Novel glutaminase-free l-asparaginase from Leuconostoc mesenteroides BN1: biochemical properties, molecular dynamics, and anticancer activity.

l-Asparaginase is an essential chemotherapeutic enzyme used to treat acute lymphoblastic leukaemia and certain lymphoid malignancies; however, its clinical use can be limited by adverse effects linked to immunogenicity and undesirable l-glutaminase activity. In this study, a novel glutaminase-free l-asparaginase from Leuconostoc mesenteroides BN1 (LmASNase) was cloned, expressed in Escherichia coli, and biochemically characterised. The recombinant enzyme appeared to form a homotetramer and demonstrated specificity towards l-asparagine, with no detectable activity towards l-glutamine. LmASNase showed optimal activity at pH 5.5-6.5 and 37 °C, and showed substantial thermal stability near physiological pH. Molecular dynamics simulations revealed stable binding of l-asparagine and predicted Pro52, Ser53, and Ala109 of LmASNase as residues involved in substrate specificity. In addition, LmASNase caused concentration-dependent reductions in the viability of Raji, Jurkat, KG-1, Kasumi-1, and K562 cells, with IC50 values of 6.27, 3.77, 0.76, 0.72, and 7.06 U mL-1, respectively, while showing limited cytotoxicity towards peripheral blood mononuclear cells. These findings support further preclinical evaluation of the anticancer activity of LmASNase.