الفهرس 
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Abstract
SUMMERY AND CONCLUSION
L-asparaginase enzyme is a widely spread that present in plant and animal tissues and microorganisms including bacteria, yeast, and fungi. L-asparaginase is an important treatment agent in the treatment of acute lymphocytic leukemia .
The objectives of this study included isolation, screening and identification of the most efficient bacterial and fungal isolates, using some cheap available agriculture by-products as nutrition sources for bacterial and fungal growth and l-asparaginase production. Subsequently ,optimization the nutritional and environmental parameters for improving L-asparaginase production by both strains using the statistical approach of response surface methodology.
Results could be summarized in the following points:
1- Twenty-two isolates (10 bacterial and 12 fungal isolates) were collected from different ecological sources (rhizosphere plant, saltwater, and Lab. of Microbiology) on M9 solid media.
2- The microbial isolates were screened using plate assay. Of the 22 isolates examined, 20 isolates gave a clear zone in plate assay and then screened for L-asparaginase production under submerged conditions. These isolates were tested for L-asparaginase activity in broth medium. The 20 isolates produced enzyme in the broth medium with activity ranged from 0.11 to 3.67 U/ml. The highest significant enzyme activity, biomass and pH value were obtained by M5 isolate (3.67 U/ml, 2.66 g/L, and 8.84) followed by A4 isolate (2.45 U/ml, 4.45 g/L, and 8.61), respectively. The lowest biomass and enzyme yield were acheived by M1 (0.08 g/L, and 0.11 U/ml) and R1 (0.08 g/L, and 0.13 U/ml), respectively.
3- Out of 22 isolates, 2 isolates of A2 and Micrococcus roseus MR were collected from marine water and Lab. of Microbiology were cannot be able to produce L-asparaginase, respectively.
4- Among 20 isolates, 2 isolates of A4 and M5 were isolated from marine water and gave the maximum enzyme activity (2.45 and 3.67 U/ml) and biomass (4.45 and 2.66 g/L of cell dry weight at pH 8.61 and pH 8.84. Both isolates were chosen for next study.
5- The most efficient bacterial and fungal isolates (A4 and M5) were identified based on phenotypic characteristics, the bacterial isolate was confirmed by 16S rRNA gene analysis (genotypic characterizes) as Bacillus velezensis M5.
6- Growth parameters:
Growth curve, specific growth rate (µ), doubling time (td), and multiplication rate (MR) were calculated for the most efficient bacterial and fungal strains using shake flasks as a batch culture.
a- The specific growth and enzyme rate (µ) were 0.20 and 0.14 h-1 for B. velezensis M5 and 0.06 and 0.04 h-1 for A. niger A4, respectively.
b- The corresponding of multiplication growth rate (MR) were 0.28 and 0.09, respectively.
c- The doubling time (td) of growth and enzyme activity were 3.55 and 4.81 h were achieved by B. velezensis M5 and 11.21 and 16.50 h were recorded by A. niger A4, respectively.
7- The optimum incubation period was found to be 20 h and 48 h incubation with enzyme productivity of 0.34 and 0.07 U/ml/h for bacteria and fungi, respectively. The pH value was increased from 6.0 or 7.0 to 8.95 or 8.96 for fungal or bacterial strains, respectively.
8- Six carbon sources sucrose, mannitol, whey, potato starchy waste and molasses were used as a sole carbon source by the tested bacteria and fungi for L-asparaginase production compared to glucose (control). The amount of 4 g /L of glucose in the base medium was replaced with an equivalent value from other carbon sources, calculated on the carbon base. The whey and molasses were the best carbon sources and had significant (p ˂0.05) for enzyme activity (10.51 and 5.24 U/ml) and productivity of 0.53 and 0.12 U/ml/h by the tested bacteria and fungi which increased in activity about 1.6- and 2.0- folds more than production in presence of glucose as a control, respectively. It was found that the whey was the best as the sole source of carbon as it gave the highest l-asparaginase activity 10.51 U/ml by B. velezensis M5 and A. niger A4 isolates, respectively.
9- Six nitrogen sources (soybean extract, corn steep liquor, NH4Cl, NH4NO3, tryptone and yeast extract) were tested plus asparagine. The maximum activity asparaginase from bacteria and fungi were more significant ((p ˂0.05) of 12.39 and 7.66 U/ml in presence of yeast extract and tryptone supplement with asparagine.
10- The asparaginase activity in different concentrations of asparagine (0.1, 0.3, 0.5, 0.7 and 1.0 g/L) was studied. The enzyme activity was maximum significant at p ˂0.05 of 13.41 and 9.13 U/ml at 0.7 g/L of asparagine in media of the tested bacteria and fungi, respectively. The enzyme activity was increased at 0.7 % of asparagine about ranged from 8.2 to 19.2% more than at 0.5% as control. The highest significantly enzyme productivity were 0.67 and 0.19 U/ml/h in presence of asparagine at 0.7 g/L for both tested bacterial and fungal strains.
11- It could be summarized the fermentation parameters for achieving maximum enzyme activity by B. velezensis M5 and A. niger A4. For B. velezensis M5, it grown in medium contained whey as carbon sources, yeast extract as nitrogen source and 0.7 g/L of asparagine as precursor, and this medium inoculated with bacteria and incubated for 20 h. While in case of A. niger A4, the suitable medium was molasses as carbon sources, tryptone as nitrogen sources and 0.7 g/L of asparagine as precursor, and this medium inoculated with tested fungi and incubated for 48 h.
12- MRR-IV statistical design was performed to screen out the factors contributing to the L-asparaginase production by B. velezensis M5 and A. niger A4. Eight variables including carbon concentration, nitrogen concentration, asparagine concentration, inoculum size, pH, incubation temperature, incubation period, and agitation speed were tested for enhancing the L-asparaginase production by B. velezensis M5 and A. niger A4.
13- The analyzed results of B. velezensis M5 suggests that out of 8 independent variables, only 3 variables of yeast extract concentration, aspargine concentration, and incubation temperature, were significantly affected the activity of enzyme. The p-values of these significant variables ranged from 0.001 to 0.046. As well as A. niger A4, out of 8 independent, three variables (tryptone concentration, pH, and inoculum size) significantly affected on enzyme activity, which had p-values ranged from 0.001- 0.021.
14- The significant factors were considered for the next optimization step using central composite design (CCD) to determine the most optimal corresponding level for each significant factor on enzyme production. L-asparaginase activity by B. velezensis M5 activity was highly and interactively influenced by all selected variables. Results clearly showed that the interaction of yeast extract concentration vs asparagine concentration with incubation temperature at 30 °C. Or interaction between yeast extract concentration vs incubation temperature with asparagine concentration at 0.7 g/L. The interaction Asparagine concentration vs incubation temperature with yeast extract concentration at 7.6 g/L. For A. niger A4, a total of 15 experimental runs with different combination of tryptone concentration (A), pH (B) and inoculum size (C) were performed and the results of experiments to study the effects of three independent variables on L-asparaginase activity.
15- L-asparaginase produced by B.velezensis M5 was purified by using ammonium sulfate precipitation followed by gel filtration on Sephacryl S-200 column and finally ion exchange chromatography on DEAE-Sepharose column.