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

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 (K(m), k(cat)) 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.

Anticancer properties of highly purified L-asparaginase from Withania somnifera L. against acute lymphoblastic leukemia.

Withania somnifera L. has been traditionally used as a sedative and hypnotic. The present study was carried out for the purification, characterization, and in vitro cytotoxicity of L-asparaginase from W. somnifera L. L-Asparaginase was purified from the fruits of W. somnifera L. up to 95% through chromatography. The purified L-asparaginase was characterized by size exclusion chromatography, polyacrylamide gel electrophoresis (PAGE), and 2D PAGE. The antitumor and growth inhibition effect of the L-asparaginase was assessed using [3-(4, 5-dimethyl-thiazol-2yl)-2, 5-diphenyl-tetrazolium bromide] (MTT) colorimetric dye reduction method. The purified enzyme is a homodimer, with a molecular mass of 72 +/- 0.5 kDa, and the pI value of the enzyme was around 5.1. This is the first report of the plant containing L-asparaginase with antitumor activity. Data obtained from the MTT assay showed a LD(50) value of 1.45 +/- 0.05 IU/ml. W. somnifera L. proved to be an effective and a novel source of L: -asparaginase. Furthermore, it shows a lot of similarity with bacterial L-asparaginases EC-2.

Withania somnifera (Ashwagandha): a novel source of L-asparaginase.

Different parts of plant species belonging to Solanaceae and Fabaceae families were screened for L-asparaginase enzyme (E.C.3.5.1.1.). Among 34 plant species screened for L-asparaginase enzyme, Withania somnifera L. was identified as a potential source of the enzyme on the basis of high specific activity of the enzyme. The enzyme was purified and characterized from W. somnifera, a popular medicinal plant in South East Asia and Southern Europe. Purification was carried out by a combination of protein precipitation with ammonium sulfate as well as Sephadex-gel filtration. The purified enzyme is a homodimer, with a molecular mass of 72 +/- 0.5 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography. The enzyme has a pH optimum of 8.5 and an optimum temperature of 37 degrees C. The Km value for the enzyme is 6.1 x 10(-2) mmol/L. This is the first report for L-asparaginase from W. somnifera, a traditionally used Indian medicinal plant.