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).