Cloning, expression and characterization of L-asparaginase from Pseudomonas fluorescens for large scale production in E. coli BL21.
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 Vmaxof 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.
Department of Biotechnology, Sapthagiri College of Engineering, Bangalore, 560 057, India.
Vijay
V
Kishore
Fermentation Technology and Bioengineering Department, Central Food Technological Research Institute (CSIR), Mysore, 570 020, India.
K P
KP
Nishita
Fermentation Technology and Bioengineering Department, Central Food Technological Research Institute (CSIR), Mysore, 570 020, India. vjykshr@gmail.com.
H K
HK
Manonmani
Cloning; E. coli; Enzyme activity; L-Asparaginase