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Reverse micellar extraction (RME), a liquid-liquid based separation is a versatile tool for protein purification. A statistical approach was employed for the purification of recombinant glutaminase free anti-cancerous enzyme viz., l-asparaginase II to evaluate the effects of RME in current study. The cationic system (CTAB/iso-octane/hexanol/butanol) was used in RME to optimize both forward and backward protein extraction efficiency. By adapting Taguchi's orthogonal array (OA), maximum forward extraction efficiency (FEE) of 86.98% with 84.82% enzyme activity recovery and 1.04 times purification fold achieved with the optimized parameters. Under the optimal levels, the back extraction efficiency (BEE) was observed to be 96.97% with 93.07% enzyme activity recovery and 1.38 times purification fold. Further, mass transfer kinetic studies of RME indicated the mass transfer coefficients of forward and backward extraction to be 0.049 min -1 and 0.036 min -1 respectively., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In this study, reverse micellar extraction of papain model system was performed using cetyltrimethylammonium bromide (CTAB)/iso-octane/hexanol/butanol system to optimize the forward and back extraction efficiency (BEE). A maximum forward extraction efficiency of 55.0, 61.0, and 54% was achieved with an aqueous phase pH of 11.0, 150 mM CTAB/iso-octane and 0.1 M NaCl, respectively. Taguchi's orthogonal array was applied to optimize the pH of stripping phase, concentration of isopropyl alcohol (IPA) and potassium chloride (KCl) for maximizing BEE. The optimal levels of stripping phase pH, concentration of IPA and KCl were found to be 6, 20% (v/v), and 0.8 M, respectively. Under these optimal levels, the BEE was found to be 88% after which enzyme activity was recovered with 2.5-fold purification. Further optimization was performed using artificial neural network-linked genetic algorithm, where the BEE was improved to 90.52% with pH 6, IPA (%) = 19.938, and KCl (M) = 0.729.

Gene encoding glutaminase-free L-asparaginase II (ans B2) from Pectobacterium carotovorum MTCC 1428 was cloned into pHT43, transformed in Bacillus subtilis WB800N and optimised the expression levels of recombinant enzyme. A three-fold higher enzyme production was observed with an efficient transformant as compared to native strain. Enzyme localization studies revealed that >90% of recombinant enzyme is secreted extracellularly, a little fraction is attached to the membrane (>6%) and localised intracellularly (3%). The expression of recombinant L-asparaginase II was confirmed by SDS-PAGE, IMAC (Immobilised metal ion affinity chromatography) purification followed by Western blotting. Process parameter optimization with OFAT (one factor at a time) revealed that rpm (120), temperature (37 °C), Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration (1 mM) and time of induction (0.8 OD600nm) plays a vital role where a maximum of 55 IU/ml was achieved. Further, consecutive induction by IPTG improved the enzyme production up to 105 IU/ml with a specific activity of 101 IU/mg of protein. Molecular modelling analysis depicted that amino acids, GLY60, GLY119 and ALA252 in the active site are responsible for the glutaminase free L-asparaginase II activity. This is the first report on enhanced expression of recombinant glutaminase-free L-asparaginase II by intermediate addition of IPTG.