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Purification of phospholipase D by two-phase affinity extraction.
1Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India.
Journal of Chromatography. A
|February 7, 2004
Summary
This study purified phospholipase D (PLD) using an aqueous two-phase system with alginate. The method achieved high enzyme recovery and purity from peanuts and carrots.
Area of Science:
- Biochemistry
- Protein Purification
- Enzyme Technology
Background:
- Phospholipase D (PLD) is an important enzyme with various applications.
- Efficient purification methods are crucial for obtaining active PLD for research and industrial use.
Purpose of the Study:
- To develop and optimize an aqueous two-phase system for the purification of PLD from peanuts and carrots.
- To utilize alginate as a macroaffinity ligand for enhanced PLD partitioning and elution.
Main Methods:
- An aqueous two-phase system composed of polyethylene glycol (PEG) and salt was employed.
- Alginate was incorporated into the PEG phase as a macroaffinity ligand for PLD.
- Enzyme elution was achieved by exploiting the reversible precipitation of alginate with Ca2+ and subsequent salt elution.
Main Results:
- Over 90% of PLD activity from both sources partitioned into the PEG phase.
- Purification factors of 78-fold for peanut PLD and 17-fold for carrot PLD were achieved.
- High activity recovery (82% for peanuts, 85% for carrots) and a single band on SDS-PAGE confirmed enzyme purity.
Conclusions:
- The developed PEG-alginate aqueous two-phase system is effective for purifying PLD from plant sources.
- This method offers a high-yield, high-purity, and efficient approach for PLD isolation.
- The findings provide a valuable purification strategy for PLD in biochemical and biotechnological applications.