Characterization and bioactivities of glutaminase-free L-asparaginase from Salinicola acorporae S4-41

The medical community has long searched for a safer and more effective cancer treatment method. Clinically, L-asparaginase derived from bacteria has shown promising results. However, it carries the risk of immunological and hypersensitive reactions due to its interaction with glutaminase. Therefore, finding a new source of glutaminase-free L-asparaginase (ASNase) is important. In this study, 35 bacterial isolates from 12 saline environmental samples were tested for ASNase, and the most promising bacterium was identified as Salinicola acroporae S4-41. This bacterium produces intracellular ASNase, which could treat cancer with fewer side effects. Agitation enhanced production by nearly five-fold compared to static conditions. The enzyme activity peaked at 40 °C and pH 8 after 96 h in a medium containing 100 g L-1 of NaCl, 2 g L-1 of glucose, and 15 g L-1 of L-asparagine. The purified enzyme was 65 kDa in size, with optimal activity in 1 M NaCl, 0.05 M L-asparagine, 0.1% DMSO, urea, and Tween 80 at pH 8 and 40 °C. The enzyme’s kinetics showed a Km of 0.007271 mM and a Vmax of 84.31 U mL⁻¹ min⁻¹. Pure ASNase significantly inhibited the growth of cancer cell lines NB4, MCF-7, and HepG-2. S. acroporae produces fewer harmful ASNase enzymes, which could improve cancer treatment. This is the first report of ASNase production from S. acroporae S4-41. Salinicola acroporae S4-41 (GenBank accession number SUB12091824; OP521775), a halotolerant bacterium isolated from the rhizosphere soil of cogon grass (Imperata cylindrica), was found to produce glutaminase-free L-asparaginase, a significant benefit for lowering treatment-related side effects, making it a promising candidate for safer therapeutic applications. Quantitative analysis revealed an activity of 10.72 U/mL, which increased significantly under optimized physicochemical conditions. Its maximal output (58.76 U/mL) attained at 40 °C and 1.5% L-asparagine, and it also showed notable pH stability at pH 8. The isolate showed improved enzyme output under shaking conditions and adaptability over a wide salinity range (0–25% NaCl). Although activity decreased with ethanol and inhibitors such SDS and EDTA, the isolated enzyme maintained significant stability in the face of heat, pH, and nonionic surfactants (Tween 80, DMSO). These findings demonstrate the enzyme’s surfactant-stable, halotolerant, and thermotolerant characteristics could serve as a safer alternative to currently used bacterial asparaginases by reducing adverse immunological reactions, highlighting its potential for use in pharmaceutical and biotechnological applications. To confirm its medicinal potential and increase its industrial utility, more research is necessary, especially in vivo assessments.