title: High yield expression of novel glutaminase free L-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Bacillus subtilis WB800N.
abstract: 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.
authors.affiliation: Biochemical Engineering Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology (IIT) Guwahati, Guwahati, Assam, 781039, India.
authors.forename: Sushma
authors.initials: S
authors.lastname: Chityala
authors.affiliation: Biochemical Engineering Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology (IIT) Guwahati, Guwahati, Assam, 781039, India. veeranki@iitg.ernet.in.
authors.forename: Veeranki
authors.initials: V
authors.lastname: Venkata Dasu
authors.affiliation: Bioengineering and Environmental Sciences, Indian Institute of Chemical Technology, Hyderabad, 500007, India.
authors.forename: Jamal
authors.initials: J
authors.lastname: Ahmad
authors.affiliation: Bioengineering and Environmental Sciences, Indian Institute of Chemical Technology, Hyderabad, 500007, India.
authors.forename: Reddy Shetty
authors.initials: RS
authors.lastname: Prakasham
mesh_terms: D001215:Asparaginase / Q000096:biosynthesis* / Q000235:genetics; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D016973:Pectobacterium carotovorum / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001215:Asparaginase
keywords: B. subtilis WB800N; Glutaminase free enzyme; IPTG induction; L-Asparaginase II; Transformation; pHT43 vector