- Title
- Bioinformatic analysis, isolation and kinetic characterisation of red algae (Gelidium capense) dehydrogenases
- Creator
- Gogela, Yanga
- Subject
- Bioinformatics Chondrus crispus
- Date Issued
- 2019
- Date
- 2019
- Type
- Thesis
- Type
- Masters
- Type
- MSc
- Identifier
- http://hdl.handle.net/10353/19164
- Identifier
- vital:39878
- Description
- Lactate and alcohol dehydrogenases have attracted much attention in various industries and scientific research for their ability to produce chirally pure compounds and be assayed for activity using more straightforward and reproducible assay methods. These enzymes have been previously isolated and purified from various plants, animals and microorganisms. So far, the molecular and biochemical properties of enzymes from these dehydrogenase families in red algae are mostly unknown. Red macroalgae have been used for centuries for the treatment of various diseases and as a source of ingredients in the food industry. The aim of this study was to identify genes in the sequenced red algae genomes that encode dehydrogenases, to use bioinformatic tools to confirm that the proteins encoded are dehydrogenases and to isolate and kinetically purify alcohol or lactate dehydrogenase from red algae species found along the coastline of the Eastern Cape Province. A combination of bioinformatics tools, molecular and biochemical techniques were used to identify, purify, and characterise ADH and LDH enzymes. Bioinformatics analysis revealed two alcohol dehydrogenase genes and two hypothetical genes encoding functional domains similar to D-lactate dehydrogenases from other species. The ADH and LDH-like genes shared low sequence identity at the protein level with medium-chain dehydrogenases/reductases (MDRs) and 2-hydroxy acid dehydrogenases, respectively. These two dehydrogenase genes showed a highly conserved NAD-binding motif (Rossmann-fold) similar to many other NAD-dependent dehydrogenases. The ADH and LDH proteins contained no signal peptides and may be located in the cytoplasm. The phylogenetic tree analysis showed that the two ADH genes belonged to cinnamyl and class III alcohol dehydrogenases, whereas the LDHlike genes were grouped with D-lactate dehydrogenases from other organisms. The ADH and LDH gene family showed cis-acting regulatory elements that are mostly involved in stress response and hormonal response. Structural analysis showed that the dehydrogenases 3D structure predicted models comprise of two domains, namely the substrate binding and the coenzyme binding domains that are rich in beta-strands secondary structure elements. The LDH from red algae was purified approximately 4-fold with a specific activity of 0.044 U/mg. The purified LDH enzyme had a molecular weight of approximately 37kDa. The LDH was active across a broad pH range from 5-9 with a pH optimum observed at 7.5. The LDH ii enzyme in red algae exhibits a temperature optimum of 40 ⁰C and heat stability up to 40 ⁰C. Above 50 °C the LDH activity rapidly decreased showing that the LDH in red algae is not thermostable. The LDH enzyme showed a Km value of 0.8 mM and Vmax of 0.0067 mM.min-1 when using sodium pyruvate as a substrate.
- Format
- 162 leaves
- Format
- Publisher
- University of Fort Hare
- Publisher
- Faculty of Science and Agriculture
- Language
- English
- Rights
- University of Fort Hare
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UFH Department of Biochemistry & Microbiology
| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | SOURCE1 | GOGELA Dissertation.pdf | 4 MB | Adobe Acrobat PDF | View Details Download |