Production, Immobilization and Anti-tumor Activity of L- Asparaginase of Bacillus sp R36

L-asparaginase is one of the known drugs in the treatment of cancer, especially acute lymphoblastic leukemia. In recent years several bio-conjugation protocols have been developed to improve the pharmacokinetic and immunological properties of anti-leukemic enzyme, L-asparaginase. In this study, fifty two bacilli species were newly isolated in our laboratory and screened for their ability to produce extracellular and intracellular Lasparaginase enzyme. Bacillus sp R36 gave the highest intracellular enzyme production. Formation physiology of the enzyme revealed that optimum culture conditions were 9:1 of air::medium ratio, with 55 x 10 CFU/mL inoculums size. The optimum incubation period was found to be 24 hours under shaking growth conditions. The initial pH value 5.6 was favorable for the highest enzyme production. Addition of 1% lactose or 1% raffinose resulted in a doubled enzyme productivity (yielded 204% and 209%, respectively). The enzyme was efficiently immobilized by covalent binding with activated carbon. Immobilized L-asparaginase activity was 33.0 U/g carrier; with immobilization yield of 73.6%. Characterization of the enzyme was performed on native and immobilized forms. Optimum pH value was 7.0 for free and immobilized forms. Optimum reaction temperature was 50 °C for native enzyme, while it was 60 °C for the immobilized enzyme preparation. The immobilization process greatly enhanced the thermal stability of the enzyme. Native L-asparaginase enzyme exhibited thermal stability up to 50 °C, while immobilized form retained 100% of its activity up to 80 °C. Anti-tumor and antioxidant activities were investigated. The enzyme inhibited the growth of two human cell lines including hepatocellular carcinoma (Hep-G2) and colon carcinoma (HCT-116) with IC50 value of 112.19 μg/mL and 218.7 μg/mL, respectively. [Journal of American Science 2010;6(8):157-165]. (ISSN: 1545-1003).