Extraction and Activity of Chymotrypsin Using AOT-DOLPA Mixed Reversed Micellar Systems

Abstract

Novel reversed micellar solutions formulated with a mixture of AOT (dioctyl sulfosuccinate) and DOLPA (dioleyl phosphoric acid) show good potential for use in reversed micellar protein extraction operations. Chymotrypsin is easily extracted from an aqueous phase into organic isooctane containing 10 mM AOT and DOLPA in a 4:1 ratio. The extraction ability of the mixed reversed micelles of 10 mM was higher than that of 200 mM AOT alone. The results of extraction indicated that the AOT-DOLPA mixed reversed micelles are very useful for separating and enriching chymotrypsin. Back-extraction of chymotrypsin from the organic phase to a fresh aqueous phase is also accomplished by adding an alcohol to the organic phase. Although the back-transfer of chymotrypsin from the reversed micelles formed by AOT alone is very slow and difficult, in the AOT-DOLPA mixed reversed micelles, the back-extraction can be achieved completely by addition of 10% (v/v) isobutyl alcohol to the reversed micellar phase. The time to attain to the equilibrium of back-extraction was reduced from more than 24 to 2 h by adding the alcohol. On the basis of the activity data, the best composition of AOT and DOLPA was a 4:1 ratio and the total surfactant concentration was 10 mM. The activity of chymotrypsin recovered from the mixed reversed micelles was higher than that of the initial protein before the forward-transfer. This result means that the novel mixed reversed micellar solutions are useful not only in separation but also in purification of proteins.

Penicillin G solubilisation into AOT reverse micelles

“…This raises the question of whether the pH can be manipulated to get better penicillin G extraction at low AOT concentration in an industrial application (it should be noted that penicillin broths are compositionally more complex than those used in this work, which will change the equilibria that can established in the extraction process). The use of a low surfactant concentration would be helpful because amphiphilic molecules are difficult to remove in subsequent downstream processing [18].…”

“…The polar heads of the surfactant molecules are directed towards the interior of a water-containing sphere, whereas the aliphatic tails are oriented toward the non-polar organic phase. Many authors have investigated the partitioning of proteins between two immiscible liquids, and the considerable success of protein partitioning has provided the motivation for using the technique for antibiotics; in our case penicillin G. Reverse micelles have the ability to extract a variety of biomolecules such as proteins [10,11,[15][16][17][18][19] and enzymes [16,19] into nanometre-size water pools surrounded by a monolayer of surfactant.…”

Extraction, Purification and Characterization of Fish Chymotrypsin: A Review

“…Back-extraction is usually very slow and CaCl 2 is added into the aqueous phase to assist the process (Hu and Gulari, 1996). The limitations of using reverse micelles in the backextraction are due to: (a) the difficulty in separating proteins from the AOT reverse micellar phase and (b) the excessive time involved in the process (Hu and Gulari, 1996;Goto et al, 1998).…”

“…This can be added to AOT to form AOT-DOLPA for use in chymotrypsin extract. Goto et al (1998) reported that the effectiveness of mixed reverse micelles increased with increasing amount of DOLPA added. He also found that 4:1 ratio of AOT: DOLPA was the best for chymotrypsin extraction and that 10 mM of mixed reverse micelles had higher extraction ability than 200 mM of AOT.…”

Electrochemical Aspects of the Reverse Micelle Extraction of Proteins

“…The reverse micelle extraction of proteins is useful for the separation and purification of proteins, 6 enzymatic reactions in organic solvents, 7,8 refolding of denatured proteins, 9,10 etc. To understand the extraction mechanism, many efforts have been made to study the effects of various experimental conditions, which include the natures and concentrations of proteins, surfactants, and aqueous electrolytes and the pH of the aqueous phase.…”