Exploring Escherichia coli EGY Type II L-Asparaginase Variant of Unique Glutaminase Activity: Cloning, Expression, Biochemical Characterization, and Molecular Docking Analysis.

Microbial L-asparaginase II is a cornerstone biopharmaceutical drug for the effective treatment of leukemia and other blood cancers. However, the associated cellular resistance mechanisms in response to the treatment require effective management and necessitate searching and developing of better versions of the enzyme. Overall, the current study described a new E. coli type II L-ASNase variant of unique L-glutaminase co-activity that was cloned into pET28a ( +) vector and expressed in E. coli BL21(DE3) pLysS as a cytosolic protein with a molecular weight of 38.390 kDa. The recombinant E. coli L-ASNase was purified by affinity chromatography and showed high specific activity of 7179.5 U/mg. The purified enzyme exhibited enormous thermal stability and its affinity for L-asparagine (L-Asn) and L-glutamine (L-Gln) was in silico analyzed by molecular docking and experimentally verified which indicated relatively similar affinity. The purified E. coli L-ASNase demonstrated high dual activities toward L-Asn (Km and Vmax 0.627 mM and 385 µmol min^-1) with 90% specificity toward L-Gln (Km and Vmax 0.715 mM and 376 µmol min^-1). The molecular docking study suggested that E. coli EGY L-ASNase follows a single displacement mechanism of catalysis based on the interacting residues analysis from a computational insight. Biochemical characterization and immunogenicity prediction were carried out to contribute for better understanding of the immunogenic determinants along with the other features of the new variant to consider its potentiality for in vitro and in vivo cytotoxicity testing in future studies.