Protein extration from an aqueous phase into a reversed micellar phase: Effect of water content and reversed micellar...

In the system composed of the cationic surfactant TOMAC (10 mM), the nonionic (co)surfactant Rewopal HV5 (2 mM), and octanol (0.1% v/v) in isooctane, reversed micelles are formed upon contact with an aqueous phase containing 50 mM ethylene diamine. alpha-Amylase can be transferred from the aqueous phase into reversed micelles in the pH range 9.5 to 10.5 and re-extracted into a second aqueous phase of different composition. The size of the reversed micelles (as reflected in the water content of the organic phase) can be varied by changes in percentage of octanol, type of counterion in the aqueous phase, or in the number of ethoxylate head groups of the nonionic surfactant. An increase in size results in transfer at lower pH values. Experiments in which the charge density in the reversed micellar interface was changed by incorporation of charged derivatives of the nonionic surfactant, without influencing the water content, revealed that an increased charge density facilitated transfer, resulting in a broader transfer profile. Replacement of TOMAC by other quaternary ammonium surfactants differing in number and length of tails revealed that, of the 14 surfactants tested, only 2 gave appreciable amounts of transfer. The amount of transfer is related to the dynamics of phase separation of the surfactants: those giving a poor phase separation inactivate the enzyme. This inactivation is caused by electrostatic interactions between the charged surfactant head groups and charged groups on the enzyme. Electrostatic interactions are the first step of transfer, and can result in either incorporation in a reversed micelle, or, if reversed micelle formation is slow, in enzyme inactivation. (c) 1995 John Wiley & Sons, Inc.