Current Microbiology

, Volume 30, Issue 5, pp 291–298 | Cite as

Purification, characterization and antitumor activity of l-asparaginase isolated from Pseudomonas stutzeri MB-405

  • Subha Manna
  • Amaresh Sinha
  • Ramkrishna Sadhukhan
  • S. L. Chakrabarty


An l-asparaginase produced by Pseudomonas stutzeri MB-405 was isolated and characterized. After initial ammonium sulfate fractionation, the enzyme was purified by consecutive column chromatography on Sephadex G-100, Ca-hydroxylapatite, and DEAE-Sephadex A-50. The 665.5-fold purified enzyme thus obtained has the specific activity of 732.3 units mg protein-1 with an overall recovery of 27.2%. The apparent Mr of the enzyme under nondenaturing and denaturing conditions was 34 kDa and 33 kDa respectively, and the isoelectric point was 6.38±0.02. It displayed optimum activity at pH 9.0 and 37°C. The enzyme was very specific for l-asparagine and did not hydrolyze L-glutaminate. The Km of the l-asparaginase was found to be 1.45×10-4m towards l-asparagine and was competitively inhibited by 5-diazo-4-oxo-l-norvaline (DONV) with a Ki of 0.03mm. Metal ions such as Mn2+, Zn2+, Hg2+, Fe3+, Ni2+, and Cd2+ potentially inhibited the enzyme activity. The activity was enhanced in the presence of thiol-protecting reagents such as DTT, 2-ME, and glutathione (reduced), but inhibited by PCMB and iodoacetamide. The tumor inhibition study with Dalton's lymphoma tumor cells in vivo indicated that this enzyme possesses antitumor properties.


Enzyme Lymphoma Glutathione Fractionation Column Chromatography 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Literature Cited

  1. 1.
    Andrews P (1964) Estimation of molecular weight of protein by Sephadex gel filtration. Biochem J 91:222–233Google Scholar
  2. 2.
    Broome JD (1963) Evidence that the l-asparaginase of guinea pig serum is responsible for its antilymphoma effects. I. Properties of the l-asparaginase of guinea pig serum in relation to those of the antilymphoma substance. J Exp Med 118:99–120Google Scholar
  3. 3.
    Capizzi R, Cheng YC (1981) Therapy of neoplasia with asparaginase. In: Enzymes as drugs, Holcenberg JS, RObert J, (eds) New York: John Wiley and Sons, pp 1–24Google Scholar
  4. 4.
    Distasio JA, Salazar AM, Nadji M, Durden DL (1982) Glutaminase-free asparaginase from Vibrio succinogenes: an antilymphoma enzyme lacking hepatotoxicity. Int J Cancer 30:343–347Google Scholar
  5. 5.
    Drainas C, Kinghorn JR, Pateman JA (1977) Aspartic hydroxamate resistance and asparaginase regulation in the fungus Aspergillus nidulans. J Gen Microbiol 98:493–500Google Scholar
  6. 6.
    Ertel IJ, Nesbit ME, Hammond D, Weiner J, Sather H (1979) Effective dose of l-asparaginase for induction of remission in previously treated children with acute lymphoblastic leukemia: a report from Children's Cancer Study Group. Cancer Res 39:3893–3896Google Scholar
  7. 7.
    Gallagher MP, Marshall RD, Wilson R (1989) Asparaginase as a drug for the treatment of acute lymphoblastic leukemia. Essays Biochem 24:1–40Google Scholar
  8. 8.
    Jaffe N, Traggis D, Das L, Kim BS, Hann HW, Moloney WC, Dohlwitz A (1972) Comparison of daily and twice-weekly schedule of l-asparaginase in childhood leukemia. Pediatrics 49:590–595Google Scholar
  9. 9.
    Jayaram HN, Cooney DA, Jayaram S, Rosenblum L (1974) A simple and rapid method for the estimation of l-asparaginase in chromatographic and electrophoretic effluents: comparison with other methods. Anal Biochem 59:327–346Google Scholar
  10. 10.
    Kidd JG (1953) Regression of transplanted lymphomas induced in vivo by means of normal guinea pig serum. I. Course of transplanted cancers of various kinds in mice and rats given guinea pig serum, horse serum, or rabbit serum. J Exp Med 98:565–582Google Scholar
  11. 11.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685Google Scholar
  12. 12.
    Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement by Folin phenol reagent. J Biol Chem 193:266–275Google Scholar
  13. 13.
    Meses JM, Gill JA, Martin JF (1990) Characterization and partial purification of l-asparaginase from Corynebacterium glutamicum. J Gen Microbiol 136:515–519Google Scholar
  14. 14.
    Raha SK, Dey SK, Roy SK, Chaudhuri S, Chakrabarty SL (1990) Antitumor activity of l-asparaginase from Cylindrocarpon obtusisporum MB-10 and its effect on the immune system. Biochem Int 21:1001–1012Google Scholar
  15. 15.
    Ramadan MEA, Asmar FEL, Greenberg DM (1964) Purification and properties of glutaminase and asparaginase from a Pseudomonad. Arch Biochem Biophys 108:143–149Google Scholar
  16. 16.
    Sallan SE, Hitchcock-Bryan S, Gelber R, Cassady JR, Frei III E, Nathan DG (1983) Influence of intensive asparaginase in the treatment of childhood non-T-cell acute lymphoblastic leukemia. Cancer Res 43:5601–5607Google Scholar
  17. 17.
    Scheetz RW, Whelan HA, Wriston JC (1971) Purification and properties of an l-asparaginase from Fusarium trieintum. Arch. Biochem. Biophys. 142:184–189Google Scholar
  18. 18.
    Tallal L, Tan C, Oettgen H, Wollner N, McCarthy M, Helson L, Burchenal J, Karnofsky D, Murphy ML (1970) E. coli l-asparaginase in the treatment of leukemia and solid tumors in 131 children. Cancer 25:306–320Google Scholar
  19. 19.
    Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354Google Scholar
  20. 20.
    Whelan HA, Wriston JC Jr (1969) Purification and properties of asparaginase from Escherichia coli B. Biochemistry 8:2386–2393Google Scholar
  21. 21.
    Wriston JC (1973) l-asparaginase: a review. Adv Enzymol 39:187–248Google Scholar

Copyright information

© Springer-Verlag New York Inc. 1995

Authors and Affiliations

  • Subha Manna
    • 1
  • Amaresh Sinha
    • 1
  • Ramkrishna Sadhukhan
    • 1
  • S. L. Chakrabarty
    • 1
  1. 1.Department of MicrobiologyBose InstituteCalcuttaIndia

Personalised recommendations