Purification of Aspartase by Aqueous Two-Phase System and Affinity Membrane Chromatography in Sequence

Abstract

A simple and rapid scheme which coupled aqueous two-phase extraction with affinity membrane chromatography for the recovery of aspartase from Escherichia coli was developed. The aspartase was recovered and purified from cell homogenate by three successive polyethylene glycol-phosphate aqueous two-phase extractions with high activity yield. During the extraction steps, cell debris, nucelic acids, and most contaminating 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-aspartate were used as support and ligand, respectively. The aspartase solution was forced to flow convectively through the pores in which ligand L-aspartate was immobilized on the surface. The affinity membrane chromatography carried out under a high flow rate resulted in a productivity 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, and a 72% recovery yield. SDS-PAGE showed little contaminating proteins were presented in the purified aspartase.