الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
L-Asparaginase is an antileukemic agent that consumes L- asparagine ‘‘a vital nutrient for cancer cells” through the hydrolysis of L- asparagine into L-aspartic acid and ammonia, leading to leukemia cell starvation and apoptosis in susceptible leukemic cell populations. Additionally, presently, bacterial L-asparaginase has been restricted by problems of lower productivity, stability, selectivity and several toxicities along with the resistance towards bacterial L-asparaginase.
This study aimed to screen new potent isolates that produce L- asparaginase and optimize fermentation parameters to maximize enzyme yields. In addition, some generalities concerning the uses of L- asparaginase in the pharmaceutical as antitumor and antioxidant.
Results could be summarized in the following points:
1. Seventy-nine microbial isolates (54 fungal isolates, 16 yeast isolates and 9 bacterial isolates) were isolated from different sources (soil, fruits and fruits juice). These isolates were tested for L-asparaginase production on MCD, MSA and M-9 agar media supplemented with asparagine as a substrate and phenol red as an indicator.
2. Sixty-eight out of the 79 microbial isolates were produced a pink zone around the growth colony on an agar plate (as a qualitative assay). They gave a diameter zone ranged from 7 to 30 mm. These isolates tested for L-asparaginase activity in broth media under SmF (as quantitative assay) with activity ranged from 2.4 to 122.6. So, the highest significant enzyme production was recorded by fungal isolate with code of F-S3 (122.6 Uml-1 of enzyme activity in broth medium and 30 mm of zone diameter on solid medium).
3. The pioneer fungal isolate (F-S3) was identified based on phenotypic characteristics as Fusarium sp. and confirmed by 18S rRNA gene analysis (genotypic characteristics). The Gen Bank accession number
for strain is OK356604, identified as Fusarium oxysporum
OK356604.1.
4. The production of L-asparaginase using agro-industrial waste (peels of orange, pea, potato, banana, lemon, pomegranate and tea waste) under SSF was studied and the results revealed that among all tested agro- industrial waste, pomegranate peel gave the highest yield of L- asparaginase, which achieved 160.3 Ugds-1.
5. The sterilization of pomegranate peel as a substrate by gamma irradiation with a dose level of 15 kGy was sufficient for decontamination and /or sterilization of a substrate and generated higher L-asparaginase production (230 Ugds-1) with an increase 1.43- Fold than the autoclaved substrate (160.3 Ugds-1).
6. Optimization of the SSF parameters using the OFAT technique resulted in a maximum L-asparaginase production of 280.4 Ugds-1 on an irradiated pomegranate peels supplemented with 0.2 % (ww-1) glucose, 1.0 % (ww-1) ammonium chloride, L-asparagine 0.6% (ww-1), 70% (vw-1) initial moisture content, adjusted pH to 7.0 and 3% inoculum volume (107 spores ml-1) for 96 h of incubation at 30°C. The enzyme activity increased 1.74-fold after cultural optimization compared to non-optimized. The results also demonstrated that enzyme production under SSF was preferred (an increase of 2.28-fold) over SmF.
7. F. oxysporum F-S3 spore suspension was exposed to gamma rays at various doses of 0.05, 0.1, 0.5, 1, 1.5, 2.0, 2.5, 3, 4, and 5 kGy. The results showed that there were no significant changes in L- asparaginase activity after exposing spore suspension to low doses of gamma rays (0.05 and 0.1 kGy), while there was a significant decrease in enzyme activity (133 and 55 Ugds-1) compared to non-irradiated F. oxysporum F-S3 when the spores were exposed to high doses of 4 and 5 kGy, respectively.
8. The fungal L-asparaginase was completely purified by the following steps:
• protein precipitation using different organic solvents (ethanol, acetone, and isopropanol), it was found that ethanol was the best solvent which achieved enzyme yield of 58.0% and specific activity of 902.7 Umg-1 protein.
• Anion exchange chromatography with DEAE-Sephadex G50 was
applied. The results showed that the specific enzyme activity was 3084.3 Umg-1 protein, and it was purified 10-fold with 9.2 % enzyme recovery.
9. The SDS-PAGE revealed double protein bands obtained with a molecular weight of 62 kDa and 59 kDa, signifying a heterodimer enzyme.
10. Characterization of purified F. oxysporum F-S3 L-asparaginase as the following:
• The maximum L-asparaginase activity was attained at pH ranged from 7 to 8, with stability at pH 7 and 8 for 60 min, reaching 97 % and 94 % of relative activity.
• The optimal temperature of enzyme activity was 37°C and the enzyme was thermal stable at 40 °C for 15 and 30 min.
• Studying the effect of various concentrations of L-asparagine ranged from 0.01 to 0.1 M as a substrate showed that 0.06 M was the best concentration for L-asparaginase activity. The Km and Vmax were calculated using Michaelis-Menten equation and Lineweaver-Burk plot equation, and they were found that 0.016 mgml-1 and 166.6 Umin-1, respectively.
• Effect of 8 different elements (metal ions and salts) on L-
asparaginase activity were tested. Results showed that the enzyme activity was enhanced in the presence of potassium chloride with relative activity of 121.8 % and slightly stimulated in the presence of sodium chloride with 101.5%. Whereas ferrous sulfate and copper sulfate showed the inhibitory effect.
11. The antioxidant activity of F. oxysporum F-S3 L-asparaginase was determined and ascorbic acid was used as a standard at varied concentrations ranged from 31.5 to 500 μgml-1. The data confirmed that L- asparaginase had shown effective dose-dependent DPPH radical scavenging activity. The IC50 of L-asparaginase was 91.6 μgml-1 compared to ascorbic acid 64.5 μgml-1.
12. The in-vitro cytotoxicity assay of L-asparaginase using HepG2, HCT-116 cancer cell lines, and OEC as a normal cell line by MTT viability assay was indicated that the enzyme exhibited anticancer activity with IC50 of 4.4, 4.5, and 13.5 Uml-1, respectively.
13. The relationships between increasing inhibition % of liver cancer cells (HepG2) and the anti-proliferative effect of F. oxysporum F-S3 L-asparaginase were detected using flow cytometry. Results revealed that the enzyme at IC50 value of 4.4 Uml-1, affected cell cycle distribution on HepG2 cells compared with the control.
14. Treatment of HepG2 cells with F. oxysporum F-S3 L asparaginase with IC50 value exhibited a significant increase of apoptosis rates to 19.5% when compared with control 2%. Consequently, L- asparaginase could meritoriously prevent the proliferation of cancer cells via apoptosis induction.
15. The expression levels of the apoptosis-related genes p53, Bcl-2 and Caspase 3 in HepG2 were assessed by quantitative real time PCR to determine the ability of L-asparaginase to induce apoptosis in HepG2 cells, compared with untreated cells. Our results indicated that the expression level of p53 and Caspase 3 were up-regulated. In contrast, the expression level of Bcl-2 gene was down-regulated.