RESUMO
Aim: The aims of the present study were to purify and characterize L-glutaminase from Penicillium brevicompactum NRC 829; and to evaluate the antitumor activity of the purified enzyme against different tumor human cell lines. Study Design: Testing of antitumor activity of L-glutaminase, purified from a filamentous fungal strain, 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 January 2011 and February 2012. Methodology: P. brevicompactum NRC 829 was grown and maintained on modified Czapek Dox agar (MCD) medium. Cell-free extract was directly used as the source of crude enzyme. L-glutaminase was purified by heat treatment for 20 min at 50ºC, followed by gel filtration on Sephadex G-100 and G-200 columns. Results: An intracellular L-glutaminase from Penicillium brevicompactum NRC 829 was purified to homogeneity (162.75 fold) with an apparent molecular mass (Mr) of 71 kDa. The purified enzyme showed its maximal activity against L-glutamine when incubated at pH 8.5 at 50ºC for 30 min. The purified enzyme retained about 92 % of its initial activity after incubation at 70ºC for 30 min indicating the thermo-stability nature of this enzyme. The highest activity was reported towards its natural substrate, L-glutamine, with an apparent Km value of 1.66 mM. The purified enzyme inhibited the growth of human cell line hepatocellular carcinoma (Hep-G2), with IC50 value of 63.3μg/ml. Conclusion: L-glutaminase purified from Penicillium brevicompactum NRC 829 is a potential candidate in food and pharmaceutical industries.
RESUMO
Aim: The aims of this study were to attempt to extract, purify and characterize of Lasparaginase, 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 crudeenzyme 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.