L-asparaginase, produced by Pseudomonas fluorescens, was purified by a three-phase partitioning method of t-butanol in the presence of ammonium sulphate. Enzyme recovery was enhanced by the optimization of process parameters (i.e., ammonium sulphate concentration, t-butanol ratio, temperature, and pH) using the Taguchi design of experiment (DOE) methodology. The enhanced recovery of 31.1% with 96.0% yield and purification of 10-fold was obtained…