Production and purification of thermostable L-asparaginase from locally isolated bacteria /
L-asparaginase has been widely studied by researchers globally. It has been proven for being an effective antitumor agent for the treatment of acute lymphoblastic leukemia (ALL) particularly in children. Currently, L-asparaginase from Escherichia coli and Erwinia carotovora are being used commercial...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
Gombak, Selangor :
Kulliyyah of Engineering, International Islamic University Malaysia,
2016
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| Subjects: | |
| Online Access: | Click here to view 1st 24 pages of the thesis. Members can view fulltext at the specified PCs in the library. |
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| Summary: | L-asparaginase has been widely studied by researchers globally. It has been proven for being an effective antitumor agent for the treatment of acute lymphoblastic leukemia (ALL) particularly in children. Currently, L-asparaginase from Escherichia coli and Erwinia carotovora are being used commercially for therapeutic treatment. But, the enzyme still has some limitations for clinical efficacy. First problem is the therapeutic response of L-asparaginase from both strains usually occurs with the evidence of high toxicity. Second limitation is the native forms of the enzyme have short half-life. It is necessary to discover alternative microbial sources to obtain high yield L-asparaginase with better enzyme properties that useful for therapeutic and industrial applications. The first part of this study was screening and isolation of new strains producing L-asparaginase from Sungai Klah Hot Spring, Perak, Malaysia. The isolated strains were then characterized and identified. Production of L-asparaginase by the isolates was deeply investigated. Experimental designs were applied to study several parameters in fermentation process including nutrients components to maximize enzyme production. Next, the production of L-asparaginase was carried out under optimal conditions and the enzyme was purified. The combinations of protein precipitation with ammonium sulfate, ion exchange chromatography followed by gel filtration were performed to obtain pure enzyme. Molecular weight of the L-asparaginase was determined by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Lastly, the purified enzyme was characterized in terms of pH and temperature. Seven potent strains producing L-asparaginase were successfully isolated. However, only one strain with good L-asparaginase activity was selected for further studies. The selected strain was identified as Bacillus anthracis by 16s rRNA sequencing. The Bacillus sp. was gram positive bacteria with rod or bacilli shape. It was unable to produce indole compound and showed negative test for catalase and oxidase activity. The selected strain managed to reduce nitrate to nitrite using anaerobic respiration. Sucrose and peptone were found as good enhancer for this L-asparaginase production. Analysis of variance (ANOVA) showed the optimal conditions for Bacillus anthracis culture were middle range of peptone concentration (1.40-1.50 g/l) with temperature of 30°C. The enzyme activity increased to 37.75% under optimal conditions. The specific activity of purified enzyme was 10.11 IU/mg and 50.07% yield with 2.21 fold purification. The purified enzyme was a dimer form, with a molecular weight of 65 kDa. The purified enzyme exhibited maximum activity at alkaline condition, pH 9 and high temperature of 60°C. |
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| Physical Description: | xv, 133 leaves : ill. ; 30cm. |
| Bibliography: | Includes bibliographical references (leaves 97-106). |