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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
Article

Abstract

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.

Keywords

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.

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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

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