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Research Article Open access CC BY 3.0

Purification, Characterization and Antitumor Activity of L-asparaginase from Penicillium brevicompactum NRC 829

Ali Mohamed Elshafei, Mohamed Mohamed Hassan, Mohamed Abd-Elmontasr Abouzeid, Dalia Ali Mahmoud, Dina Helmy Elghonemy

Microbiology Research Journal International · pp. 158–174 · Published 8 Dec 2012

10.9734/BMRJ/2012/1735

Abstract

Aim: The aims of this study were to attempt to extract, purify and characterize of L-asparaginase, an antitumor agent, from Penicillium brevicompactum NRC 829. Study Design: Testing of antitumor activity of L-asparaginase against four different tumor human cell lines. Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and Biotechnology Division, National Research Centre (NRC), Cairo, Egypt, between June 2010 and November 2011. Methodology: Penicillium brevicompactum NRC 829, a local isolated strain from Culture Collection of the National Research Centre of Egypt, was grown and maintained on modified Czapek Dox medium. The fresh fungal biomass was thoroughly ground with washed cold sand. The cell contents were extracted with cold 0.1M Tris-HCl pH 8.0, thereafter, the slurry obtained was centrifuged at 5500 rpm for 15 min and the supernatant was directly used as the source of enzyme. The purification of L-asparaginase from crude-enzyme extracts of P. brevicompactum was achieved by a sequential multi-steps process starting by heat treatment for 20 min at 50ºC, followed by gel filtration on Sephadex G-100 column, and the most active fractions of L-asparaginase were dialyzed out, lyophilized and then loaded on a Sephadex G-200 column. Results: An intracellular glutaminase-free-L-asparaginase from Penicillium brevicompactum NRC 829 was purified to homogeneity with an apparent molecular mass (Mr) of 94 kDa. The purified enzyme was 151.12 fold with a final specific activity of 574.24 IU/mg protein and about 40% yield recovery. The purified L-asparaginase showed its maximal activity against L-asparagine when incubated at pH 8.0 at 37ºC for 30 min. The enzyme was more stable at alkaline pH than the acidic one and thermally stable up to 60 min at 50-60ºC. L-asparaginase was highly specific for its natural substrate, L-asparagine with a Km value of 1.05 mM. The activity of L-asparaginase is activated by mono cations and various effectors including K+, Na+, 2-mercaptoethanol (2-ME), and reduced glutathione (r-GSH), whereas it is moderately inhibited by various divalent ions including Hg2+, Cu2+, and Ag+. Results indicated the involvement of sulfhydryl group(s) in the enzyme active site(s). The purified enzyme inhibited the growth of human cell line hepatocellular carcinoma (Hep-G2), with IC50 value of 43.3μg/ml. Conclusion: L-asparaginase purified from Penicillium brevicompactum NRC 829 is a potential candidate for medical applications.

L-asparaginase Penicillium brevicompactum NRC 829 purification kinetic properties antitumor activity

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