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Separation of endo-polygalacturonase using aqueous two-phase partitioning
Y T Wu1, M Pereira, A Venâncio
1Centro de Engenharia Biológica-IBQF, Universidade do Minho, Braga, Portugal.
Journal of Chromatography. A
|October 12, 2001
Summary
Aqueous two-phase extraction effectively purifies endo-polygalacturonase (endo-PG) from fermentation broth. This method achieves high enzyme recovery and concentration, with recyclable polymers for sustainable bioprocessing.
Area of Science:
- Biochemistry
- Biotechnology
- Separation Science
Background:
- Enzyme purification is crucial for industrial applications.
- Aqueous two-phase extraction (ATPS) offers a scalable method for biomolecule separation.
- Endo-polygalacturonase (endo-PG) is a valuable enzyme requiring efficient purification strategies.
Purpose of the Study:
- To investigate the partitioning behavior of endo-PG in ATPS.
- To develop and evaluate a multi-step ATPS scheme for endo-PG purification.
- To assess the recovery and purification efficiency of the proposed method.
Main Methods:
- Studied endo-PG partitioning in polyethylene glycol (PEG)-polyvinyl alcohol (PVA) and PEG-hydroxypropyl starch systems.
- Developed a two-step ATPS purification scheme using PEG-PVA followed by PEG-ammonium sulfate.
- Performed laboratory-scale experiments to validate the separation efficiency.
Main Results:
- Endo-PG preferentially partitioned to the top phase in PEG-PVA systems (partitioning coefficient of 6).
- The two-step ATPS scheme achieved 91% enzyme recovery and a 1.9 purification factor.
- A concentration factor exceeding 5 was obtained, with over 90% PEG recovery and recycling.
Conclusions:
- ATPS is a viable technique for purifying and concentrating endo-PG from fermentation broth.
- The developed two-step ATPS scheme is efficient and allows for polymer recycling.
- This purification strategy enhances the economic feasibility of endo-PG production.