L-asparaginase is an enzyme catalyzing the hydrolysis of L-asparagine into L-aspartate and ammonia, which is of great therapeutic importance in tumor treatment. However, commercially available enzymes are associated with adverse effects, and searching for a new L-asparaginase with better pharmaceutical properties was the aim of this work. The coding sequence for Mycobacterium smegmatis L-asparaginase (MsA) was cloned and expressed. The recombinant protein showed high activity toward L-asparagine, whereas none was detected for L-glutamine. The enzymatic properties (Kₘ = 1.403 ± 0.24 mM and kcat = 708.1 ± 25.05 s‾¹) indicate that the enzyme would be functional within the expected blood L-asparagine concentration, with good activity, as shown by kcat. The pH and temperature profiles suggest its use as a biopharmaceutical in humans. Molecular dynamics analysis of the MsA model reveals the formation of a hydrogen bond network involving catalytic residues with L-asparagine. However, the same is not observed with L-glutamine, mainly due to steric hindrance. Additionally, the structural feature of residue 119 being a serine rather than a proline has significant implications. These findings help explain the low glutaminase activity observed in MsA, like what is described for the Wolinella succinogenes enzyme. This establishes mycobacterial asparaginases as key scaffolds to develop biopharmaceuticals against acute lymphocytic leukemia.