Steady-state fluorescence studies on lipase-vesicle interactions.

The interaction of lipase from Candida cylindracea (CCL) with positively charged polymerizable surfactant vesicles was studied by the use of steady-state fluorescence techniques. The phase transition of vesicles composed of nonpolymerized and polymerized N-allylbis[2-(hexadecanoyloxy)ethyl]methylammonium bromide (ABHEMA Br) was determined in the absence of lipase, by measuring the change in fluorescence anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The phase transition temperature for nonpolymerized vesicles is 49 degrees C and for the polymerized analogues 45 degrees C. Fluorescence anisotropy and resonance energy transfer measurements were used to illustrate the incorporation of the lipase in the vesicle membrane. These studies demonstrated that CCL is incorporated into the hydrophobic bilayer of the vesicle. By using an interfacial membrane probe 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene p-toluene sulphonate, TMA-DPH) and an internal membrane probe (DPH), it could be determined that the enzyme is incorporated more efficiently into nonpolymerized vesicles, and that the penetration of the enzyme into the bilayer is less deep in the case of the polymerized vesicles.

Lipase activity in vesicular systems: characterization of candida cylindracea lipase and its activity in polymerizable dialkylammonium surfactant vesicles.

Lipase from Candida cylindracea (CCL) was incorporated into polymerizable positively charged dialkylammonium bromide surfactant vesicles. The enzyme was incorporated by the use of the dehydration-rehydration method or by incubation. In the latter case, trapping efficiencies of up to 100% could be obtained. Activities of free and vesicle-incorporated CCL were tested for three triglycerides: triacetin, tributyrin, and tricaprylin. Enzyme activity was lowest in homogeneous mixtures (triacetin and small concentrations of tributyrin) and highest in heterogeneous mixtures (tricaprylin and high concentrations of tributyrin). Entrapment in vesicular systems is advantageous, especially in homogeneous reaction mixtures and in the case of the production of insoluble fatty acid (caproate), because inhibition by the acid can be suppressed. The influence of several surface-active additives, including vesicles, on the activity of lipase in triglyceride assays was tested. Vesicles have a positive influence on the activity, whereas other positively charged additives act as inhibitors. In the case of tricaprylin assays, the positively charged additives increase the activity. Finally, tryptic digestion for free and incorporated CCL were compared. Free CCL is readily inactivated, whereas incorporated enzyme is protected from proteolytic degradation.