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.
Cite this paper
C. Battiston Loureiro, K. Silva Borges, A. Faria Andrade, L. Gonzaga Tone and S. Said, "Purification and Biochemical Characterization of Native and Pegylated Form of L-Asparaginase from Aspergillus terreus and Evaluation of Its Antiproliferative Activity," Advances in Microbiology, Vol. 2 No. 2, 2012, pp. 138-145. doi: 10.4236/aim.2012.22019.
 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.
 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.
 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.
 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.
 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.
 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.
 H. Blum, H. Beier and H. J. Gross, “Improved Silver Staining of Plant Proteins, RNA and DNA in Polyacrylamide Gels,” Electrophoresis, Vol. 8, No.2, 1987, pp. 93-99. doi:10.1002/elps.1150080203
 R. C. Stong, S. J. Korsmeyer, J. L. Parkin, D. C. Arthur and J. H. Kersey, “Human Acute Leukemia Cell Line with the t(4:11) Chromosomal Rearrangement Exhibits B Lineage and Monocytic Characteristics,” Blood, Vol. 65, No. 1, 1985, pp. 21-31.
 V. C. Foster and S. Said, “The Influence of Nitrogen Source on L-Asparaginase Production by Strain Isolated from Soil,” VII Seminário Brasileiro de Tecnologia Enzimática, Caxias do Sul, 21-24 May 2006, p. 124.
 V. I. Avramis and E. H. Panosyan, “Pharmacokinetic/Pharmacodynamic Relationships of Asparaginase Formulations: The Past, the Present and Recommendations for the Future,” Clinical Pharmacokinetics, Vol. 44, No. 4, 2005, pp. 367-393.
 E. H. Panosyan, R. S. Grigoryan, I. A. Ayramis, N. L. Seibel, P. S Gaynon, S. E. Siegel, H. J. Finger and V. I. Ayramis, “Deamination of Glutamine Is a Prerequisite for Optimal Asparagine Deamination by Asparaginases in Vivo (CCG-1961),” Anticancer Research, Vol. 24, No. 2C, 2004, pp. 1121-1125.
 G. Ollenschlager, E. Roth, W. Linkescht, S. Jansen, A. Simmel and B. Modder, “Asparaginase-Induced Derangements of Glutamine Metabolism: The Pathogenetic Basis for Some Drug-Related Side-Effects,” European Journal of Clinical Investigation, Vol. 18, No. 5, 1988, pp. 512-516. doi:10.1111/j.1365-2362.1988.tb01049.x
 M. N. Offman, M. Krol, N. Patel, S. Krishnan, J. Z. Liu, V. Saha and P. A. Bates, “Rational Engineering of L-Asparaginase Reveals Importance of Dualactivity for Cancer Cell Toxicity,” Blood, Vol. 117, No. 5, 2011, pp. 1614-1621. doi:10.1182/blood-2010-07-298422
 A. M. Aslanian, B. S. Fletcher and M. S. Kilberg, “Asparagine Synthetase Expression Alone Is Sufficient to Induce L-Asparaginase Resistance in MOLT-4 Human Leukaemia Cells,” Biochemical Journal, Vol. 357, No. Pt 1, 2001, pp. 321-328. doi:10.1042/0264-6021:3570321
 A, M. Aslanian and M. S. Kilberg, “Multiple Adaptive Mechanisms Affect Asparagine Synthetase Substrate Availability in Asparaginase-Resistant MOLT-4 Leukaemia Cells,” Biochemical Journal, Vol. 358, No. Pt1, 2001 pp. 59-67. doi:10.1042/0264-6021:3580059