الفهرس 
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Abstract
Twenty six fungal isolate and eight bacterial species were screened for their potency for production of extracellular L- arginine deiminase (ADI). All of them had the potency for production of ADI, with reliable fluctuation. The most potent fungal isolate to production of ADI was selected and identified according to their morphological and molecular identification as A. oryzae jx006239. Also B. subtilis exhibited the largest increase in ADI activity. Different culture conditions to maximize the production of ADI by A. oryzae and B. subtilis were assessed. The maximum production of ADI from A. oryzae was achieved by adding 0.3%w/v glucose, 0.5% arginine and 10 mM CaCl2 to the growth medium at pH 8 and incubation for 7 days at 30OC. While 0.5% w/v glucose, 0.5% arginine, and 10mM FeSO4 optimized at pH 7.4, the ADI production by B. subtilis after incubation for 36 h. at 37OC was increased. Production of ADI by A. oryzae using various agricultural by-products as substrates under SSF was assessed. Wheat bran with 0.5% arginine and 1% sucrose achieved the maximum enzyme production under SSF. ADI was purified from A. oryzae and B. subtilis by several steps lead to increase the activity of A. oryzae ADI by 3.53 folds with 16.92 % under SF and 3.09 folds with 13.26% under SSF, also B. subtilis ADI was increased by 2.58 fold with 13.07 % compared to crude enzyme activity. Studying the biochemical properties of A. oryzae ADI revealed that, the enzyme had optimum temperature activity at 40oC, thermal stability below 45 oC and its T1/2 was 10.35, 8.27, 6.89, 3.91, 3.04 and 1.97 h. at 30, 35, 45, 55, 65 and 75ºC, respectively. Its Tm was 84.31ºC and Kr was 0.87x10-3 1.2X10-3, 1.57X10-3, 2.7X10-3, 3.11X10-3 and 4.6X10-3 min. at 30, 35, 45, 55, 65 and 75ºC respectively. The optimum pH for maximum A. oryzae ADI activity was 8. Its catalytic stability was at pH ranged from 6.4 to 9.4. NaCl, CoCl2, Hydroxylamine, CdCl2, and KCl stimulated the enzyme activity by 28.66, 25,89, 11.30, 4.33 and 1.70 % , respectively. While EDTA, SDS, CuSO4, LiSO4 and NiCl2 reduce the enzyme activity by 65.74, 57.65, 71.16, 57.65 and 84.63%, respectively. Tween 80 and HgCl2 completely inhibited the enzyme activity. A. oryzae ADI had a highest affinity for L- arginine (100%) as substrate followed by L- alanine (75%) and L-lysine (69.44%). The maximum affinity of A. oryzae ADI was for L-arginine with Km10.03mM, Vmax 208.2 U/mg/min, Kcat 2.083 min-1 and Kcat/Km 0.21 mM-1min-1 . Also the biochemical properties of B. subtilis ADI were evaluated. They revealed that, optimum temperature was 40oC, the enzyme had a thermal stability below 45ºC. Its T1/2 was 5.24, 3.31, 3.1, 1.74 and1.14 h. at 35, 45, 55, 65 and 75ºC, respectively. Tm of enzyme was 76.53ºC, Kr was 1.77X10-3, 3.3X10-3, 3.5X10-3, 5.9X10-3 and 7.9X10-3 min. at, 35, 45, 55, 65 and 75ºC, respectively. Optimum pH for maximum enzyme activity was 7.4, with catalytic stability at pH ranged from 6.4 to 8.6. The higher maximum activity was achieved by presence of SDS, EDTA, NaCl, ZnSO4 and KCl by (51.55, 30.93, 20.62, 10.31, and 4.12 %), respectively. While CdCl2, CoCl2, CuSO4, FeSO4 and LiSO4 led to loss more than 50% of the enzyme activity, moreover HgCl2 inhibited its activity. B. subtilis ADI had a highest affinity for arginine (100%) as substrate followed by L-lysine (66.10%) and L- alanine (34.46%). The maximum affinity of B. subtilis ADI was for L-arginine with Km5.2mM, Vmax 227.1U/mg/min, Kcat 2.27min-1 and Kcat/Km 0.44 mM-1min-1. A.oryzae ADI and B. subtilis ADI had anticancer potency against HEPG-2 with IC50 (29.15 and 47.17U/ml), respectively and MCF-7with IC50 (48 and 48.41 U/ml), respectively. Ethanol and methanol plant extracts were tested for their anticancer potency against HEPG-2, it was found that ethanol bay laurel extract was the most effective with IC50 (0.97mg/ml). Also, anticancer potency of the combination ethanol bay laurel extract with A. oryzae ADI was more effective (with IC50 0.0022mg/ml) than ethanol bay laurel extract with B. subtilis ADI (with IC50 0.0035 mg/ml).