Plant pectin methylesterase and its inhibitor: A quantitative interaction study in a food processing context

iv On the other hand, the effect of extrinsic process parameters, namely high temperature and high pressure, on the interaction between plant PME and kiwi PMEI was investigated, with focus on the individual interaction partners (i.e., PME and PMEI) as well as on the PME-PMEI complex as a whole. The effect of a high-temperature(55 95 °C) or high-pressure(500 800 MPa, 25 °C) induced denaturation of kiwi PMEI on its interaction with plant PME was analysed by measuring residual inhibitory capacity (towards carrot PME) after treatment and estimating inactivation kinetic parameters. Even upon prolonged thermal or pressure processing, a certain fraction of the PMEI inhibitory capacity was conserved (contrary to the PME activity under the same conditions). In fact, the inhibition capacity was only minimally affected by high pressure at low pH values (ca. pH 4). SPR-based interaction analysis with immobilised PMEI and size exclusion chromatography were applied to study the PME-PMEI interaction ensuing (partial) denaturation of carrot PME induced by temperature (50 60 °C) or pressure (800 MPa, 25 °C). Both strategies revealed that the PME-PMEI binding is highly associated with PME inactivation: binding was restricted to the active (i.e., non-inactivated) PME subpopulation, whereas PMEI did not engage a complex with the denatured enzyme fraction. To examine the behaviour of the PME-PMEI complex at elevated temperature or pressure levels, product formation (i.e., methanol) in a mixture of PME, PMEI and pectin as a function of treatment time was followed. The results obtained suggested the complex not to dissociate upon the application of heat (T ≤ 65 °C) and relatively mild pressure (p < 400 MPa at 25 °C), whereas exposure to higher pressure levels caused the complex to separate. These initial experiments were complemented with HPSEC analyses of equimolar PME-PMEI mixtures after treatment at various intensities. The earlier observations were confirmed. At elevated temperature (55 65 °C), enzyme and inhibitor remained united and denatured as a single entity. High-pressure treatment (800 MPa, 25 °C), conversely, induced dissociation of enzyme and inhibitor, followed by gradual inactivation of PME and PMEI and subsequent reunion of the non-inactivated partners upon decompression. PMEI showed distinct pH-dependent inactivation characteristics, being strikingly baroresistant at low pH values (ca. pH 4). Finally, in the quest of obtaining a molecular probe for in situ detection of plant PME in model or food systems, PMEI was biotinylated and the biotinylated PMEI (bPMEI) extensively characterised. The bPMEI retained its complex forming capacity towards PME and gained the ability to strongly bind an avidin species. The PMEI-based probe was successfully tested and its versatility demonstrated in dot-blot binding assays (with native and denatured PMEs) and tissue prints of various plant organs (i.e., carrot root, broccoli stem and tomato fruit). In agreement with the earlier results, the PMEI probe only recognised the noninactivated PME. The whole of the data collected in this thesis endorses the putative application of PMEI in PME inhibition and detection. In the mean time, the results highlight the opportunities and define the application boundaries. However, further research will be needed to translate the findings to real food matrices and to fully exploit the inherent potential of the PME inhibitor.