Molecular Characterization of a Stable and Robust L-Asparaginase from Pseudomonas sp. PCH199: Evaluation of Cytotoxicity and Acrylamide Mitigation Potential

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Abstract

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 Pseudomonas sp. PCH199, with a high affinity for L-asparagine, was cloned and expressed in Escherichia coli BL21(DE3). Recombinant L-asparaginase (Pg-ASNase II) was purified with a monomer size of 37.0 kDa and a native size of 148.0 kDa. During characterization, Pg-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 kd values were 625.15 min and 1.10 × 10−3 min−1 at 37 °C. The kinetic constant Km, Vmax, kcat, and kcat/Km values were 0.57 mM, 71.42 U/mg, 43.34 s−1, and 77.90 ± 9.81 s−1 mM−1 for L-asparagine, respectively. In addition, the enzyme has shown stability in the presence of most metal ions and protein-modifying agents. Pg-ASNase II was cytotoxic towards the MCF-7 cell line (breast cancer) with an estimated IC50 value of 0.169 U/mL in 24 h. Further, Pg-ASNase II treatment led to a 70% acrylamide reduction in baked foods. These findings suggest the potential of Pg-ASNase II in therapeutics and the food industry.

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Kumar, S., Darnal, S., Patial, V., Kumar, V., & Singh, D. (2022). Molecular Characterization of a Stable and Robust L-Asparaginase from Pseudomonas sp. PCH199: Evaluation of Cytotoxicity and Acrylamide Mitigation Potential. Fermentation, 8(10). https://doi.org/10.3390/fermentation8100568

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