چكيده به لاتين
Abstract:
Reverse micelle extraction (RME) is one of the liquid-liquid extraction methods. RME is a fast and simple method with high efficiency. With the progress in production of recombinant proteins by microbial hosts, separation and purification of these productions from fermentation media and cell debris has become the main challenge of production of the proteins. For this purpose, insulin was taken as a low molecular weight protein model and CTAB/ Isooctane/n-Hexanol/n-Butanol was used for RME process. Extraction using reverse micelle methods contains two main parts of forward and backward steps which the protein transfer to an organic phase in forward extraction and then to a new aqueous phase during backward extraction step. Effective factor was determined using one factor at a time design and the optimum conditions was obtain as: 30mM of surfactant concentration, 50mM of NaCl conc., 10% v/v of each Hexanol as cosurfactant and Butanol as co-solvent, and KCl conc. of 0.6M at constant pH 7 for both steps and contact time of 90s and 15min for forward and backward steps, respectively. The extraction yields of these conditions are 97 and 76% for forward and backward processes, respectively. Two methods were used to evaluate the selectivity of this process. First, insulin was added to fermentation medium in the presence of other bacterial proteins. Insulin selectivity extraction was investigated at optimum condition obtained from one factor at a time design and insulin selectivity extraction related to other proteins was validated using electrophoresis gel. Then, in order to compare the selectivity extraction of insulin to a molecule with similar isoelectric point value and different molecular size, α-amylase was used. Ful factorial design was firstly done for determining of optimum conditions of insulin extraction and then the selectivity was investigated at the obtained optimum condition (surfactant concentration of 40mM, 10% v/v n-butanol as solvent and and KCl conc. of 0.8M at constant pH 7 for both steps and contact time of 5min and 20min) in the presence of equal two molecules concentartions. At this condition, insulin extraction yield was 65 and 55% in forward and backward extractions, respectively and amylase concentration in backward step became zero that represents a complete separation of the two biomolecules.
Keywords: liquid-liquid extraction, reverse micelles, CTAB, Insulin, selectivity, amylase
