Purification and Biochemical Characterization of Native and Pegylated Form of L-Asparaginase from <i>Aspergillus terreus</i> and Evaluation of Its Antiproliferative Activity
- 1 Departamento de Ciências Farmacêuticas, FCFRP-USP, Ribeir?o Preto, Brazil
- 2 Departamento de Genética, FMRP-USP, Ribeir?o Preto, Brazil
- 3 Departamento de Genética, FMRP-USP, Ribeir?o Preto, Brazil
- 4 Departamento de Genética, FMRP-USP, Ribeir?o Preto, Brazil; Departamento de Puericultura e Pediatria, FMRP-USP, Ribeir?o Preto, Brazil
- 5 Departamento de Ciências Farmacêuticas, FCFRP-USP, Ribeir?o Preto, Brazil
Abstract
L-asparaginase is a chemotherapeutic drug used in the treatment of lymphoblastic leukemia. In the present study, the extracellular L-asparaginase produced by strain (PC-1.7A) of Aspergillus terreus was purified, characterized, and modified with polyethylene glycol. Moreover, its antiproliferative activity was evaluated. The apparent molecular weight of the enzyme was found to be 136 kDa. The optimal pH and temperature for the enzyme were 9.0℃ and 40℃, respectively. The enzyme retained 100% of the activity at 40℃ for 120 min. Pegylated L-asparaginase was more thermostable and more resistant to trypsin than native enzyme. Native L-asparaginase against human normal cells did not show cytotoxicity. However, in the leukemia cell lines RS4;11 and HL60 the antiproliferative effects of native L-asparaginase were observed after 96 and 72 h of incubation, respectively. For the first time, an L-asparaginase from fungus was evaluated as an antitumor agent in human cells lines and further investigations should be conducted to improve the knowledge about this enzyme.
- U. K. Narta, S. S. Kanwar and W. Azmi, “Pharmacological and Clinical Evaluation of L-Asparaginase in the Treatment of Leukemia,” Critical Reviews in Oncology/Hematology, Vol. 61, No. 3, 2007, pp. 208-221. doi:10.1016/j.critrevonc.2006.07.009
- S. Patil, J. Coutsouvelis and A. Spencer, “Asparaginase in the Management of Adult Acute Lymphoblastic Leukemia: Is It Used Appropriately?” Cancer Treatment Reviews, Vol. 37, No. 3, 2011, pp. 202-207. doi:10.1016/j.ctrv.2010.08.002
- J. C. Panetta, A. Gajjar, N. Hijiya, L. J. Hak, C. Cheng, W. Liu, C. H. Pui and M. V. Relling, “Comparison of Native E. coli and PEG-Asparaginase Pharmacokinetics and Pharmacodynamics in Pediatric Acute Lymphoblastic Leukemia,” Clinical Pharmacology and Therapeutics, Vol. 86, No. 6, 2009, pp. 651-658. doi:10.1038/clpt.2009.162
- M. I. Sarquis, E. M. Oliveira, A. S. Santos and G. L. Costa, “Production of L-Asparaginase by Filamentous Fungi,” Memorias do Instituto Oswaldo Cruz, Vol. 99, No. 5, 2004, pp. 489-492. doi:10.1590/S0074-02762004000500005
- A. Mishra, “Production of L-Asparaginase, an Anticancer Agent, from Aspergillus niger Using Agricultural Waste in Solid State Fermentation,” Applied Biochemistry and Biotechnology, Vol. 135, No. 1, 2006, pp. 33-42. doi:10.1385/ABAB:135:1:33
- C. Drainas and J. A. Pateman, “L-Asparaginase Activity in the Fungus Aspergillus nidulans,” Biochemical Society Transactions, Vol. 41, 1977, pp. 1365-1371.
- R. Pieters, S. P. Hunger, J. Boos, C. Rizzari, L. Silverman, A. Baruchel, N. Goekbuget, M. Schrappe and Ch.-H. Pui, “L-Asparaginase Treatment in Acute Lymphoblastic Leukemia,” Cancer, Vol. 117, No. 2, 2011, pp. 239-249. doi:10.1002/cncr.25489
- A. Imada, S. Igarasi, K. Nakahama and M. Isono, “Asparaginase and Glutaminase Activities of Microorganisms,” Journal Genetics Microbiology, Vol. 76, No. 1, 1973, pp. 85-99.
- M. Bradford, “A Rapid and Sensitive Method for the Quantification of Microgram Quanties of Protein Utilizing the Principle of Protein Dye Binding,” Analitical Biochemistry, Vol. 72, No. 1-2, 1976, pp. 248-254. doi:10.1016/0003-2697(76)90527-3
- A. L. Soares, G. M. Guimar?es, B. Polakiewicz, R. N. M. Pitombo and J. Abrah?o-Neto, “Effects of Polyethylene Glycol Attachment on Physicochemical and Biological Stability of E. coli L-Asparaginase,” International Journal of Pharmaceutics. Vol. 237, No. 1-2, 2002, pp. 163-170. doi:10.1016/S0378-5173(02)00046-7
- U. K. Laemmli, “Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4,” Nature, Vol. 227, No. 5259, 1970, pp. 680-685. doi:10.1038/227680a0