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JARMS. 2012; 4(4): 293-301


Production, Purification and Characterization of extracellular L-Asparaginase having anti neoplastic activity from Fusarium sp.

*Asha Abraham,*Pallavi Baliga.

Abstract
The present investigation, deals with the production, purification and evaluation of extracellular L-Asparaginase produced by Fusarium sp. and its effect on EAC cell lines and human lymphocytes in vitro. Evaluation of various media for optimal production of the enzyme was carried out. Submerged fermentation in Czapek medium with L-asparagine as a nitrogen source was found to give better yield. The enzyme was purified to near homogeneity by ammonium sulphate precipitation, dialysis, gel filtration on Sephadex G-100 column, DEAE cellulose column and SDS-PAGE. The enzyme was purified 242.65 fold and showed a final specific activity of 2.9118 IU/ml/mg protein with 1.5% yield. SDS PAGE of purified enzyme showed that the purified enzyme consists of 3 subunits of molecular weights 79.4 KDa, 70.8KDa, 44.7 KDa respectively. A Lineweaver Burk analysis showed a Km value of 443.98mM and Vmax of 40 IU. The enzyme showed maximum activity at pH 9 when incubated at 37C for 10 min. Also the L-Asparaginase demonstrated significant cytotoxic effect on Ehrlich Ascites Carcinoma cells in vitro. It is noteworthy that the enzyme did not elicit any immunostimulatory response in human lymphocytes in vitro unlike most of the reported prokaryotic asparaginases. These properties of Fusarium asparaginase is ideal for cancer therapy.

Key words: L-Asparaginase activity; Fusarium; anti neoplastic; submerged fermentation; characterization.



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