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Abstract
Various fungal strains have been screened for L- glutaminase synthesis. Aspergillus oryzae NRRL 32657 proved to be the highest producer (24.19 U.ml-1) with the largest pink zone diameter (8 mm). Production of L- glutaminase (E.C. 3.5.1.2. L- glutamine amidohydrolase) by Aspergillus oryzae in batch submerged system was optimized. Several factors: incubation period, source and concentration of carbon, source and concentration of nitrogen, initial pH level, incubation temperature, amino acids source and concentrations and sodium chloride concentrations were tested for their effects on enzyme synthesis. The highest L- glutaminase synthesis (46.53 U. ml-1) was achieved when Aspergillus oryzae was allowed to grow aerobically (30 {u00BA}C for 3 days) in a buffered medium (pH 7.0) containing 2.5 % lactose, 2 % yeast extract, 0.5% L- glutamine and 0.5% sodium chloride under shaking conditions (100 rpm). Glutaminase was purified by ammonium sulfate fractionation followed by gel filtration on sephadex G-75. Purification fold was 10.2. SDS-PAGE of the purified glutaminase showed the presence of one band with a molecular weight of 68 KDa. Optimum pH and temperature were 7.0 and 30 to 40{u00BA}C, respectively. Whereas the highest pH stability and thermostability were obtained at pH 7.0 and 30 to 40{u00BA}C, respectively. The apparent Km value was calculated from the Linenweaver-Burk plots and was found to be 4.5 mM. Glutamine was the favorable substrate for the enzyme and was hydrolysed at the highest rate. The maximum relative activity was observed at 2.5% and 5% salt (130% and 120%, respectively). Potassium showed a slight increase in activity of glutaminase especially at the concentration of 0.5 mM. While, Ferric ions followed by ferrous showed the highest inhibition effect on glutaminase.