Extraction and purification of Ipomoea peroxidase employing three-phase partitioning.
Three-phase partitioning (TPP) is a novel separation process used for the extraction and purification of biomolecules. The biomolecules are recovered in a purified form at the interface (precipitate), while the contaminants partition in t-butanol and aqueous phases. Peroxidase from the leaves of Ipomoea palmata was purified by using TPP. The ratio of the crude extract to t-butanol of 1:1 and 30% ammonium sulfate at 37 degrees C resulted in about 160% activity recovery and twofold purification in the aqueous phase of the first cycle of TPP. On subjecting the aqueous phase to the second cycle of TPP, a purification of 18-fold was achieved with about 81% activity recovery. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed substantial purification, and the molecular weight of peroxidase was found to be 20.1 KDa. The present study shows a higher degree of purification and activity yield as a primary purification process in comparison with existing literature values, thus demonstrating TPP as an attractive downstream process for the purification of peroxidase.
Narayan
A V
AV
Department of Biotechnology, RV College of Engineering, Bangalore, India.
Madhusudhan
M C
MC
Raghavarao
K S M S
KS
D000645:Ammonium Sulfate; D005591:Chemical Fractionation / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D027741:Ipomoea / Q000201:enzymology*; D007526:Isoelectric Point; D009195:Peroxidase / Q000302:isolation & purification*; D018515:Plant Leaves / Q000201:enzymology; D013696:Temperature; D020002:tert-Butyl Alcohol
D009195:Peroxidase; D020002:tert-Butyl Alcohol; D000645:Ammonium Sulfate