Abstract

ASSESSMENT OF ION EXCHANGE CHROMATOGRAPHY PARAMETERS FOR DESIGNING AN OPTIMUM PURIFICATION PROFILE OF L-ASPARAGINASE FROM PSEUDOMONAS FLUORESCENS

Hrishikesh Mungi, Rohan Dighe and V.G.Shanmuga Priya*

Abstract

Purification is a vital step in the downstream processing of any industrially important compound. The purification profile must be designed by considering the efficacy of each step along with economics involved. The current study is based on designing an optimum purification profile for L-asparaginase, comprising ammonium sulphate fractionation, diafiltration and ion exchange chromatography. Maximum enzyme recovery in the first step was observed at 60% saturation of ammonium sulphate. The removal of ammonium ions and concentration of the sample was carried out using a 30 kDa poly ether sulphone diafiltration membrane. The optimization of purification profile was mainly concentrated on assessment of static and dynamic binding capacity of Seralite SRC 120 ion exchange resin to L-asparaginase. Optimum adsorption of enzyme to resin was observed at pH 4 within 70 minutes. The adsorption pattern was best explained by Langmuir isotherm. The adsorbed enzyme was eluted by using 0.2 M NaCl. The analysis of Height Equivalent to Theoretical Plates (HETP) shows 77% column performance, indicating the set parameters are optimum for performance of the column.

Keywords: L-asparaginase, Purification, optimization, ion exchange chromatography, adsorption, HETP.