Separation of endo-polygalacturonase using aqueous two-phase partitioning.
The partitioning of endo-polygalacturonase (endo-PG) in polyethylene glycol (PEG)-polyvinyl alcohol (PVA10000) 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-PVA10000 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.
Wu
Y T
YT
Centro de Engenharia Biológica-IBQF, Universidade do Minho, Braga, Portugal.
Pereira
M
M
Venâncio
A
A
Teixeira
J
J
D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D011096:Polygalacturonase / Q000302:isolation & purification*; D011506:Proteins / Q000302:isolation & purification; D014867:Water / Q000737:chemistry
D003470:Culture Media; D011506:Proteins; D014867:Water; D011096:Polygalacturonase