Immobilized pineapple stem bromelain activity in a wine-like medium: Effect of inhibitors

Abstract

The catalytic activity of stem bromelain, covalently immobilized on chitosan beads, was characterized towards a synthetic substrate (Bz-Phe-Val-Arg-pNA), in a wine-like acidic medium containing wine inhibitors (ethanol, sulphur dioxide, grape skin and seed tannins), at their average concentration range.For all tested substances, the inhibition constant (Ki) values of immobilized bromelain were significantly higher than the corresponding values obtained in a previous work for free enzyme, thus indicating that direct covalent immobilization on chitosan beads makes protease more resistant to the inhibition effect.Immobilized protease was affected by ethanol inhibition (competitive type) only if present at abnormal concentration for real wine. Grape skin and seed tannins exerted uncompetitive inhibition to the same extent, with a Ki value close to 1 g lgallic acid eq−1. The effect of free sulphur dioxide on immobilized bromelain changed respect to free enzyme, both becoming uncompetitive inhibitor and dramatically lowering its strength.These results show that immobilized stem bromelain could have productive biotechnological applications in winemaking, even though further studies will be necessary to test its proteolytic activity towards wine proteins in real matrices.