High Efficiency, Selectivity against Cancer Cell Line of Purified L-Asparaginase from Pathogenic Escherichia coli

Authors: Hazim Saadoon Aljewari, Mohammed Ibraheem Nader, Abdul Hussain M. Alfaisal, NatthidaWeerapreeyakul, Sahapat

Abstract:

L-asparaginase was extracted from pathogenic Escherichia coli which was isolated from urinary tract infection patients. L-asparaginase was purified 96-fold by ultrafiltration, ion exchange and gel filtration giving 39.19% yield with final specific activity of 178.57 IU/mg. L-asparaginase showed 138,356±1,000 Dalton molecular weight with 31024±100 Dalton molecular mass. Kinetic properties of enzyme resulting 1.25×10-5 mM Km and 2.5×10-3 M/min Vmax. L-asparaginase showed a maximum activity at pH 7.5 when incubated at 37 ºC for 30 min and illustrated its full activity (100%) after 15 min incubation at 20-37 ºC, while 70% of its activity was lost when incubated at 60 ºC. L-asparaginase showed cytotoxicity to U937 cell line with IC50 0.5±0.19 IU/ml, and selectivity index (SI=7.6) about 8 time higher selectivity over the lymphocyte cells. Therefore, the local pathogenic E. coli strains may be used as a source of high yield of L-asparaginase to produce anti cancer agent with high selectivity.

Keywords: L-asparaginase, Purification, Cytotoxicity, selectivity index

Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1085742

References:

[1] El-Bessoumy A A, Sarhan M, and Mansour J. Production, Isolation, and Purification of L-asparaginase from Pseudomonas aeruginosa 50071 Using Solid-state Fermentation. J Biochem Mol Biol. 2001;37:387- 393.
[2] Borek, D. and JaskÓlski, M., Sequence analysis of enzymes with asparaginase activity. Acta Bioch Pol. 2001;48:893-902.
[3] Yun M K, Nourse A, White S W, Rock C O, and Heath R J. Crystal structure and allosteric regulation of the cytoplasmic Escherichia coli Lasparaginase I. J Mol Biol. 2007;369:794-811
[4] Sieciechowicz K A, Joy K W, and Ireland R J. The metabolism of asparagine in plants. Phytochemistry. 1988;27:663-671.
[5] Borek D, and JaskÓlski M. Sequence analysis of enzymes with asparaginase activity. Acta Bioch Pol. 2001;48:893-902.
[6] Swain A L, JaskÓlski M, Housset D, Rao J K M, and Wlodawer A. Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy. Proc Natl Acad Sci. 1993;90:1474-1478.
[7] Broome J D. Evidence that the L-asparaginase activity of guinea pig serum is responsible for its antilymphoma effects. Nature. 1961;191:1114-1115.
[8] Mashburn L T, and Wriston j c. Tumor inhibitory effect of Lasparaginase from E. coli. Arch Biochem Biophys. 1964;105: 450-452.
[9] Asselin B L, Lorenson M Y, Whitin J C, Coppola D J, Kende A S, Blakley R L, and Cohen H J. Measurement of serum L-asparagine in the presence of L-asparaginase requires the presence of an L-asparaginase inhibitor. Cancer Res. 1991; 51: 6568-6573.
[10] Sarqis M I de M, Oliveira E M M, Santos A S, and da Costa G L. Production of L-asparaginase by filamentous fungi. Mem Inst Oswaldo Cruz, 2004;99:489-492.
[11] Cedar H, and Schwartz J H. Production of L-asparaginase II by Escherichia coli. J Bact. 1968;96: 2043-2048.
[12] Mokrane S A, Production, Purification and characterization of Lasparaginase II from a local isolated of Escherichia coli. PhD thesis. College of Science, Baghdad University, Iraq. 2003.
[13] Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem. 1976;72: 248-254.
[14] Alazawe A M. Production of L-asparaginase from a Local Isolated of Serratia marcescens. MSc thesis. College of Science, Baghdad University, Iraq. 2005.
[15] Garfin, D. E., One-dimensional electrophoresis. In: Methods in enzymology, Deutscher, M.P.(ed.). Academic press, New York, 1990;182:425-441.
[16] David K, and David G. L-asparaginase production by the rumen anaerobe Vibrio succinogenes. J Appl. Microbial., 1972;27:206-209.
[17] Prayong P, Barusrux S, and Weerapreeyakul N. Cytotoxic activity screening of some indigenous Thai plants. Fitoterapia. 2008;79:598- 601.
[18] Mossman T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity. J Immunol Methods. 1983;65:55-63.
[19] Alhilli Z A. Inhibitory effect of Cyperus rotundus L. crude extracts on cancer cell lines. MSc thesis. College of Science, Baghdad University, Iraq. 2004.
[20] Cedar H, and Schwartz J H. Localization of two L-asparaginase in an aerobically grown Escherichia coli. J Biol Chem. 1967;242: 3753-3755.
[21] Roberts J, Burson G, and Hill J M. New procedures for purification of L-asparaginase with high yield from Escherichia coli. J Bacteriol. 1968;95:2117-2123.
[22] Frank B H, Pekar A H, Veros A J, and Ho, P. K. Crystalline Lasparaginase from Escherichia coli B. J Biol Chem. 1970;245:3716- 3724.
[23] Scheetz R W, Whelan H A, and Wriston J C. Purification and Properties of an L-asparaginase from Fusarium tricinctum. Arch Biochem Biophys. 1971;142:184-189.
[24] Irino T, Kitoh T, Koami K, Kashima T, Mukai K, Takeuchi E, Hongo T, Nakahata T, Schuster S, and Osaka M. Establishment of real-time polymerase chain reaction method for quantitative analysis of asparagine synthetase expression. J Mol Diagn. 2004;6: 217-224.