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 antineoplastic
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 lM/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.