COUPLING OF TWO PURIFICATION METHODS: AMMONIUM SULPHATE PRECIPITATION AND AQUEOUS TWO PHASE SYSTEMS

Aqueous two phase systems are specific and economic to be used for enzyme purification before high cost techniques such as chromatography. Using a commercially viable enzyme, horse radish peroxidase, as a model enzyme, a scheme was chalked out for its purification from crude extracts of horse radish roots employing precipitation and aqueous two phase systems, comprising polyethylene glycol (PEG) and ammonium sulphate. Based on results of the precipitation, two polyethylene glycol/ammonium sulphate systems were selected and using binodal curve diagrams, three compositions each of the two systems, were investigated. The enzyme showed preference for the top phase when PEG 400 was used whereas with PEG-1500, no definite trend was followed. In one of the compositions of PEG 1500/ammonium sulphate, a greater partition of the enzyme into the top phase, with restoration and enhancement of enzyme activity was observed. A purification fold of 34 was obtained with this coupled purification method