Enzyme activity in bicontinuous microemulsions

Abstract

The thesis deals with enzymatic catalysis in bicontinuous microemulsions, which consist of a dynamic network of oil and water domains separated by a monolayer of surfactant molecules, i.e. the interfacial layer. Hence, a microemulsion with the composition buffer – n-octane – nonionic surfactant was tested as a reaction medium for enzyme-catalysed reactions with the emphasis on the conversion of hydrophobic substrates, which are difficult to convert in aqueous buffer solutions. The first part of the thesis focuses on the activity of the lipase B from Candida antarctica (CalB) in bicontinuous microemulsions. First, the optimum reaction conditions determined by temperature, pH and ionic strength were evaluated. Second, it was found that CalB concentrations which showed fast adsorption at an oil-water interface also displayed fast reaction rates. Additionally, no saturation was found for substrate concentrations up to 40 mM of p-nitrophenyl laurate, which according to Michaelis-Menten suggests a Km >> 40 mM. Third, the composition of the interfacial layer had a distinct influence on CalB activity, e.g. the presence of sugar surfactants (b-C10G1) or phospholipids (DOPC) enhanced or decreased CalB activity, respectively. The second part of the thesis describes the activity of the squalene-hopene cyclase from Alicyclobacillus acidocaldarius (Aac SHC) converting its natural substrate squalene in bicontinuous microemulsions. The Aac SHC activity studies revealed a linear dependence on enzyme concentration and a hyperbolic curve for the substrate concentration, with a saturation of Aac SHC at substrate concentrations above 20 mM. The composition of the interfacial layer was found to have neither a significant influence on the activity nor on the conformation of Aac SHC. In summary, good turnover rates were achieved for interfacially-active enzymes (CalB) due to enhanced enzyme-substrate contact at the interfacial layer. For water-soluble enzymes (Aac SHC), a distinctly enhanced selectivity was discovered, although no faster reaction rate was found. The main difference in the catalytic turnover was explained by the adsorption of CalB at the interfacial layer, whereas Aac SHC stays in the aqueous phase of the microemulsion. To conclude, bicontinuous microemulsions were suitable for enzymatic catalysis and are thus interesting in terms of reaction medium engineering to optimise biocatalytic processes.