الفهرس 
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Abstract
The present study aimed to produce microbial extracellular L-glutaminase enzyme and using this enzyme as a powerful antioxidant and anticancer agent.
Twelve recommended microbial strains were screened for the production of extracellular L-glutaminase enzyme, namely (seven fungi and five bacteria). The bacterial strain Escherichia coli ATCC 8739 was obtained from Microbiological Resources Centre (Cairo Mircen), Faculty of Agriculture, Ain Shams University, Egypt. While, Bacillus cereus NRC-20, Aspergillus terreus NRC-7, Aspergillus oryzae NRC-34 and Penicillium janthinellum Biourge are local strains obtained from the Culture Collection of the Department of the Natural and Microbial Products Chemistry, National Research Centre, Dokki, Egypt. In addition, Bacillus subtilis NRRL 1315, Trichoderma reesei NRRL 11460 and Trichoderma viride NRRL 6418 were obtained from the National Center for Agricultural Utilization Research, Peoria, Illinois 61604, U.S.A. Other four strains Pseudomonas aeruginosa ASU-1, Staphylococcus aureus, Aspergillus niger ASU-a6 and Penicillium cyclopium were local isolated strains provided from Microbiology Department, Faculty of Science, Ain Shams University.
The present study could be summarized as follows:
1. Screening of twelve microorganisms for the production of extracellular L-glutaminase enzyme was carried out. Of the tested microorganisms, the bacterial strain Bacillus subtilis NRRL 1315 was the most potent and produced the highest effective extra-cellular L-glutaminase (27.13 U/reaction) after 48 hr. of incubation
applying the shaken technique, accordingly this strain was chosen for the succeeding studies.
2. To optimize the productivity of L-glutaminase by the chosen strain Bacillus subtilis NRRL 1315, it was inoculated and followed up for its growth and enzyme productivity in Mineral- Salt Glutamine medium. Different factors such as; different culture media, carbon and nitrogen sources, production pH, production temperature, inoculum size and agitation speed were optimized by a single factor of varying parameters one at a time. Different culture media were tested in order to enhance the L-glutaminase production, of all the culture media investigated, Mineral Salts Glutamine offered the highest enzyme productivity (27.13 U/reaction) and had the following composition g/L: L-glutamine, 10; D-Glucose, 5; NaCl, 3; KH2PO4, 1; MgSO4.7H2O, 0.5; CaCl2 ,0.1; NaNO3, 0.1; trisodium citrate, 0.1 and distilled water, pH 7.0. On the other hand, glucose and sodium nitrate in the basal production medium were the most proper carbon and nitrogen
sources, respectively. In addition, Bacillus subtilis NRRL 1315 produce maximally L-glutaminase (28.04 U/reaction) when pH of the production medium was adjusted to 7.5, also the maximum enzyme productivity was at 37oC. On the other hand, the enzyme productivity increased with increasing L-glutamine concentration till 1.5%, (w/v) and led to the considerable productivity (30.08 U/reaction), inoculum size 18 (% ,v/v) was the most suitable and afforded the highest L-glutaminase productivity (30.97 U/reaction) and the agitation speed of 150 rpm was the most favorable for Bacillus subtilis NRRL 1315 yielding the highest enzyme productivity reached to (31.60 U/reaction).
3. Utilization of some agro-industrial residues (wheat bran & rice straw) for L-glutaminase production by Bacillus subtilis NRRL 1315, unfortunately rice straw was unsuitable for the enzyme production by Bacillus subtilis NRRL 1315 and led to no enzyme productivity at all. On the other hand, wheat bran led to high enzyme productivity (22.15 U/reaction), after applying all the optimized conditions.
4. The partial purification of Bacillus subtilis NRRL 1315 crude L-glutaminase enzyme was achieved by fractional precipitation by ammonium sulphate, ethanol or acetone. In addition, batch precipitation by ethanol or acetone was employed. The whole precipitation processes offered a total of twelve fractions, these
included two fractions obtained by the fractional precipitation with ammonium sulphate (40-90% saturation). Four fractions obtained by the fractional precipitation with ethanol (40-90% concentration), four fractions obtained by the fractional precipitation with acetone (40-90% concentration) and two fractions obtained by the batch precipitation by acetone or ethanol. All fractions obtained were assayed for both L-glutaminase activity & protein content and all exhibited enzyme activity despite of low protein and activity recovery exhibited by some fractions. Of all the fractions obtained by the applied methods, it was found that fraction 80% acetone gave the maximum yields of specific enzyme specific activity, recovered activity and purification fold of L-glutaminase enzyme (40.15 U/mg protein, 33.50 %, 2.017, respectively). It was considered as the partially purified L-glutaminase preparation fraction which was used in further experimental studies.
5. The crude Bacillus subtilis NRRL 1315 L-glutaminase preparation exhibited its maximum activity at enzyme protein concentration of 2 mg/mL enzyme, 2 mg enzyme protein was sufficient to afford the maximal enzyme activity yielding 18.79 U/reaction. Also, a direct relationship was shown between the substrate (L-glutamine) concentration and the L-glutaminase activity which attained its maximum (18.79 U/reaction) at 40 mM L-glutamine concentration. Furthermore, the crude L-glutaminase
preparation exhibited its maximal activity at 37°C and pH 7.5 (18.77 & 19.56 U/reaction, respectively).
6. The partially purified L-glutaminase preparation exhibited its maximum activity at enzyme protein concentration of 2 mg/mL enzyme (37.06 U/reaction), substrate concentration of 40 mM (37.06 U/reaction), reaction temperature 37oC (37.30 U/reaction) and pH 7.5 (37.95 U/reaction).
7. Applying the recommended Hanes-Woolf plot, each of Michaelis constant (Km) and maximum velocity constant (Vmax) were identified as 2.6 mM and 37.14 U/reaction, respectively.
8. The partially purified L-glutaminase preparation exhibited an excellent stability profile at 37°C and kept all its original activity after 15 min. incubation and after extended periods of incubation (30, 60 and 120 min). The enzyme retained more than 99.8, 94.4 and 84.4 % of its original activity, respectively. On the other hand, heating the enzyme at 45oC for 15, 30, 60 & 120 min., it retained 94.4, 89.6, 86.5 and 74% of its original activity, respectively. At the elevated temperature 55oC, the enzyme retained 91.9, 84.8, 81.7 & 66.6% of its original activity after 15, 30, 60 &120 min of incubation, respectively.
9. The partially purified L-glutaminase enzyme was highly stable at the neutral pH 7.0 and retained more than 99.2, 96, 86.8 and 81.5 % of its original activity after 15, 30, 60 and 120 min of incubation,
respectively. On the other hand, at pH of 3.0 the enzyme also retained more than 85.1, 82.1 and 71.9% after 15, 30 and 60 min. of incubation, respectively but at extended period of incubation (120 min) the enzyme lost more than 53% of its activity. On the other hand, the enzyme was labile when incubated at pH 10.0 after 120 min. incubation and lost more than 48% of its original activity, but incubation for 15, 30 and even 60 min. the enzyme exhibited an excellent stability and it retained more than 86.2, 79 & 68.3% of its activity, respectively.
10. Among all the substances tested, at concentration of 100 mM, the metal ions Mg+2 & Mn+2 had very good stimulatory effects on the enzyme activity and led to relative activities amounted to 202.56 and 179.77 %, respectively. While, Fe+2, Zn+2 & Ca+2 had good stimulatory effects on the enzyme activity, which led to relative activity amounted to 130.39, 124.45 and 113.94%, respectively. In addition, K+ & Cu+2 slightly stimulate the enzyme and led to relative activity amounted to 109.64 & 106.60%, respectively. On the other hand, EDTA and Hg+2 at 100 mM inhibited the enzyme activity, which retained 79.01 and 77.98%, respectively of its original activity. Na+ in each of NaCl & NaN3 slightly inhibited the enzyme, which retained most of its activity (93.32 and 91.66% of the original activity, respectively).
Concerning the other metals Mg+2, Mn+2 & Zn+2 at 10 mM had good stimulatory effects on the enzyme activity, and the relative activity amounted to 138.68, 126.72 & 114.21%, respectively. While, K+ & Fe+2 had slight stimulatory effects on the enzyme activity and led to relative activity amounted to 105.60 & 105.33%, respectively. It was worth noting that any of Ca+2 & Cu+2 had no effect at 1-10 mM and the enzyme kept all of its activity.
11. The antimicrobial activity of both the crude and the partially purified enzyme preparations was investigated and unfortunately, there were no antifungal or antibacterial activities of both enzyme forms and this was followed with other important activities, like antioxidant and anticancer.
12. Each of the crude and the partially purified L-glutaminase preparations exhibited excellent free radical scavenging activity comparable to the standard ascorbic acid. It is worthy to decide here that both the crude and the partially purified L-glutaminase preparations afforded scavenging activity very close to that of the standard ascorbic acid at concentration ranged from 9-10 mg/mL and from 0.25 to 1 mg/mL.
13. The partially purified L-glutaminase enzyme has cytotoxic activity against the three human tumor cell lines examined, the highest antitumor activity was against HCT-116 and 65.1% growth
of the cells was inhibited at 100 µg/mL enzyme and IC50 was calculated to be 70.6 µg/mL. While the least antitumor activity was obtained towards MCF-7 and 27.7% cells growth was inhibited at 100 µg/mL enzyme. The antitumor activity of the partially purified L-glutaminase against Hep-G2 occurred 40.9% inhibition of cells growth and IC50 was 102.6 µg/mL enzyme.