A HIGHLY PURIFIED L-GLUTAMINASE FROM IMMOBILIZED PSEUDOMONAS SP. RAS123 CULTURES WITH ANTITUMOR AND ANTIBACTERIAL ACTIVITIES

Abstract

L-glutaminase (E.C.3.5.2.1) is an antineoplastic enzyme and in the present study, an extracellular L-glutaminase was produced from a marine local strain identified as Pseudomonas sp. RAS123. The enzyme was produced from free cultures and from cultures immobilized on and in different supports. Pseudomonas sp. RAS123 L-glutaminase produced from immobilized cultures was purified to homogeneity. The specific activity of the enzyme reached 698.655 U/mg protein, with Km and Vmax value of 3.2 mg/ml and 2000 U/ml, respectively. A single band with a molecular weight of about 32.0 kDa was produced by the purified enzyme on SDS-PAGE. Further findings indicated that the pure enzyme's maximum activity occurred at 50°C and pH 9. The enzyme was stable at 60°C for 60 min and in the pH range of 8.0 to 10.0, The effect of chemicals showed that Mn²⁺, Mg²⁺, Ni²⁺ and Fe²⁺activated the enzyme, while SDS (10% w/v) strongly inhibited the activity of the enzyme. The purified enzyme showed cytotoxic activity against HCT-116, HepG2, MCF-7, HeLa, and CCL-86 cell lines tested with IC₅₀ values of 122, 175, 195, 306, and > 500 µg/ml, respectively. Also, the antibacterial effect of the enzyme showed activity against Staphylococcus aureus, Bacillus subtilis, Streptococcus mutants, Enterobacter cloacae and Escherichia coli. These findings demonstrate that L-glutaminase might be used in numerous biotechnological applications, particularly food and pharmaceutical processing.