Lipid Self-Assembly and Lipase Action

In this thesis the action of the Thermomyces lanuginosa lipase (TLL) on "real" substrates, that is, lipid aggregates - vesicles, micelles, emulsions, gels and liquid crystals is discussed. Even for a pure triolein, the system becomes quite complex as the lipolytic process starts, both in terms of structure and composition, involving 5 additional components - diolein (DO), monoolein (MO), oleic acid (OA), sodium oleate (NaO) and glycerol . Therefore the focus has been on the product end of the system, that is DO, MO, OA and NaO. The objective of the thesis was to relate the lipid self-assembly structures formed during the lipolysis to the initial substrate structure as well as to determine how the self-assembled structure affect the lipase activity. For this purpose the isothermal ternary phase diagrams for aqueous mixtures of MO with DO, OA and NaO have been determined. These phase diagrams have been used as maps to navigate through the changes of the lipid self-assembly structures that occur during the lipolytic process. All ternary systems formed a reversed hexagonal liquid crystalline phase, HII , in the water-poor part of the phase diagram and the bicontinuous cubic (C) phase formed by the aqueous MO has very limited capability to solubilize DO, OA and NaO within the stability of the phase. NaO mixed with MO and 2H2O produced a large lamellar phase (La), a C phase and stable vesicles at high water content, whereas, a micellar type of cubic phase (Cmic) was only observe in the MO-OA system and then at high OA content. The observed sequence of phase structures due to lipase addition was pH dependent and correlated very well with the ternary MO-OA-2H2O at acid to neutral pH and with the MO-NaO-2H2O systems at alkaline pH. The phase transitions induced by the lipase action on C- and HII phases (acid to neutral pH) followed the same pathway and the transitions occurred at similar time and composition, following the order: (C) -> HII -> Cmic -> reverse micellar phase and the transitions corresponded to those observed for the phase behaviour of the MO-OA-2H2O system. The changes also correlated with the lipid compositions obtained by 13C NMR and HPLC measurements as well as with changes of the lipid and water self-diffusion coefficients. Addition of lipase to the dispersed cubic phase at high water content lead to the formation of cubosomes, hexasomes and defective vesicles. On addition of lipase to the La phase from the MO-NaO aqueous system (alkaline pH), the phase transitions followed the order La -> normal hexagonal phase (HI) which follows the MO-NaO-2H2O system. Lipase activity did not change the morphology of vesicles, formed from the corresponding La phase of MO-NaO at high water content, as observed by light microscopy and cryo-TEM. The specific activity of TLL on the C and HII substrates seemed to be the same, in spite of their different nano-structures