Partitioning of protease using an ionic liquid based aqueous two phase system: Experimental study and thermodynamic modeling

The aqueous two-phase system (ATPS) as a liquid-liquid fractionation technique has gained an interest because of great potential for recovery and purification of many biological materials such as proteins, enzymes, and nucleic acids. Ionic liquids (ILs), are a group of organic salts comprising entirely of ions and can be suggested as a suitable solvent for a specific purpose, especially separation processes. Among the various ILs, the alkyl-sulfate derivatives have the great advantages such as synthesizing easily in a halide-free way at a reasonable cost with relatively low viscosity and considering as a promising IL in ATPSs. In the present study, the partitioning of protease using the ATPS [1-Ethyl-3-Methyl-Imidazolium][EthylSulfate]([emim][eSO4])/Na2CO3 was evaluated. The effect of parameters, such as the initial concentration of enzyme (protease), concentration of ionic liquid (IL) ([emim][eSO4]) and concentration of salt (sodium carbonate) on the purification of protease was investigated.

The design experiment of BOX-Behnken as a method of the response surface methodology (RSM) was applied to minimize the number of runs and the process optimization.

The optimum conditions were enzyme/IL of 0.210 and enzyme/salt of 0.381 and concentration of enzyme in the top phase (IL rich phase) and separation yield were obtained, 4 ppm and 94%, respectively.

The results showed that the protein tends to be extracted in a top phase, hence the protein concentration in the bottom phase (salt rich phase) was negligible. Furthermore, the experimental results are correlated successfully using non-random two liquid non-random factor (NRTL-NRF) equation as a short-range contribution.