Hydroxynitrile lyase adsorption at liquid/liquid interfaces

Abstract

The adsorption behavior of hydroxynitrile lyase from Prunus amygdalus ( p-Hnl) at liquid/liquid interfaces has been investigated using dynamic-interfacial-tension measurements. Unfolding of proteins at organic/aqueous interfaces results in a decrease of interfacial tension due to an increasing number of interfacial contacts of hydrophobic parts of the protein molecule. Seven organic solvents with different physical properties, including hexadecane, heptane, diisopropyl ether (DIPE) and ethyl acetate, were studied. It is known that p-Hnl loses its activity very quickly in a variety of solvents, but not in DIPE and ethyl acetate. For these two organic solvents, dynamic interfacial tensions remain constant over a period of 20 h. However, slow decline in the dynamic interfacial tension is found for the less polar solvents suggesting that the protein adsorbs and denatures. Additionally, experiments have been carried out with the aqueous buffer phase at low pH values down to 3.5 with DIPE as the organic phase, simulating conditions that have been previously used for the synthesis of cyanohydrins. There, it is known that the enzyme loses its activity very quickly. Correspondingly, the interfacial tension decreases under these conditions indicating that the bulk enzyme denatures and adsorbs at the interface in an unfolded configuration.