PURIFICATION, CHARACTERIZATION AND EVALUATION OF L-ASPARAGINASE FROM ENDOPHYTIC FUNGI FUSARIUM SP. LCJ273 FOR ITS ANTICANCER PROPERTY

Abstract

L-Asparaginase (L-asparagine amido-hydrolase, EC 3.5.1.1) is an industrially and pharmaceutically important enzyme that catalyses the hydrolysis of asparagine into aspartic acid and ammonia. In this study, L-asparaginase was produced from Fusarium sp. LCJ273. The crude L-asparaginase produced was precipitated using ammonium sulphate precipitation followed by purification using ion exchange chromatography. The molecular weight of L-asparaginase was found to be approximately 66 kDa by SDS-PAGE. The culture filtrate, ammonium sulphate precipitate and partially purified Lasparaginase exhibited optimum activity in pH 7.0 at 30˚C. The partially purified L-asparaginase from Fusarium sp. LCJ273 showed 100% relative activity towards the natural substrate L-asparagine. Further, the kinetics studies of partially purified L-asparaginase revealed that Km value was 24.2 mM and Vmax value was 125 mM min-1. The in vitro anticancer activity of partially purified L-asparaginase was investigated. Different concentration of partially purified enzyme was tested on three different cell lines namely MCF-7 (breast cancer cell line), Hep-2 (human laryngeal carcinoma cells) and vero cell lines by MTT assay (3-4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide). Purified L-asparaginase reduced cell viability of MCF-7 (16.61%), Hep-2 (8.12%) and vero cell line (88.73%) after 72 hours with IC50 value of 33.21, 9.28 and 622.99 μg/mL respectively and showed better toxicity on the cell lines at a concentration of 120 μg/mL.