Aqueous two-phase extraction using thermoseparating polymer : a new system for the separation of endo-polygalacturonase

Abstract

Aqueous two-phase partitioning of endo-polygalacturonase (endo-PG) produced by Kluyveromyces marxianus strains was studied, on systems containing the thermoseparating polymer Ucon 50-HB-5100 (a random copolymer of 50% ethylene oxide (EO) and 50% propylene oxide (PO)) as one of the phase-forming compounds. Ucon 50-HB-5100 (Ucon)–polyvinyl alcohol (PVA 10,000), Ucon 50-HB-5100–hydroxypropyl starch (Reppal PES100) and Ucon 50-HB-5100–(NH4)2SO4 systems were tested. Ucon recycling was also investigated. Ucon–(NH4)2SO4 system proved to be the most efficient system for aqueous two-phase extraction (ATPE) of endo-PG as when compared to total protein partition, the enzyme was strongly partitioned to the salt-rich phase. Using Ucon–(NH4)2SO4 system, a separation scheme consisting of three stages was proposed and tested at laboratory scale. In the first and second stages, operated in series, extractions were performed using the same Ucon–(NH4)2SO4 system. Ucon recycling was done in each stage. In the third stage, removal of the Ucon and salt residues present in the enzyme-rich phase of the second extraction stage was done. The proposed scheme allowed for a 10-fold enzyme concentration and a purification factor close to the expected maximum while maintaining more than 95% of the initial enzyme activity.