Document Details
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Document Type |
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Thesis |
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Document Title |
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Biosynthesis, purification and characterization of L-asparaginase from actinomycetes, isolated from Kingdom Saudi Arabia الإنتاج الحيوي وتنقية وتوصيف إنزيم اسبرجينيز من الاكتينوميسيتات المعزولة من المملكة العربية السعودية |
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Subject |
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Faculty of Science |
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Document Language |
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Arabic |
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Abstract |
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Strains of actinomycetes were isolated from different sources viz. fresh water, marine water, sand, soil, marine fishes, shrimps and sediments using starch nitrate medium with either bekanamycin or amphotericin B. The samples were collected from Makkah, Al-Madinah and Jeddah. Among the samples, soil harbored highest number of actinomycetes population followed by sand, water and marine animals. Among the media used for isolation of actinomycetes, starch nitrate agar medium was found to be the most efficient for isolation. Addition of bechanamycin decreased the number of actinomycetes recovered and allowed to more resistance isolates to be developed. Amphertricin B suppressed the growth of fungi and enhanced actinomycetes isolation. Out of the 100 strains isolated, only eleven isolates produced varying quantities of L-asparaginase in solid and liquid media. The most active isolate ATRM47 that produced the highest quantities of intracellular L-asparaginase was from rhizosphere of palm tree, grown in Al-Madinah. It was taken up for further studies. Impact of various physical and chemical factors such as carbon sources, nitrogen source, amino and organic acids, incubation temperatures, initials pH, shaking rates and inoculums size on the growth of isolate ATRM47 and intracellular and extracellular L-asparaginase activity were also studied. Optimum growth and enzyme activity was noticed under initial pH 6.5, temperature 30°C and 100 rpm for 5 days, dextrose 0.5% and L-asparagine 1.5% as carbon and nitrogen sources respectively, inoculum size of 4x105 CFU/ml and without any other amino acids addition. The enzyme was purified using column chromatography. The molecular weight for the extracellular enzyme was 120 kDa and it was140 kDa for intracellular enzyme. The purified intracellular L-asparaginase showed antitumor activity against Ehrlich Ascites Carcinoma with LD50 of 150U/ml. The optimum pH was 8.5; optimum temperature was 37°C, substrate specificity L-asparagine at 30 mmole. The enzyme activity was activated by the presence of glycerol, manitol sorbitol, and CaCl2 and inhibited by ETDA, isopropanol , mercapoethanol, FeSO4 and NiSO4. Analysis of the cell components of the isolated strains has revealed the wall type-I (the wall type-I is typical for the genus Streptomyces) and the strains were micromorphologically similar to the genus Streptomyces. Hence, the morphological, physiological, biochemical and genetic results obtained for the L-asparaginase producing strain was compared, and the strain was tentatively identified as Streptomyces filipinensis ATRM47. |
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Supervisor |
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Dr. Magda Mohamed Aly |
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Thesis Type |
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Doctorate Thesis |
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Publishing Year |
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1431 AH
2010 AD |
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Co-Supervisor |
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Dr.Mohammed Gurban Kuchari |
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Added Date |
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Saturday, December 4, 2010 |
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Researchers
| سامية درويش جستنيه | Jastaniah, Samyah Darwish | Researcher | Doctorate | |
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