| Summary: | L-asparaginase is an important industrial enzyme widely used to treat acute lymphoblastic leukemia (ALL) and to reduce acrylamide formation in food products. In the current study, a stable and robust L-asparaginase from <i>Pseudomonas</i> sp. PCH199, with a high affinity for L-asparagine, was cloned and expressed in <i>Escherichia coli</i> BL21(DE3). Recombinant L-asparaginase (<i>Pg</i>-ASNase II) was purified with a monomer size of 37.0 kDa and a native size of 148.0 kDa. During characterization, <i>Pg</i>-ASNase II exhibited 75.8 ± 3.84 U/mg specific activities in 50.0 mM Tris-HCl buffer (pH 8.5) at 50 °C. However, it retained 80 and 70% enzyme activity at 37 °C and 50 °C after 60 min, respectively. The half-life and <i>k</i><sub>d</sub> values were 625.15 min and 1.10 × 10<sup>−3</sup> min<sup>−1</sup> at 37 °C. The kinetic constant <i>K</i><sub>m</sub>, <i>V</i><sub>max</sub>, <i>k</i><sub>cat</sub>, and <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> values were 0.57 mM, 71.42 U/mg, 43.34 s<sup>−1</sup>, and 77.90 ± 9.81 s<sup>−1</sup> mM<sup>−1</sup> for L-asparagine, respectively. In addition, the enzyme has shown stability in the presence of most metal ions and protein-modifying agents. <i>Pg</i>-ASNase II was cytotoxic towards the MCF-7 cell line (breast cancer) with an estimated IC<sub>50</sub> value of 0.169 U/mL in 24 h. Further, <i>Pg</i>-ASNase II treatment led to a 70% acrylamide reduction in baked foods. These findings suggest the potential of <i>Pg</i>-ASNase II in therapeutics and the food industry.
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