Rapid process for purification of an extracellular beta-xylosidase by aqueous two-phase extraction

A rapid process for purification of an extracellular beta-xylosidase with high purity was developed. The manipulation involved the precipitation of protein from culture medium and the extraction of enzyme from the resuspended crude protein solution by an aqueous-two phase separation. A linear random copolymer, PE62, with 20% ethylene oxide and 80% propylene oxide was employed in both stages of the purification. The enzyme was precipitated effectively by using 10% (w/v) PE62 and 5% (w/v) Na2HPO4. The aqueous two-phase extraction was performed with PE62 (10%)-NaH2PO4 (15%) as phase-forming reagent. SDS-PAGE analysis revealed that the purified enzyme is near homogeneity. The yield is about 100% with a purification factor of 8.8-fold. The whole process can be completed within an hour without any column chromatography.