Active actinidin retains function upon gastro-intestinal digestion and is more thermostable than the E-64-inhibited counterpart

J Sci Food Agric. 2014 Nov;94(14):3046-52. doi: 10.1002/jsfa.6656. Epub 2014 Apr 10.


Background: Actinidin is a cysteine protease and major allergen from kiwi fruit. When purified under specific native conditions, actinidin preparations from fresh kiwi fruit contain both an active and inactive form of this enzyme. In this study, biochemical and immunological properties upon simulated gastro-intestinal digestion, as well as thermal stability, were investigated for both active and E-64-inhibited actinidin.

Results: Active actinidin retained its primary structure and proteolytic activity after 2 h of simulated gastric digestion, followed by 2 h of intestinal digestion, as assessed by SDS-PAGE, zymography and mass spectroscopy. Immunological reactivity of active actinidin was also preserved, as tested by immunoelectrophoresis. The E-64 inhibited actinidin was fully degraded after 1 h of pepsin treatment. Differential scanning calorimetry showed that active actinidin has one transition maximum temperature (Tm ) at 73.9°C, whereas in the E-64-actinidin complex the two actinidin domains unfolded independently, with the first domain having a Tm value of only 61°C.

Conclusion: Active actinidin is capable of reaching the intestinal mucosa in a proteolytically active and immunogenic state. Inhibitor binding induces changes in the actinidin molecule that go beyond inhibition of proteolytic activity, also influencing the digestion stability and Tm values of actinidin, features important in the characterisation of food allergens.

Keywords: E-64; actinidin; digestion; kiwi fruit; stability.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actinidia / chemistry
  • Actinidia / enzymology
  • Allergens
  • Cysteine Endopeptidases / chemistry
  • Cysteine Endopeptidases / pharmacology*
  • Cysteine Proteinase Inhibitors / pharmacology
  • Digestion / physiology*
  • Enzyme Stability / drug effects
  • Enzyme Stability / physiology*
  • Leucine / analogs & derivatives*
  • Leucine / pharmacology
  • Models, Biological


  • Allergens
  • Cysteine Proteinase Inhibitors
  • Cysteine Endopeptidases
  • actinidain
  • Leucine
  • E 64