Abstract
l-Asparaginase (E.C. 3.5.1.1) is used as an anti-neoplastic drug in the treatment of acute lymphoblastic leukemia. l-Asparaginase from Pseudomonas fluorescens was cloned and overexpressed in E. coli BL21. The Enzyme was found to be a Fusion protein-asparaginase complex which was given a lysozyme treatment and sonication, and then was purified in a Sepharose 6B column. The enzymatic properties of the recombinant enzyme were studied and the kinetic parameters were determined with kilometre of 109.99 mM and Vmax of 2.88 µM/min. Recombinant enzyme showed pH optima at 6.3 and temperature optima at 34 °C. Asp gene was successfully cloned into E. coli BL21 which produced high level of asparaginase intracellularly with 85.25 % recovery of enzyme with a specific activity of 0.94 IU/mg protein. The enzyme was a tetramer with molecular weight of approximately 141 kDa.
Highlights
L-Asparaginase enzymes (L-asparagine amidohydrolase) catalyse the hydrolysis of L-asparagine to L-aspartate and ammonia, and to a lesser extent, the hydrolysis of L-glutamine to L-glutamate (Ebrahiminezhad et al 2011)
The DNA yield obtained through Lysozyme method from Pseudomonas fluorescens was of good quantity and suitable for further use
We have produced L-asparaginase by cloning the asparaginase gene from Pseudomonas sp. into E. coli, for that we started our work with isolation of DNA, primer designing for asparaginase gene, cloning into pET101 for expression and pTZ57 R/T vector for sequencing, expression of the Asparaginase gene into E. coli BL21, obtaining large quantity of enzyme using 10 L fermenter, purifying the enzyme and characterizing the cloned purified Asparaginase was carried out
Summary
L-Asparaginase enzymes (L-asparagine amidohydrolase) catalyse the hydrolysis of L-asparagine to L-aspartate and ammonia, and to a lesser extent, the hydrolysis of L-glutamine to L-glutamate (Ebrahiminezhad et al 2011). Bacterial L-asparaginase are of two types: type 1 and type 2; type 2 showed antitumor activity because of which interest in Lasparaginase arose (Lee et al 1989). LAsparaginases with high asparaginase activity and negligible glutaminase activity are reported to be less troublesome during the course of antitumor therapy (Hawkins et al 2004). The search for other asparaginase sources, with new immunological characteristics can lead to enzyme with less adverse effects. Introduction of new fermentation and purification protocols for production of L-asparaginase II will be mandatory to satisfy these demands (Aghaeepoor et al 2011). In this study we will be describing the cloning, expression, purification and characterization of recombinant L-asparaginase from P. fluorescens into E. coli BL21
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