الفهرس 
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Abstract
Information of asparaginase has been increasingly forthcoming in recent years because of their applications as therapeutic agents in the treatment of certain types of human cancer. In our present study, we report for the first time, the production of asparaginase from Bacillus subtilis DALX2 using submerged fermentation, its very effective technique as the yield of the product, and it also offers many other advantages.
1- The screening experiments were carried out in two lines; the first goal was to select the most potent microorganism that produced the highest amount of asparaginase enzyme in the culture filtrate. Second was to find out the most suitable medium and type of fermentation; The tested bacteria exhibited different capacities to produce the tested enzyme.
2- Fermentation medium that received an inoculum size of 2ml/ flask, attained the asparaginase activity.
3- The bacterial isolates under static conditions grown on basal medium M9 containing asparagine at pH 7.0, after 24hr, at 37°C. Bacillus subtilisDALX2 supported the highest enzyme synthesis 139.3 U/ml. Followed by Bacillus licheniformis a fairly high asparaginase activity 56.6 U/ml. The lowest values were recorded with Bacillus deformans and Bacillus circulans 20.1 and 14.4 U/ml, respectively. The above mentioned results indicate that Bacillus subtilisDALX2 was the most efficient bacterium from is point of view, and was therefore selected for further experiments.
4- Bacillus subtilis DALX2 was able to produce high asparaginase activity 166.8 U/ml under shaked conditions, also synthesis asparaginase enzyme activity 143.2 U/ml under static conditions when grown on medium containing ammonium sulphate.
5- Medium containing ammonium sulphate exerted a marked effect on asparaginase enzyme formation. Optimal enzyme activity titre (166.8 U/ml) occurred with 24 hrs. of incubation.
6- Asparaginase activity in culture filtrates of Bacillus subtilis DALX2 was detected over a broad pH range (2.0-8.8), the optimal (166.8 U/ml ) at an initial pH 7.0.
7- Asparaginase activity in culture filtrates of Bacillus subtilis DALX2 was detected over a broad temperature range ( Refrigeratore, 20, 30, 37, 40, 45, 50 and 55°C), the optimal (166.8 U/ml ) at 37°C.
8- Some of the tested medium additives attained a better production of asparaginase enzyme.
a- The effect of medium control containing ammonium sulphate produced highest asparaginase activity was recorded (166.8 U/ml). Also medium containing soybean and the medium containing whey mixed with ammonium sulphate produced a fairly high asparaginase activity, (158.6 and 146.1 U/ml, respectively).
b- On the other hand, the medium containing peanut and medium containing wheat bran produced a relatively high asparaginase activity recorded 134.2 and 128.3 U/ ml, respectively . Also medium containing potatoe peels and potatoes produced a relatively low asparaginase activity 93.2 and 77.3 U/ml, respectively.
c- from these results, a high production of asparaginase enzyme was obtained in the medium containing ammonium sulphate without any other additives.
9- On equal nitrogen basis, it was indicated that the biosynthesis of asparaginase was affected by the nature of nitrogen source. The medium containing ammonium sulphate proved to be the most satisfactory nitrogen source when recording the highest asparaginase titre 166.8 U/ml compared to the other medium nitrogen source, whereas, cells grown on the glucose as the best carbon source.
10- Moreover, increasing physical and chemical stability to avoiding the chance of leakage of cells or contamination in fermentation medium, the cells entrapment on Ca- alginate produce asparaginase activity recorded 211.6 U/ml. Also cells adsorption on sponge porous material, produce asparaginase activity of 196.3 U/ ml.
11- Due to Ca-alginate entrapped cells and sponge adsorbed cells of Bacillus subtilis DALX2 yield asparaginase activity higher than free cells, these used to evaluate efficiency of repeated batch fermentation. Even after 6 cycles, without disintegration of beads till the 4th run.
12- The optimum values of the selected variables were obtained by conducting 14 experiments to identify the best combinations of the parameters which were involved in the production of biomass to obtain high yield of crude extract. The data obtained from 14 experiments, were used to find out the optimum point of the process parameters by using Plackett- Burman design.
13- Partial purification of the crude asparaginase produced by Bacillus subtilis DALX2 was carried out by fractional precipitation with 85% ammonium sulphate. The specific activity after precipitation ( 47.06 U/mg protein) was 3.73 fold- purification of the original specific activity in the culture filtrate.
14- Further purification of the partially purified enzyme was achieved by using gel - filtration chromatography technique on Sephadex G-75 column. Stepwise elution with phosphate buffer yielded three protein components. Most of the asparaginase activity was present in the 1st major protein component compared to the culture filtrate. The fractions containing high asparaginase activities was rechromatographed on CM - Sephadex C-50 column. Stepwise elution with phosphate buffer with sodium chloride gradient. The enzyme activity yield was characterized by one sharp peak coinciding with one protein peak indicating its purity and homogeneity.
15- The different purification fractions were examined by electrophoretic techniques using SDS/PAGE. On using SDS- PAGE, the final fraction showed one band with electrophoretic, indicating that the molecule consists of one peptide chain with molecular weight of 92.000 Daltons.
16- The enzyme kinetics studies indicated that the enzyme activity was dependent on the concentration of the enzyme and the substrate. When the relation between the enzyme activity and the substrate concentration was treated by lineweaver – Burk analysis, it was found that Km was 0.443 mM and Vmax.