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  • Title: Characterization and applications of glutaminase free L-asparaginase from indigenous Bacillus halotolerans ASN9.
    Author: Shafqat I, Shahzad S, Yasmin A, Chaudhry MT, Ahmed S, Javed A, Afzal I, Bibi M.
    Journal: PLoS One; 2023; 18(11):e0288620. PubMed ID: 38015853.
    Abstract:
    L-asparaginase (L-ASNase) is a versatile anticancer and acrylamide reduction enzyme predominantly used in medical and food industries. However, the high specificity of L-asparaginase formulations for glutamine, low thermostability, and blood clearance are the major disadvantages. Present study describes production, characterization, and applications of glutaminase free extracellular L-asparaginase from indigenous Bacillus halotolerans ASN9 isolated from soil sample. L-asparaginase production was optimized in M9 medium (containing 0.2% sucrose and 1% L-asparagine) that yielded maximum L-ASNase with a specific activity of 256 U mg-1 at pH 6 and 37°C. L-asparaginase was purified through acetone precipitation and Sephadex G-100 column, yielding 48.9 and 24% recovery, respectively. Enzyme kinetics revealed a Vmax of 466 mM min-1 and Km of 0.097 mM. Purified L-ASNase showed no activity against glutamine. The purified glutaminase free L-ASNase has a molecular mass of 60 kDa and an optimum specific activity of 3083 U mg-1 at pH 7 and 37°C. The enzyme retains its activity and stability over a wide range of pH and temperature, in the presence of selected protein inhibitors (SDS, β-mercaptoethanol), CoCl2, KCl, and NaCl. The enzyme also exhibited antioxidant activity against DPPH radical (IC50 value 70.7 μg mL-1) and anticancer activity against U87 human malignant glioma (IC50 55 μg mL-1) and Huh7 human hepatocellular carcinoma (IC50 37 μg mL-1) cell lines. Normal human embryonic kidney cells (HEK293) had greater than 80% cell viability with purified L-ASNase indicating its least cytotoxicity against normal cells. The present work identified potent glutaminase free L-ASNase from B. halotolerans ASN9 that performs well in a wide range of environmental conditions indicating its suitability for various commercial applications.
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