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Appl Environ Microbiol. 1977 March; 33(3): 508-514
Copyright © 1977 American Society for Microbiology. All Rights Reserved.

Purification and Some Biological Properties of Asparaginase from Azotobacter vinelandii

S. A. Gaffar1 and Y. I. Shethna

a Microbiology and Cell Biology Laboratory, Indian Institute of Science, Bangalore-560012, India

ABSTRACT

Asparaginase was found in the soluble fraction of cells of Azotobacter vinelandii, and its activity remained the same during growth of the organism in a nitrogen-free medium. The specific activity and the yield of A. vinelandii increased twofold in the presence of ammonium sulfate. Within limits, the temperature (30 to 37°C) and pH (6.5 to 8.0) of the medium showed little effect on the levels of enzyme activity. The enzyme was purified to near homogeneity by standard methods of enzyme purification, including affinity chromatography, and had optimum activity at pH 8.6 and 48°C. The approximate molecular weight was 84,000. The apparent Km value for the substrate was 1.1 x 10-4 M. Metal ions or sulfhydryl reagents were not required for enzyme activity. Cu2+, Zn2+, and Hg2+ showed concentration-dependent inhibition, whereas amino and keto acids had no effect on the enzyme activity. Asparaginase was stable when incubated with rat serum and ascites fluid. The enzyme had no effect on the membrane of sheep erythrocytes and did not inhibit the incorporation of radioactive precursors into deoxyribonucleic acid, ribonucleic acid, and protein in Yoshida ascites sarcoma cells. Asparaginase activity was not detected in the tumor cells.

FOOTNOTES

1 Present address: Laboratory of Immunodiagnosis, National Cancer Institute, Bethesda, MD 20014.

Appl Environ Microbiol. 1977 March; 33(3): 508-514
Copyright © 1977 American Society for Microbiology. All Rights Reserved.





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Copyright © 1977 by the American Society for Microbiology. All rights reserved.