Investigating the in situ localisation of pectin methylesterase and its inhibitor: Development of an immunological toolbox

Investigating the in situ localisation of pectin methylesterase and its inhibitor: Development of an immunological toolboxDuring processing of plant-based food products the aim is to guarantee food safety and quality among which texture forms one of the most prominent quality attributes. The plant cell wall, one of the characteristic components of plant-based food products, greatly contributes to texture. Amongst the numerous compounds building the structurally heterogeneous cell wall, pectin is of interest because of its functional properties in the cell wall with regard to tissue integrity and rigidity. Moreover, pectin is an important target in texture engineering during food processing, in which also the enzyme pectin methylesterase (PME) and its inhibitor (PMEI) play an important role. In order to fully exploit the possibilities of the pectin conversions imposed by PME in texture engineering, it is important to understand these mechanisms prior to and during processing. Ex situ analysis alone is subjected to some restrictions and should ideally be complemented with in situ analysis. Thus, it is in this context that probes have been developed in the current work to enhance the in situ insight into PME and PMEI. During processing, endogenous PME in plant-based foods plays an important role in attaining the desired food structure. Because of the interest in endogenous PME, plant PME was here purified from red ripe tomato fruit (Solanum lycopersicon) and used for the production of monoclonal antibodies (MAs) as probes. Two isoenzymes were fractionated from tomato fruit (t1PME and t2PME), both having a molar mass of 34.5 kDa and displaying differences in amino acid sequence. t1PME was identified as the major isoenzyme of PME in tomato fruit. Both isoenzymes were used for the generation of MAs, resulting in a panel of six interesting MAs designated MA-TOM1-12E11, MA-TOM1-41B2, MA-TOM2-9H8, MA-TOM2-20G7, MA-TOM2-31H1 and MA-TOM2-38A11. Characterisation of these antibodies, including the evaluation of the cross-reactivity towards PME from tomato, carrot, strawberry and Aspergillus aculeatus, indicated an immunological difference between t1PME and t2PME and also revealed a conserved region on t2PME, carrot PME and strawberry PME. The PME specificity of the developed antibodies makes them excellent probes for immunolocalisation of PME. Tomato fruit tissue printing revealed a pronounced co-localisation of t1PME and t2PME on tissue level, especially in the perica