Partitioning of Nitralase Enzyme from Pseudomonas putida in Polymer/Salt Aqueous Two Phase System

Abstract

Nitralases which belongs to class of hydrolase are an interesting group of enzymes which catalyzes the conversion of nitriles to carboxylic acids and ammonia. The partitioning behavior of nitralase produced from Pseudomonas putida has been evaluated in aqueous two phase systems (ATPS) comprised of different PEG with salts (Na2SO4, NaH2PO4, Na3C6H5O7, K3C6H5O7). The binodal and phase equilibrium data were generated experimentally for all the salt combination with PEG 1000. The tie line compositions were further estimated and are correlated using othmer-Tobias and Bancroft equations. The partition behaviour of the enzyme was investigated by studying the system parameters such as molecular weight and concentration of PEG, type of salt and their concentration, Tie line length (TLL), broth loading, pH and volume ratio. It was observed that PEG 1000 of 15 % and sodium sulfate of 10 % was found as a suitable ATPS at a TLL of 35.09 (%w/w), pH 7.5, 50 % broth loading and volume ratio of 1.5, which favors the partitioning of nitralase to top phase with maximum yield and partition coefficient.