Optimizing the Production of Soluble Form of Recombinant L-asparaginase isolated from Halophilic Bacterium in Escherichia coli

L-asparaginase has an essential role in the treatment of cancers, especially Acute Lymphoblastic Leukemia. The rapid removal of the enzyme from the bloodstream, glutaminase activity, and also inducing an immune response in the body are disadvantages of commercial enzymes, therefore, the identification of new L-asparaginase with desirable properties is essential. Previously, the coding sequence of L-asparaginase isolated from Halomonas elongata had been cloned in E. coli and had shown to have advantageous properties as a chemotherapeutic agent. However, it was mostly expressed as inclusion bodies.

In this study, soluble enzyme expression was examined in two E. coli hosts, BL21 (DE3) and ArcticExpress strains. After selecting the host, the effects of various factors such as culture media, inducer concentration, inoculum size, induction duration, temperature, and also aeration rate were optimized using the one-factor-at-a-time approach.

Based on the results, the highest amount of active L-asparaginase production was related to Escherichia coli host Bl21 (DE3) strain in LB culture medium by adding 1% inoculum size, after 9 h induction with 0.5 mM IPTG at 37 ˚C, and shaking at 150 rpm. In the next step, the effect of various additives on the culture medium was investigated and it was observed that the addition of ethanol 2% (v/v) or arginine (200 mM) has a significant effect on increasing the specific activity of the enzyme.

Finally, the optimization could increase soluble L-asparaginase production from 1000 U/mg in the basal condition up to approximately 3500 U/mg (more than three times).