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Purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from saline strain Idiomarina loihiensis

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Author(s): Ilham Mardad | Tarik Baibai | Emna Ammar | Abdelaziz Soukri

Journal: Advances in Biological Chemistry
ISSN 2162-2183

Volume: 03;
Issue: 02;
Start page: 170;
Date: 2013;
Original page

Keywords: Glyceraldehyde-3-Phosphate Dehydrogenase | Idiomarina loihiensis | Purification | NAD+ | Kinetics | Saline Strain

ABSTRACT
Idiomarina loihiensis was isolated from the salt works in Sfax (Tunisia), until now, the characterization of the GAPDH phosphorylante was never studied. Here, we report the isolation and the biochemical characterization of glyceralehyde-3-phosphate dehydrogenase (GAPDH) fromI. loihiensis saline’s bacteria on the basis of the apparent native and subunit molecular weights, physico-chemical and kinetic characterizations. The purification method consisted of two steps, ammonium sulfate fractionation followed by one chromatographic step, namely dye-affinity on Blue Sepharose CL-6B. Polyclonal antibodies against the purified enzyme were used to recognize theI. loihiensis GAPDH by Western blotting. The optimum pH of the purified enzyme was 8.5. Studies on the effect of temperatures revealed an enzyme increasing activity of about 45?C. The molecular weight of the purified enzyme was 36 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yield a molecular weight of 147 kDa. The Michaelis constants for NAD+ and D-glyceraldehyde-3-phosphate estimated was 19 μM and 3.1 μM, respectively. The maximal velocity of the purified enzyme was estimated to be 2.06 U/mg, approximately 6-fold increase in specific activity and a final yield of approximately 32.5%. The physicochemical properties of this GAPDH, being characterized, could be used in further studies.

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