Cloning, expression and characterization of l-asparaginase from Withania somnifera L. for large scale production

Abstract

l-Asparaginase (E.C. 3.5.1.1) is used as a therapeutic agent in the treatment of acute childhood lymphoblastic leukemia. It is found in a variety of organisms such as microbes, plants and mammals. In plants, l-asparaginase enzymes are required to catalyze the release of ammonia from asparagine, which is the main nitrogen-relocation molecule in these organisms. An Indian medicinal plant, Withania somnifera was reported as a novel source of l-asparaginase. l-Asparaginase from W. somnifera was cloned and overexpressed in E. coli. The enzymatic properties of the recombinant enzyme were investigated and the kinetic parameters (Km, kcat) for a number of substrates were determined. The kinetic parameters of selected substrates were determined at various pH and the pH- and temperature-dependence profiles were analyzed. WA gene successfully cloned into E. coli BL21 (DE3) showed high asparaginase activity with a specific activity of 17.3 IU/mg protein.

Biochemical Characterization of a Novel l -Asparaginase with Low Glutaminase Activity from Rhizomucor miehei and Its Application in Food Safety and Leukemia Treatment

“…The specific activity of RmAsnase was similar to that of L-asparaginase from Pectobacterium carotovorum (2,020.9 U/mg) (11) but much higher than the specific activities of most bacterial and fungal L-asparaginases (14,(30)(31)(32). The optimum pH and temperature of RmAsnase are similar to those reported for many L-asparaginases (2,31,33). Moreover, the enzyme showed negligible activity with L-glutamine as the substrate.…”

“…RmAsnase, the fungal L-asparaginase from R. miehei, showed antiproliferative activity against cells from several human leukemia cell lines and displayed very low L-glutaminase activity. Thus, RmAsnase may also be useful as an alternative to bacterial L-asparaginases in leukemia treatment (2,6,11,32,35).…”

“…The enzyme is widely distributed among plants, animals, and microorganisms (1)(2)(3). It can be used to reduce the formation of acrylamide in baked goods (4)(5)(6), and its antileukemic capacity has garnered much attention for its application in the treatment of malignancies of the lymphoid system, acute lymphoblastic leukemia, and non-Hodgkin's lymphoma (7).…”

In vitro cytotoxic study for partially purified L-asparaginase from fresh leaves, unripe and ripe fruits of Withania somnifera plant

“…L-asparaginase (E.C. 3.5.1.1) is used as a therapeutic agent in the treatment of acute childhood lymphoblastic leukemia (Oza et al, 2011). L-aspara-ginases are known chemotherapeutic agents against cancer, such as acute lymphoblastic leukemia and lymphosarcoma.…”

“…L-aspara-ginases are known chemotherapeutic agents against cancer, such as acute lymphoblastic leukemia and lymphosarcoma. Several recent reviews are available concerning the use of L-asparaginase in cancer therapy (Muller and Boos, 1998;Oza et al, 2011). Leukaemic cells lacking the mammalian asparagine-synthetase enzyme depend on exogenous sources of asparagine for protein synthesis and survival.…”