PJB-2018-1017
PURIFICATION, CHARACTERIZATION AND ANTICANCER EFFICIENCY OF L-GLUTAMINASE FROM ASPERGILLIUS FLAVUS
Medhat Ahmed Abu-Tahon
Abstract
The aim of this study is to purify L-glutaminase from Aspergillus flavus. The enzyme has been purified 12.47-fold from cell-free extract with a final specific activity 613.3 U/mg and yield was 51.11%. The molecular weight of enzyme as determined by SDS-PAGE was found to be 69 kDa. Maximal activity of L-glutaminase was recorded at pH 8 and 40oC. The highest activity was reported towards its natural substrate, L-glutamine, with an apparent Km value of 4.5 mmol and Vmax was 20 Uml-1. The enzyme was activated by Na+ and Co+2 while it was strongly inhibited by Iodoacetate, NEM, Zn+2 and Hg+2 at 10 mM. L-glutaminase activity increased gradually with the increase of NaCl concentrations 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, kidney functions and hematological parameters on rates. Purified A. flavus L-glutaminase had neither an appreciable effect on human platelet aggregation nor hemolytic activity. In addition, MTT assay showed that the purified L-glutaminase has high toxic effect to Hela and Hep G2 cell lines with IC50 value 18 and 12 μg/ml, respectively and a moderate cytotoxic effect to HCT-116 and MCF7 cells with IC50 value 44 and 58 μg/ml, respectively
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