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

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 with an overall recovery of 27.2%. The apparent M 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 K of the l-asparaginase was found to be 1.45×10 m towards l-asparagine and was competitively inhibited by 5-diazo-4-oxo- l-norvaline (DONV) with a K of 0.03 mm. Metal ions such as Mn, Zn, Hg, Fe, Ni, and Cd 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. [ABSTRACT FROM AUTHOR]