Separation of endo-polygalacturonase using aqueous two-phase partitioning

• Author(s): You-Ting Wu ; Pereira, Martinha ; Venâncio, Armando ; Teixeira, J. A.
• Date: 2001
• Persistent ID: http://hdl.handle.net/1822/1776
• Origin: RepositóriUM - Universidade do Minho
• Subject(s): Partitioning; Aqueous two-phase systems; Enzymes; Endo-polygalacturonase
• Document Type: Article
• Language: English

Description

The partitioning of endo-polygalacturonase (endo-PG) in polyethylene glycol (PEG)–polyvinyl alcohol (PVA10 000) and PEG–hydroxypropyl starch (Reppal PES100) aqueous two-phase systems was studied, and revealed the possibility of using aqueous two-phase extraction to purify and concentrate endo-PG from its clarified fermentation broth. For the PEG8000– PVA10 000 system, endo-PG presented in the fermentation broth (at concentration that is more than 40% of total protein) mainly dominates in the top phase with a partitioning coefficient of 6, while total protein concentrates in the bottom phase. A separation scheme consisting of two consecutive aqueous two-phase extraction steps was proposed: a first extraction in polyethylene glycol (PEG8000)–polyvinyl alcohol system, followed by a second extraction in PEG8000–(NH4)2SO4 system. This allowed the separation of endo-PG from polymer and the recycling of PEG polymer, since endo-PG was very strongly partitioned into the bottom phase of the PEG8000–(NH4)2SO4 system. Laboratory-scale experiments were performed to test the efficiency of this scheme. It was found that enzyme recovery was up to 91% with a total purification factor of about 1.9 and a concentration factor of more than 5. About 90% of the total PEG added into the systems can be recovered, and no reduction was obtained in the purification factor using recycled PEG.