Immobilization of L-glutaminase from Bacillus arachidis E12 on glutamic acid activated aluminum oxide nanoparticles (Al2O3 NPs): Purification, biochemical characterization, and anticancer activity

L-glutaminase (L-GLNase) was initially purified from Bacillus arachidis E12 using a three-phase partitioning (TPP) technique. The enzyme was then immobilized on glutamic acid-activated aluminum nanoparticles (GluAl₂O₃-GLNase NPs) for further characterization and evaluation of biological activity. Optimal TPP conditions—20 % (w/v) ammonium sulfate, a 1:1 (v/v) crude extract to t-butanol ratio, pH 7.0, and temperature of 25 °C—were determined using response surface methodology (RSM), resulting in a 192 % yield with 6.68-fold purification. Immobilization was most effective (96 % yield) with 1.5 U/mL enzyme, 0.1 g nanoparticles, and 24h of incubation. The immobilized enzyme demonstrated enhanced thermal and pH stability and retained 65 % of its activity after 10 reuse cycles. In vitro assays revealed no cytotoxicity against L929 normal cells, while significant anticancer activity was observed against A549 lung cancer cells. These properties support the potential of GluAl₂O₃-GLNase NPs for sustainable industrial and therapeutic applications.