Application of Reverse Micelles for the Extraction of Amino Acids and Proteins

Abstract

The use of water-in-oil microemulsions for the solubilization and extraction of amino acids and proteins is investigated, using both the liquid-liquid extraction and the solid-liquid extraction method. The solubilization behaviour of small monomeric proteins such as cytochrome c, lysozyme, ribonuclease A, trypsin, ?-chymotrypsin, papain, pepsin, and bovine serum albumin (BSA) into mainly AOT (the anionic surfactant bis(2-ethylhexyl) sodium sulfosuccinate)/H2O/isooctane is studied by varying several external parameters, such as wo (wo = [H2O]/[AOT]), surfactant type and concentration in the organic phase, and, pH, ionic strength, salt type in the H2O phase. In the liquid-liquid extraction method, the present biphasic system consists of an aqueous and a supernatant reverse micellar solution. The compounds are transferred from the H2O phase into the organic micellar phase. This process is investigated also in view of the simultaneously occurring H2O transfer. Studies of transfer kinetics give an information about the connection between the H2O and protein extraction. In the second technique, the compounds are given in their solid state and suspended in the organic micellar solution which contains already a known amount of solubilized water.With the liquid-liquid phase-transfer method, it is shown that, under certain conditions, a quantitative and quite remarkable transfer can be achieved, whereby the salt type and its concentration in the H2O phase, as well as the aqueous pH, play the most important role in determining the extent mass transfer. The significance of electrostatic interactions between the micelles and the extracted molecules is evidenced also by comparing extraction results obtained with AOT and CTAB (cetyltrimethylammonium bromide; is a cationic surfactant) water-in-oil microemulsions. Moreover, the extraction yield of a series of positively, negatively charged, and neutral amino-acid derivatives is investigated in the two surfactant systems, in order to characterize the significance of electrostatic and hydrophobic interactions with the surfactant molecules. This series of amino-acid derivatives is also studied with the solid-phase extraction method. In the case of proteins transferred from the solid state, the solubilization yield is affected by the protein and micellar droplet size. For small proteins, i.e. with a molecular weight below ca. 15 000 D, the extraction curve as a function of wo shows surprisingly enough a peak in the small wo region (between 6 and 12). By increasing the H2O content above this wo limit, the transfer efficiency of the H2O-soluble proteins decreases. For proteins having a molecular weight above 20 000 D, the extraction curve as a function of wo shows a monotonous increase with increasing wo without any sharp wo maxima. A qualitative model is offered to explain this different behaviour. It is also shown that the ionization state of commercially available protein powders markedly affects the extraction behaviour. The micellar exclusion effect is finally applied in the extraction of proteins from a crude bacterial protein mixture.