| Summary: | L-asparaginases from bacterial sources have been used in antineoplastic treatments and the food industry. A type II L-asparaginase encoded by the N-truncated gene <i>ansZP21</i> of halotolerant <i>Bacillus subtilis</i> CH11 isolated from Chilca salterns in Peru was expressed using a heterologous system in <i>Escherichia coli</i> BL21 (DE3)pLysS. The recombinant protein was purified using one-step nickel affinity chromatography and exhibited an activity of 234.38 U mg<sup>−1</sup> and a maximum catalytic activity at pH 9.0 and 60 °C. The enzyme showed a homotetrameric form with an estimated molecular weight of 155 kDa through gel filtration chromatography. The enzyme half-life at 60 °C was 3 h 48 min, and L-asparaginase retained 50% of its initial activity for 24 h at 37 °C. The activity was considerably enhanced by KCl, CaCl<sub>2</sub>, MgCl<sub>2</sub>, mercaptoethanol, and DL-dithiothreitol (<i>p</i>-value < 0.01). Moreover, the <i>V</i><sub>max</sub> and <i>K</i><sub>m</sub> were 145.2 µmol mL<sup>−1</sup> min<sup>−1</sup> and 4.75 mM, respectively. These findings evidence a promising novel type II L-asparaginase for future industrial applications.
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