Three phase partitioning a novel technique for purification of peroxidase from orange peels (Citrus sinenses)

The present work describes the work on three phase partitioning (TPP) of a peroxidase enzyme from orange peels (Citrus sinenses). TPP is a bioseparation technique which has been used for the extraction, concentration and purification of enzymes as well as natural products. Three phase system is formed with simultaneous addition of ammonium sulfate directly to crude broth/feed followed by addition of t-butanol. Effect of different process parameters such as time, ammonium sulfate concentration, pH, broth to t-butanol ratio, temperature and speed of agitation have been studied on purification fold and activity recovery. Maximum purity of 18.20-fold with 93.96% recovery has been obtained with optimized parameters. Optimized system comprised of solution saturated with 50% ammonium sulfate at pH 6 and temperature 30 degrees C with broth to t-butanol ratio 1:1.5 (v/v) with 200 rpm with for 80 min. SDS PAGE analysis of partially purified enzyme shows molecular weight of 26 kDa. TPP was found to be an attractive technique for the extraction and partial purification of peroxidase from orange peels. (C) 2014 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.