Optimum production of Mycobacterium smegmatis phytase by recombinant Escherichia coli DH5 a /
Phytase, also known as phytate-degrading enzyme, catalyzes the hydrolysis of phytate (inositol hexakisphosphate) with sequential release of phosphate and lower inositol phosphate.In the present work, the ~1.1 kb gene encoding for the novel phytase from Mycobacterium smegmatis (Msphy gene) has been i...
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| Format: | Thesis |
| Language: | English |
| Published: |
Kuala Lumpur :
Kulliyyah of Engineering, International Islamic University Malaysia,
2012
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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: | Phytase, also known as phytate-degrading enzyme, catalyzes the hydrolysis of phytate (inositol hexakisphosphate) with sequential release of phosphate and lower inositol phosphate.In the present work, the ~1.1 kb gene encoding for the novel phytase from Mycobacterium smegmatis (Msphy gene) has been isolated and amplified from the genomic DNA by the polymerase chain reaction (PCR) and cloned into pBAD-TOPO expression vector. The newly constructed plasmid MSuia was then transformed into Eschericha coli DH5α as a host for protein over-expression. The ~45 kDa active Msphy phytase was expressed and confirmed by spectrophotometric study and Western blotting. Plackett-Burman design, One-Factor-at-ATime (OFAT) method and central composite design (CCD) under response surface methodology (RSM) were deployed to optimize the medium components to improve recombinant phytase production in E. coli DH5α. Six variables showed positive effects on phytase production by Plackett-Burman; nitrogen sources (yeast extract, peptone and ammonium phosphate dibasic), dipotassium hydrogen phosphate and minerals (Mg and Fe). Based on the OFAT method, the optimum levels of the variables were, 30 g/L yeast extracts, 1 g/L (NH4)2HPO4, 3 g/L MgSO4.7H2O, 0.3 g/L FeSO4.7H2O, 0.1 % (v/v) glycerol. Yeast extract, MgSO4.7H2O and FeSO4.7H2O were selected for optimization by central composite design, whereas the other factors were maintained at their optimum level. Statistical analysis showed that the optimum media containing 34.06 g/L yeast extract, 3.6 g/L MgSO4.7H2O and 0.32 g/L FeSO4.7H2O gave the maximum phytase production of 118.9 U/mL. The analysis showed that the yeast extract and MgSO4.7H2O were significant compared to the FeSO4.7H2O for phytase production. The optimization of process conditions such as temperature, agitation speed and seed age was carried out by using the Full Factorial Experimental (FUFE) Design formulated by statistical software MINITAB. Optimization of process conditions was carried out with different ranges of temperature, agitation speed and seed age with fixed media compositions. A regression model was developed to determine the optimum variables. Several techniques such as ANOVA, t-test and p-values were observed to evaluate the model as well as the optimization process. The maximum phytase production (200 U/mL), while the model equation predicted phytase production with 193 U/mL using the optimum condition (temperature of 37°C, 500 RPM agitation speed and seed age of 1.55 at OD600nm). The results depicted that both temperature and agitation speed was significantly affecting phytase production. The optimum process conditions improved the phytase production and plasmid stability. In large-scale production, constant tip speed suit the best for scale-up strategy compare to the constant power number. Lastly, the recombinant phytase was purified to homogeneity and biochemically characterized with respect to its molecular characterization,specific activity, pH activity profile and substrate specificity. |
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| Item Description: | Abstracts in English and Arabic. "A dissertation submitted in fulfilment of the requirements for the degree of Master of Science (Biotechnology Engineering)."--On t.p. |
| Physical Description: | xx, 168 leaves : ill. ; 30cm. |
| Bibliography: | Includes bibliographical references (leaves 138-153). |