Aqueous Two-phase Extraction Of Serum Albumin And Enzymes

Proteins and enzymes are important promising biomacromolecules which have been widely used in our daily life, treatment and prevention of disease and industrial production. At present, the main primary separation of proteins and enzymes are salting out, precipitation at isoelectric point and ethanol precipitation. Although these methods can obtain target proteins to a certain extent, many disadvantages still exist, for instance, tedious process, severe operating conditions and easy denaturation of proteins during separation. For these reasons, aqueous two-phase systems (ATPS) composed of hydrophilic organic solvent and inorganic salt were investigated to separate proteins or enzymes in this paper.Firstly, the phase separation and extraction ability of different ATPSs of monohydroxy-alcohol/salt were determined to extract serum albumin. Many systems showed good extraction results. In order to increase separation efficiency and for safety consideration, K₂HPO₄/ethanol and Na₂CO₃/ethanol ATPS have been selected to be further researched. When the system was composed of 18%(w/w) K₂HPO₄ and 18% (w/w) ethanol or 13%(w/w) Na₂CO₃ and 18%(w/w) ethanol, the highest recovery was 94.02% and 93.61%, respectively. These results are similar to traditional ATPS of salt and PEG with lower molecular weight.Secondly, ATPS of K₂HPO₄/ethanol was applied to separate serum albumin from blood and fermentation broth. For human recombinant serum albumin expressed in yeast, the recovery and removal ratio of cells were 93.50% and 99.79%, respectively, in an ATPS of 20%(w/w) K₂HPO₄ and 18% (w/w) ethanol on a scale of 0.2L. Furthermore, the recovery of serum albumin in the top phase was 106.30%, 113.20%, 98.30%and 96.60%, respectively, when the ATPE was scaled up from 2L to 73L. The purification of HSA was further conducted in column chromatography, including cation exchange column,hydrophobic chromatography and anion exchange column. According to the HPLC analysis of HSA, the purity was up to 99.71%. At the aspect of serum, BSA was inclined to the top phase with a highest recovery of 84.41%, whiles IgG was precipitated at the interphase. This is a good case for separation of BSA from IgG. Finally, ATPS of polyalcohol/salt was used to extract proteins and enzymes. The recoveries of proteins were all above 80% on the primary stability of BSA and high activity of enzyme above 73%.The experimental results showed that ATPS of hydrophilic organic solvent/salt is suitable for the separation of proteins and enzymes and has a wide application on an industrial scale.