Purification and characterization of rhodanese from a bacterium isolated from soil in a cassava processing site.
| dc.contributor.author | IDOWU Clement Oluwaseun | |
| dc.date.accessioned | 2025-09-02T10:27:44Z | |
| dc.date.available | 2025-09-02T10:27:44Z | |
| dc.date.issued | 2022 | |
| dc.description | xvi, 106p | |
| dc.description.abstract | This study determined the cyanide content in the soil of cassava waste dumping site, isolated and identified a rhodanese-producing bacteria and optimized conditions for rhodanese production. It also purified and characterized the rhodanese. These were with a view to providing information on form of rhodanese for use in pretreatment of cyanide polluted environment. Soil samples were collected at garri processing site located at Tonkere gate, Obafemi Awolowo University, Ile-Ife, Nigeria. The soil samples were soaked in distilled water at 1:3 (w/v) for 72 h. This was followed by centrifugation at 4000 rpm for 10 min. Cyanide concentration determination was carried out by the standard titrimetric method. The soil sample mixture was serially diluted with sterilized water and plated on Luria-Bertani (LB) agar medium to isolate bacteria. The bacterial isolates were screened for their ability to degrade free cyanide and the best rhodanese-producing strain was selected for further study. Optimization of culture conditions was carried out by varying incubation time, inoculum volume, temperature, pH and carbon and nitrogen sources. Rhodanese was assayed according to a standard method and activity expressed as Rhodanase Unit (RU). Rhodanese produced was partially purified with ion-exchange chromatography on CM-Sephadex C-50. The kinetic studies were carried out by varying the concentration of potassium cyanide and sodium thiosulphate, one at a time. Effects of pH, temperature and salts on the activity of the partially purified rhodanese were carried out. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out to determine the subunit molecular weight of the enzyme. Three rhodanese-producing strains were identified based on cell morphology, cultural and biochemical characteristics. The optimized conditions for the growth of the bacterium include 1% casein, 50 mM potassium cyanide (KCN) (as sources of nitrogen and carbon respectivelyand 0.1 ml inoculum size for 24 h. The specific activity of the rhodanese enzyme was 152.8 RU/mg of protein with 35.7% yield and 2.08 purification fold. The subunit molecular weight was 25,990 Da. The kinetic studies gave the values of Km as 2.4108 ± 0.3697 mM and 3.0303 ± 0.5216 mM, Vmax as 13.44 ± 0.1735 RU and 13.97 ± 0.2354 RU for varied concentration of KCN and sodium thiosulphate (Na2S2O3) respectively. The activity of the enzyme was not affected by CaCl2, CuCl2, KCl, MgCl2, MnCl2 and NaCl, but inhibition occurred with CoCl2 at concentration as low as 0.01 mM. The optimum temperature for the activity of the enzyme was found to be 40 oC and optimum pH was 7.0. The enzyme was heat stable at 30 to 50 oC and was less stable at 60 oC. The Arrhenius graph was biphasic and the activation energies were 12.5 kJ/mol/K and 14.85 kJ/mol/K from the two phases. The study concluded that the rhodanese from a bacterium of the soil of cassava processing sites possessed good catalytic properties and could be used for bioremediation of cyanide polluted soil. | |
| dc.identifier.citation | IDOWU, C.O.(2022) Purification and characterization of rhodanese from a bacterium isolated from soil in a cassava processing site. Department of Biochemistry and Molecular Biology, Faculty of Science, Obafemi Awolowo University. | |
| dc.identifier.uri | https://ir.oauife.edu.ng/handle/123456789/6747 | |
| dc.language.iso | en | |
| dc.publisher | Department of Biochemistry and Molecular Biology, Faculty of Science, Obafemi Awolowo University. | |
| dc.title | Purification and characterization of rhodanese from a bacterium isolated from soil in a cassava processing site. | |
| dc.type | Thesis |