Publications scientifiques
BARTHELEMIE GUEBOTO
PAUL FEWOU MOUNDIPA
Cameroon Journal of Biological and Biochemical Sciences
Urease is an industrially important enzyme whose production remains limited due to the low availability of the raw material (urease source). Controlling the production of this enzyme can, in view of its many applications, promote the development of importing countries such as Cameroon. This study aimed to extract and characterize urease from Centrosema pubescens for its future valorization. This work is part of the search for local sources of this enzyme through its purification and characterization from Centrosema pubescens. Seeds were germinated at 2, 4, 6, 8, and 10 days, and crude extracts of the enzyme were prepared in an extraction solvent consisting of a mixture of phosphate buffer (pH 7; 0.1 M)/acetone 80/20 v/v to obtain homogenates at 25% w/v, and then centrifuged. Protein content and urease activity were assessed spectrophotometrically. The day of germination on which the germinated seeds showed high enzymatic activity was selected. The crude extract was partially purified using ammonium sulphate precipitation followed by desalting through exclusion chromatography. The presence of urease in the mixture of active fractions was verified by denaturing gel electrophoresis (SDS-PAGE). The kinetic parameters (Vmax and Km) of urease were determined using the kinetic method. The results showed that the 4th day of germination showed the highest specific activity (0.140±0.004 µmol NH4+/min/mg protein) and was chosen as the germination day for the rest of the work. The crude extract from the 4th day was selected for having a purification percentage of 18.78 and a degree of purification of 27.79. The kinetic parameter values for urease under these conditions were: 0.052 ± 0.01 µmol NH4+/min and 0.48 ± 0.153mM for Vmax and Km, respectively, and those for Jack Bean urease were 0.53 ± 0.01 µmol NH4+/min and 0.030 ± 0.01 mM. These observations show that Centrosema pubescens may be a good source of urease. Key words: Urease, Centrosema pubescens, kinetic parameters, plant.
