probes were used for PME localization in tissue-printing experiments. bPMEI proved its versatility by staining prints of carrot root, broccoli stem, and tomato fruit. Applying the tissue-printing technique on carrot roots after thermal treatment demonstrated the complementarity of bPMEI and anti-PME antibodies, with the former selectively detecting the remaining active PME and the latter staining both native and inactivated PME molecules.

Development and evaluation of monoclonal antibodies as probes to assess the differences between two tomato pectin methylesterase isoenzymes.

The enzyme pectin methylesterase (PME) was purified from red ripe tomatoes (Lycopersicon esculentum) and through affinity chromatography two isoenzymes were fractionated (t1PME and t2PME). Further analysis of these two isoenzymes, both having a molar mass of 34.5kDa, revealed a difference in the N-terminal sequence and in amino acid composition. t1PME was identified as the major isoenzyme of PME in tomato fruit. In this study the aim was to develop a toolbox, consisting of monoclonal antibodies, that allows to gain insight into the in situ localization of PME in plant based food systems like tomatoes. A panel of six interesting monoclonal antibodies was raised against both isoenzymes, designated MA-TOM1-12E11, MA-TOM1-41B2, MA-TOM2-9H8, MA-TOM2-20G7, MA-TOM2-31H1 and MA-TOM2-38A11. The differences in epitopes between these monoclonal antibodies were determined using affinity tests towards denatured PME, cross-reactivity tests and inhibition tests. Characterization of these antibodies indicated an immunological difference between t1PME and t2PME, also revealing a conserved epitope on t2PME, carrot PME and strawberry PME. Different epitopes are recognized by the generated antibodies making them excellent probes for immunolocalization of PME by tissue printing. In tomato, t1PME and t2PME showed a pronounced co-localization, especially in the pericarp and the radial arms of the pericarp. Three of the generated antibodies could be used for immunolocalization of PME in carrots (Daucus carota L.), which was only present in the cortex and not in the vascular cylinder of carrots.

Advances in understanding pectin methylesterase inhibitor in kiwi fruit: an immunological approach

In order to gain insight into the in situ properties and localisation of kiwi pectin methylesterase inhibitor (PMEI), a toolbox of monoclonal antibodies (MA) towards PMEI was developed. Out of a panel of MA generated towards kiwi PMEI, three MA, i.e. MA-KI9A8, MA-KI15C12 and MA-KI15G7, were selected. Thorough characterisation proved that these MA bind specifically to kiwi PMEI and kiwi PMEI in complex with plant PME and recognise a linear epitope on PMEI. Extract screening of green kiwi (Actinidia deliciosa) and gold kiwi (Actinidia chinensis) confirmed the potential use of these MA as probes to screen for PMEI in other sources. Tissue printing revealed the overall presence of PMEI in pericarp and columella of ripe kiwi fruit. Further analysis on the cellular level showed PMEI label concentrated in the middle lamella and in the cell-wall region near the plasmalemma. Intercellular spaces, however, were either completely filled or lined with label. In conclusion, the developed toolbox of antibodies towards PMEI can be used as probes to localise PMEI on different levels, which can be of relevance for plant physiologists as well as food technologists.

Size exclusion chromatography to gain insight into the complex formation of carrot pectin methylesterase and its inhibitor from kiwi fruit as influenced by thermal and high-pressure processing.

A size exclusion chromatography (HPSEC) method was implemented to study complex formation between carrot pectin methylesterase (PME) and its inhibitor (PMEI) from kiwi fruit in the context of traditional thermal and novel high-pressure processing. Evidence was gained that both thermal and high-pressure treatments of PME give rise to two distinct enzyme subpopulations: a catalytically active population, eluting from the size exclusion column, and an inactive population, aggregated and excluded from the column. When mixing a partly denatured PME sample with a fixed amount of PMEI, a PME-PMEI complex peak was observed on HPSEC, of which the peak area was highly correlated with the residual enzyme activity of the corresponding PME sample. This observation indicates complex formation to be restricted to the active PME fraction. When an equimolar mixture of PME and PMEI was subjected to either a thermal or a high-pressure treatment, marked differences were observed. At elevated temperature, enzyme and inhibitor remained united and aggregated as a whole, thus gradually disappearing from the elution profile. Conversely, elevated pressure caused the dissociation of the PME-PMEI complexes, followed by a separate action of pressure on enzyme and inhibitor. Remarkably, PMEI appeared to be pressure-resistant when compressed at acidic pH (ca. 4).

High pressure processing to optimise the quality of in-pack processed fruit and vegetables.

Publisher Summary This chapter highlights that High Pressure (HP) technology has been known for more than 100 years, but was adopted and adapted for industrial food applications in the early 1990s. HP processing at room/moderate temperature has been introduced for industrial food processing and preservation. Currently, in countries such as Japan, United States, Spain, France, Czech Republic and United Kingdom, this technique is being applied to produce high-quality foods, including ham, sea food, jam, mixed fruit and vegetables, juices and sauces, etc. Typically, foods are pre-packed in flexible containers or in plastic packs before HP treatment. Small molecules, such as vitamins, pigments, and volatile compounds, are less affected by HP processing compared with proteins/enzymes, which are often characterized by a complex three-dimensional architecture stabilized by various covalent and non-covalent interactions. Consequently, HP processing allows the processing of foods while preserving their “fresh-like” properties, due to the limited loss of flavor, color, and nutritional value. From an engineering point of view, HP processing is faced with limitations of heat transfer, although it applies pressure isostatically, that is, instantaneously and uniformly throughout a mass of food material irrespective of size, shape, and composition. HP processing has been extended, on a laboratory scale, to applications at elevated temperatures, referred to as High Pressure Sterilization (HPS) to obtain spore inactivation. This application takes advantage of adiabatic heating during pressurization.