High yield expression of novel glutaminase free l-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Bacillus subtilis WB800N

摘要

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 A degrees C), Isopropyl beta-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.