Recombinant production and characterization of L-glutaminase (glsA) as a promiscuity therapeutic enzyme.

Because of the therapeutical impacts of hydrolytic enzymes in different diseases, in particular malignancies, we aimed to produce a recombinant putative L-glutaminase (GLS A SL-1 ) from a recently characterized halo-thermotolerant Bacillus sp. SL-1. For this purpose, the glsA gene was identified and efficiently overexpressed in the Origami™ B (DE3) strain. The yield of the purified GLS A SL-1 was ~ 20 mg/L, indicating a significant expression of recombinant enzyme in the Origami. The enzyme activity assay revealed a significant hydrolytic effect of the recombinant GLS A SL-1 on L-asparagine (Asn) (i.e., K m 39.8 μM, k cat 19.9 S -1 ) with a minimal affinity for L-glutamine (Gln). The GLS A SL-1 significantly suppressed the growth of leukemic Jurkat cells through apoptosis induction (47.5%) in the IC 50 dosage of the enzyme. The GLS A SL-1 could also change the Bax/Bcl2 expression ratio, indicating its apoptotic effect on cancer cells. The in silico analysis was conducted to predict structural features related to the histidine-tag exposure in the N- or C-terminal of the recombinant GLS A SL-1 . In addition, molecular docking simulation for substrate specificity revealed a greater binding affinity of Asn to the enzyme binding-site residues than Gln, which was confirmed in experimental procedures as well. In conclusion, the current study introduced a recombinant GLS A SL-1 with unique functional and structural features, highlighting its potential pharmaceutical and medical importance. GLS A SL-1 represents the first annotated enzyme from Bacillus with prominent asparaginase activity, which can be considered for developing alternative enzymes in therapeutic applications. KEY POINTS: • Hydrolytic enzymes have critical applications in different types of human malignancies. • A recombinant L-glutaminase (GLS A SL-1 ) was produced from halo-thermotolerant Bacillus sp. SL-1. • GLS A SL-1 displayed a marked hydrolytic activity on L-asparagine compared to the L-glutamine. • GLS A SL-1 with significant substrate promiscuity may be an alternative for developing novel pharmaceuticals.