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

• First report on three phase partitioning (TPP) of peroxidase from oranges peels.
• Oranges peel used as source of enzyme which is waste and easily available.
• Effects of various process parameters on purity and recovery have been studied.
• Process has been optimized to get maximum purity and activity recovery.
• Peroxidase after TPP is characterized by SDS-PAGE.

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 °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.