Abstract
The aim of this work was to purify L-glutaminase from Aspergillus flow's. The enzyme was purified 12.47-fold from a cell-free extract with a final specific activity of 613.3 U/mg and the yield was 51.11%. The molecular weight of the enzyme, as estimated by SDS-PAGE, was found to be 69 kDa. The maximal activity of L-glutaminase was recorded at pH 8 and 40 degrees C. The highest activity was reported towards L-glutamine as substrate, with an apparent Km value of 4.5 mmol and V-max was 20 Uml(-1). The enzyme was activated by Na+ and Co2+, hile it was greatly suppressed by iodoacetate, NEM, Zn2+ and Hg2+ at 10 mM. L-glutaminase actiN ity increased w ith a gradual increase of sodium chloride concentration up to 15%. In vivo, the median lethal dose (LD50) was approximately 39.4 mg/kg body weight after intraperitoneal injection in Sprague Dawley rats. Also, L-glutaminase showed no observed changes in liver and kidney functions and hematological parameters on rates. Purified A. flavus L-glutaminase had neither a cognizable effect on human platelet aggregation nor hemolytic activity. In addition, MTT assay sh o wed that the purified L-glutaminase has a high toxic impact on Hela and Hep G2 cell Hues with an IC50 value 18 and 12 mu g/ml, respectively, and a moderate totoxic effect on HCT-116 and MCF7 cells, w ith an IC50 value 44 and 58 mu g/ml, respectively.