PURIFICATION OF ASPARTASE BY AQUEOUS 2-PHASE SYSTEM AND AFFINITY MEMBRANE CHROMATOGRAPHY IN SEQUENCE

A simple and rapid scheme which coupled aqueous two-phase extraction w ith affinity membrane chromatography for the recovery of aspartase fro m Escherichia coli was developed. The aspartase was recovered and puri fied from cell homogenate by three successive polyethylene glycol-phos phate aqueous two-phase extractions with high activity yield. During t he extraction steps, cell debris, nucelic acids, and most contaminatin g proteins were removed. The aspartase was recovered in the phosphate- rich phase. The enzyme was further purified by affinity chromatography in which the regenerated microporous cellulose membrane and L-asparta te were used as support and ligand, respectively. The aspartase soluti on was forced to flow convectively through the pores in which ligand L -aspartate was immobilized on the surface. The affinity membrane chrom atography carried out under a high flow rate resulted in a productivit y of 17 L/L/h. The overall purification scheme yielded aspartase with a specific activity of 27.3 units/mg, a 32-fold increase in purity, an d a 72% recovery yield. SDS-PAGE showed little contaminating proteins were presented in the purified aspartase.