Recombinant kiwi pectin methylesterase inhibitor: Purification and characterization of the interaction with plant pectin methylesterase during thermal and high-pressure processing

• Recombinant pectin methylesterase inhibitor (rPMEI) was expressed in P. pastoris.
• Compared to kiwi PMEI (kPMEI), rPMEI had a slightly higher Mw.
• The inhibitory activity of rPMEI was similar to kPMEI.
• The interaction of rPMEI with PME under processing conditions was similar to kPMEI.

Recombinant kiwi pectin methylesterase inhibitor (rPMEI) with a six-histidine tag at the C-terminus was successfully expressed in Pichia pastoris GS115 strains. rPMEI was purified using pectin methylesterase (PME)-CNBr-affinity chromatography or nickel affinity chromatography. The purified rPMEI had a slightly higher molecular weight than the kiwi PMEI probably due to the presence of added six histidines and some glycosylation. The expression in P. pastoris did not result in changes in the inhibitory activity of rPMEI, nor did it significantly change the complexation with PME under thermal and high pressure processing conditions compared to kiwi PMEI. While thermal treatment of an equimolar PME–rPMEI complex resulted in the aggregation of PME and rPMEI as an entity, high-pressure processing led to the dissociation of PME–rPMEI complex resulting in the presence of free rPMEI after processing. The obtained results presented a viable option for utilization of recombinant PMEI for control of PME activity.Industrial relevanceThe discovery of a pectin methylesterase inhibitor (PMEI) in kiwi fruit opened an additional way to control PME activity, although the industrial application is inhibited by the low yield obtainable from kiwi. For a possible utilization of PMEI in large scale production, recombinant PMEI (rPMEI) was modified with an additional six-histidine tag, expressed and studied. Our results showed that the expression of rPMEI with the additional six-histidine tag allows a feasible large scale purification of the inhibitor using nickel affinity chromatography without negatively affecting the inhibitory activity nor significantly changing the complexation with PME compared to kiwi PMEI. Thus the combination of rPMEI with high-pressure treatment can be exploited to process food systems where cloud stability is of importance but the high pressure itself is insufficient to completely inhibit the PME.