Studies on the incorporation of lipase in synthetic polymerisable vesicles

This thesis describes studies on the suitability of synthetic polymerisable vesicles for the incorporation and stabilisation of lipase for the bioconversion of organic chemical compounds.

In chapter 1 , some characteristics are reviewed of hydrolytic enzymes, and more specific those of lipases. In chapter 2 an overview is presented of the features and properties of surfactants and vesicles.

In chapter 3 , the incorporation is described of lipase from Candida cylindracea (CCL) into polymerisable positively charged dialkylammonium bromide surfactant vesicles.

Before incorporation the lipase has been purified and characterised. The enzyme has a molecular weight of 58.5 kD and an isoelectric point of 4.1; the pH optimum is broad, ranging from pH 4 to 6 and the optimal temperature is 45°C

The synthesis of several polymerisable surfactants and the preparation of nonpolymerised and polymerised vesicles from these surfactants are described. The vesicle systems were characterised in terms of morphology (electron microscopy) and stability. It appeared that polymerised vesicles are considerably more stable than their nonpolymerised analogues.

The enzyme was incorporated in the vesicle by the use of the dehydration- rehydration method or by incubation. In the latter case, trapping efficiencies are obtained of up to 100%. Activities of free and vesicle incorporated CCL are tested for three triglycerides: triacetin, tributyrin and tricaprylin and for 2,4-dinitrophenyl butyrate. Enzyme activity is lowest in homogeneous mixtures (triacetin and relatively low concentrations of tributyrin) and highest in heterogeneous mixtures (tricaprylin and relatively high concentrations of tributyrin and 2,4-dinitrophenyl butyrate). Incorporation of the enzyme in vesicular systems is advantageous for the activity, especially in homogeneous reaction mixtures, due to the presence of hydrophobic sites of the vesicles. Moreover, in the case of the production of insoluble fatty acid (caproate), inhibition by the acid is suppressed.

The influence of several surface active additives is tested on the activity of lipase. Vesicles have a positive influence on the activity, whereas positively charged surfactant addenda act as inhibitors. In the case of tricaprylin assays, the positively charged surfactant addenda increase enzymatic activity.

In addition, the sensitivity for tryptic digestion of free and incorporated CCL is compared. Free CCL is readily inactivated, whereas incorporated enzyme is protected from proteolytic degradation.

In chapter 4 the stability of vesicle incorporated Candida cylindracea lipase is described.
For this purpose, the enzyme was incorporated into vesicles of the polymerisable zwitterionic surfactant bis[2-(pentacosa-10,12-diynoyloxy)ethyll-2-aminoethanesulfonic acid WAS). Vesicle systems of BPAS were characterised
in terms of morphology (electron microscopy) and stability. Polymerisation of vesiculated BPAS surfactants does not alter the vesicle morphology. Polymeric vesicles are considerably more stable than the monomeric analogues. CCL incorporated into the vesicle membrane by the incubation method remains fully active; especially in homogeneous assay mixtures the vesicle incorporated enzyme shows an increased activity when compared to the free lipase. The stability of free and incorporated lipase was determined by measuring the residual activity of the various systems when mixed with ethanol (50% v/v) or 2-(n-butoxy)ethanol (37.5% v/v), at 50°C and 60°C and in the presence of the proteolytic enzyme trypsin. In all cases the vesicle incorporated enzyme displays an increased stability against denaturating conditions.

The interaction of lipase from Candida cylindracea with positively charged polymerisable surfactant vesicles was studied by the use of steady state fluorescence techniques. The results of these studies are described in chapter 5 .
The phase transition of vesicles composed of nonpolymerised and polymerised N- allylbis[2-(hexadecanoyloxy)ethyllmethylammonium bromide was determined by measuring the change in fluorescence anisotropy of the membrane probe diphenylhexatriene. The phase transition temperature for nonpolymerised vesicles is 49°C and for the polymerised analogues 45°C. Fluorescence anisotropy and energy transfer measurements were used to demonstrate that Candida cylindracea lipase is readily incorporated into the hydrophobic bilayer of the vesicle. By using an interfacial membrane probe (trimethylammonium diphenylhexatriene) and an internal membrane probe (diphenylhexatriene), it could be determined that the lipase is incorporated more efficiently into the nonpolymerised vesicles, and that the penetration of the enzyme into the bilayer is less deeply in the case of polymerised vesicles.

In chapter 6 , a rapid and sensitive assay for the detection of lipase activity is described. The method is based upon the increase in absorbance at 360 nm due to the formation of the 2,4-dinitrophenolate anion during the enzymatic hydrolysis of 2,4- dinitrophenyl esters. Several esters with different acyl chain length have been tested. 2,4-Dinitrophenyl butyrate proved to be a suitable standard substrate. This substrate can be used in homogeneous reaction systems and in emulsified form. In the latter case, a correction can be made for absorbance changes due to clearance of the emulsion during hydrolysis by using a diode array spectrophotometer with internal referencing. The small reaction volume and the high extinction coefficient of the product makes this method suitable for assay mixtures of low substrate and low enzyme concentration.

In chapter 7 the results from the preceding chapters are reviewed in a general discussion.