en Biology Research (Original) <span style="font-size:11pt"><span style="background:white"><span style="line-height:normal"><span style="unicode-bidi:embed"><span sans-serif="" style="font-family:Calibri,"><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times=""><span style="color:#0f1115">L‑Asparaginase (EC 3.5.1.1) is a therapeutic enzyme that is widely used in the treatment of acute lymphoblastic leukemia (ALL) and also serves as a processing aid in the food industry. However, its clinical application is limited by poor stability, a short plasma half‑life, and significant immunogenicity. In this study, the&nbsp;<i>AsnZ</i>&nbsp;gene encoding L‑asparaginase from&nbsp;<i>Bacillus subtilis</i>&nbsp;was cloned into pET26b(+) and expressed in&nbsp;<i>Escherichia coli</i>&nbsp;BL21(DE3). The recombinant His-tagged enzyme was purified by Ni‑NTA affinity chromatography and characterized biochemically. The purified enzyme exhibited a total activity of 4.63 U/mL and a specific activity of 18.52 U/mg protein at 37 &deg;C and pH 6.0, with optimal activity observed at 37 &deg;C and pH 6.0. The enzyme retained over 80% of its activity between 30‑45 &deg;C and at pH 5.5‑7.0. Subsequently, the recombinant enzyme was successfully immobilized in chitosan‑alginate nanoparticles prepared by ionic gelation, yielding nanoparticles with an average size of 185.4 nm and a zeta potential of +32.7 mV. The nanoparticles exhibited pH-responsive swelling behavior at physiological pH (7.4), reaching approximately 180% within 8 hours, indicating their potential for controlled release. Our results demonstrate that recombinant L‑asparaginase from&nbsp;<i>B. subtilis</i>&nbsp;can be effectively produced in&nbsp;<i>E. coli</i>&nbsp;and immobilized in biodegradable polymeric nanoparticles, offering a promising platform for developing improved therapeutic formulations for leukemia treatment.</span></span></span></span></span></span></span></span><br> &nbsp; L Asparaginase, Recombinant production, Bacillus subtilis, Chitosan alginate nanoparticles, Enzyme immobilization, Acute lymphoblastic leukemia 47 56 http://tbsrj.mazums.ac.ir/browse.php?a_code=A-10-3639-2&slc_lang=en&sid=1 fateme hashemi juybari Fatemehashemi7591@gmail.com 100319475328460032135 100319475328460032135 No sareh rezaie sarhrezaee9@gmail.com 100319475328460032136 100319475328460032136 Yes