Protein expression of alkyl hydroperoxide reductase subunit F (salmonella typhimurium) in E. coli
Nicotinamide Adenine Dinucleotide reduced form (NADH) serves an important role as an electron carrier in numerous cellular pathways, for instance, in the transformation of malate to oxaloacetate in the Krebs cycle involves the conversion of NADH to NAD+. The enzyme that catalyzes this reaction is...
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| Format: | Final Year Project |
| Language: | English |
| Published: |
2009
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| Online Access: | http://hdl.handle.net/10356/16413 |
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| Institution: | Nanyang Technological University |
| Language: | English |
| Summary: | Nicotinamide Adenine Dinucleotide reduced form (NADH) serves an important role as an electron carrier in numerous cellular pathways, for instance, in the transformation of malate to oxaloacetate in the Krebs cycle involves the conversion of NADH to NAD+.
The enzyme that catalyzes this reaction is H¬2O2 – forming NADH oxidase produced by transcription from the gene, Alkyl Hydroperoxide Reductase subunit F (AhpF), found commonly in Salmonella typhimurium.
To investigate how NADH binds to the active site of the enzyme, the enzyme was cloned, overexpressed and purified and its activity was measured based on its ability to take up the substrate, NADH or NAPDH. The activity result was compared against with the activity of other mutants done by other FYP students. There was a marked reduction in the activity of the mutated enzyme in taking up NADH. However, there was no significant difference in the specific activity between the wildtype enzyme and the other mutants. |
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