الفهرس 
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Abstract
L-Glutaminase is an amidohydrolase which catalyses the hydrolytical deamination of L-glutamine resulting in the production of L-glutamic acid and amMonia. L-Glutaminase is gaining marked importance due to its application potential in cancer therapy, food industry and of high value chemicals like threonine. Fifty-eight isolates were isolated from the soil rhizosphere plants samples of wheat, clover and corn, 31 isolates were capable to grow on the medium and produce L-glutaminase by change the colour of the medium from yellow to pink. 13 isolates gave the maximum amount of enzyme and one isolate (W6) was selected as the most efficient isolate for L-glutaminase production and identified based on
16S rRNA sequence as Ochrobactrum intermedium with similarity of 94.99%. Carbon and nitrogen sources were studied for the tested bacterial
L-glutaminase optimization using one at a time method. Fructose and amMonium sulphate were used as a sole carbon and nitrogen sources. The interaction between variables of fermentation process was studied using response surface methodology. Three out of 7 variables (glutamine concentration, fructose concentration, amMonium sulphate concentration, pH, temperature, inoculum size and incubation period) were evaluated by Plackett-Burman design as the most significant variables being glutamine concentration, fructose concentration and amMonium sulphate concentration. The most positive significant variables were further optimized by using response surface methodology (RSM) based central composite design (CCD). By using the surface plots and response optimizer of statistical software package Design-Expert software 12.0.0 (Stat-Ease, Inc., Minneapolis, MN 55413, USA 2014), the maximum L-glutaminase activity of 175.5 U/mL was predicted at temperature (37°C), inoculum size (4%), incubation period (8 days), and pH (8) in the presence of 5.8284 (gm) glutamine, 17.5 (gm) fructose and 0.231805 (gm) amMonium sulphate.
O.intermedium L-glutaminase was fractional precipitated by amMonium sulphate, Sephacryl S-300 column chromatography and
CM-Sepharose column chromatography.
Kinetic studies showed that L-glutaminase had specific activity 2192.1 U/mg with purification fold 9.52, and molecular weight of 51 kDa by
SDS-PAGE. As well as, optimal and stability of pH and temperature were 8.0 and 50ºC respectively , Ca2+(5mM), Ca2+(10mM), Cu2+(5mM), Cu2+(10mM), Mg2+(5mM) and Mg2+(10mM) had inhibitory effect on enzyme while cations (Na2+ (10mM) , K+ (10mM) , Mn2+(10mM) , K+ (5mM), Co2+(5mM), and Co2+(10mM)) markedly activated the enzyme.
Keywords: L-glutaminase production; Ochrobactrum intermedium; Identification; 16S rRNA; carbon and nitrogen sources; response surface methodology, enzyme purification, enzyme characterization.