The kinetic separation of protein mixtures using reverse micelles

Commercial interest in cost-effective methods that can separate, concentrat e, and purify proteins continuously, and be easily scaled-up, has increased markedly in recent years. Liquid-liquid extraction using reverse micellar organic solvents has been found to have this potential. The overall objecti ve of this work was to investigate the kinetic separation of protein mixtur es using reverse micelles. The kinetics of simultaneous forward extraction of lysozyme, cytochrome c, and ribonuclease A were investigated in a stirre d cell (Lewis cell). In addition, a Graesser contactor was used for the sim ultaneous extraction of lysozyme and cytochrome c from a buffer solution as well as from diluted hen egg white to which cytochrome c had been added. T he Lewis cell experiments showed that the mutual effect on the overall mass transfer coefficient, k(0), of the three simultaneously extracted proteins at pH 5.5 was significant, and in some cases quite large. The presence of cytochrome c or ribonuclease A helped to prevent lysozyme precipitation at the interface, while the addition of cytochrome c altered the pH dependency pattern of lysozyme, and the presence of lysozyme reduced the k(0)-value o f cytochrome c. The kinetic separation of lysozyme and cytochrome c was pos sible with a Graesser contactor, and maximized at low rotor speed (2-3 rpm) , low temperature (4 degrees C), and a pH close to the pI of both proteins (pH 10); after 30 minutes about 80% of the lysozyme and only 10% of the cyt ochrome c were extracted into the reverse micellar phase. The extraction ra te measured in the Graesser contactor differed from that measured in the Le wis cell, and this observation indicates that different steps of the revers e micellar transfer mechanism are controlling the transfer. Using hen egg w hite (the natural source of lysozyme), the kinetics of lysozyme and added c ytochrome c were different from those with buffer solutions; cytochrome c e xtraction was faster. making kinetic separation more difficult. In addition , a stable emulsion formed with hen egg white, which is not desirable in an industrial application since it results in an additional separation step.