BARTHELEMIE GUEBOTO, FREDERIC NICO NJAYOU* & PAUL FEWOU MOUNDIPA
Laboratory of Pharmacology and Toxicology, Department of Biochemistry, Faculty of Science, University of Yaoundé I, PO Box 812, Yaoundé, Cameroon
Corresponding Author: njayou@yahoo.fr
Received: 17 Jul 2025, Reviewed: 03 Sep 2025, Revised: 22 Sep 2025, Accepted: 03 Sep 2025, Published: 09 Jan 2026.
https://doi.org/10.63342/cjbbs2026.34.25024.en
ABSTRACT
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.
RÉSUMÉ
L’uréase est une enzyme d’importance industrielle dont la production reste limitée en raison de la faible disponibilité de la matière première (source d’uréase). Compte tenu de ses nombreuses applications, la maîtrise de la production de cette enzyme peut favoriser le développement des pays importateurs comme le Cameroun. Le but de cette étude était d’extraire et de caractériser l’uréase de Centrosema pubescens en vue d’une valorisation future. Ce travail s’inscrit dans le cadre de la recherche des sources locales de cette enzyme à travers la purification et la caractérisation de l’uréase de Centrosema pubescens. Des extraits bruts ont été préparés dans un mélange de tampon phosphate (pH 7; 0.1 M)/acétone 80/20 v/v en vue d’obtenir un homogénat à 25% m/v pour chaque jour de germination des graines (2, 4, 6, 8 et 10 jours) par centrifugation. La teneur en protéines et l’activité uréasique ont été évaluées par spectrophotométrie. Le jour de germination au cours duquel les graines germées ont montré une activité enzymatique élevée a été sélectionné. L’extrait brut préparé a été utilisé pour la purification partielle de l’uréase par précipitation au sulfate d’ammonium suivie d’une chromatographie d’exclusion après dessalage. La présence d’uréase dans le mélange de fractions actives a été vérifiée par électrophorèse sur gel dénaturant (SDS-PAGE). Les paramètres (Vmax et Km) de l’uréase ont été déterminés par la méthode de cinétique enzymatique. Les résultats ont montré une activité spécifique plus élevée (0,140±0,004 µmol NH4+/min/mg de protéine) au 4e jour de germination et les graines à cette période physiologique ont été utilisées comme matériel pour la suite du travail. L’uréase a été purifiée dans les graines germées au jour 4 avec un pourcentage et un degré de purification respectifs de 18,78 et de 27,79. Les valeurs des paramètres cinétiques pour l’uréase dans ces conditions étaient : 0,052 ± 0,01 µmol NH4+/min et 0,48 ± 0,153 mM pour Vmax et Km respectivement, et celles de l’uréase de Jack Bean étaient de 0,53 ± 0,01 µmol NH4+/min pour la Vmax et 0,030 ± 0,01 mM pour le Km. Ces observations montrent que Centrosema pubescens peut être une bonne source d’uréase.
Mots-clés : Uréase, Centrosema pubescens, paramètres cinétiques, plante.
REFERENCES
Altın S, Tohma H, Gülçin İ, & Köksal E. (2017). Purification, characterization, and inhibition sensitivity of peroxidase from wheat (Triticum aestivum ssp. Vulgare). International Journal of Food Properties https://www.tandfonline.com/doi/abs/10.1080/10942912.2016.1225308
Biglar M, Soltani K, Nabati F, Bazl R, Mojab F, & Amanlou M. (2012). A Preliminary Investigation of the Jack-Bean Urease Inhibition by Randomly Selected Traditionally Used Herbal Medicine. Iranian Journal of Pharmaceutical Research 11(3), 831–837.
Burbank M, Weaver T, Lewis R, Williams T, Williams B, & Crawford R. (2013). Geotechnical Tests of Sands Following Bioinduced Calcite Precipitation Catalyzed by Indigenous Bacteria. Journal of Geotechnical and Geoenvironmental Engineering 139(6), 928–936. https://doi.org/10.1061/(ASCE)GT.1943-5606.0000781
Dikhtyarev SI, Yanina MM, Kuznetsova RG. (1983). Isolation of a urease from watermelon seeds and the study of its properties. Chem Nat Compd 19, 587–591. https://doi.org/10.1007/BF00576090
EL-Hefnawy ME, Sakran M, Ismail AI, & Aboelfetoh EF. (2014). Extraction, purification, kinetic and thermodynamic properties of urease from germinating Pisum Sativum L. seeds. BMC Biochemistry 15(1), 15. https://doi.org/10.1186/1471-2091-15-15
Follmer C (2008). Insights into the role and structure of plant ureases. Phytochemistry 69(1):18-28. doi: https://10.1016/j.phytochem.2007.06.034
Gornall AG, Bardawill CJ, & David MM. (1949). Determination of serum proteins by means of the biuret reaction. The Journal of Biological Chemistry 177(2), 751–766.
Hamdan N, Kavazanjian E, & O’Donnell S. (2013). Carbonate cementation via plant-derived urease: 18th International Conference on Soil Mechanics and Geotechnical Engineering, ICSMGE 2013. 18th International Conference on Soil Mechanics and Geotechnical Engineering, 3, 2489–2492.
Javadi N, Khodadadi H, Hamdan N, & Kavazanjian E. (2018). EICP Treatment of Soil by Using Urease Enzyme Extracted from Watermelon Seeds. Geotechnical Special Publication 2018-March (GSP 296), 115-124. https://doi.org/10.1061/9780784481592.012
Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680-685. https://doi.org/10.1038/227680a0
Lim K, Leverenz H. Characterization of Urease Derived from Citrullus lanatus (Watermelon) Seeds to Estimate Total Kjeldahl Nitrogen in Human Urine. (2019). International Journal of Environmental Analytical Chemistry 99(5), https://doi.org/10.6084/m9.figshare.7998416
Lineweaver H, & Burk D. (1934). The Determination of Enzyme Dissociation Constants. Journal of the American Chemical Society, 56(3), 658–666. https://doi.org/10.1021/ja01318a036
Mobley HL, Island MD, Hausinger RP (1995). Molecular biology of microbial ureases. Microbiol Rev. 59(3):451-80. https://doi.org/10.1128/mr.59.3.451-480
Pervin S, Jahan GS, & Rana AYK, Rana M, Sana NK, Rahman H, Shaha RK (2013). Effects of some environmental variables on urease in germinating chickpea (Cicer arietinum L.) seed. Journal of Stress Physiology and Biochemistry 9(3), 345-356.
Polacco JC, Sparks RB. (1982). Patterns of urease synthesis in developing soybeans. Plant Physiol. 70(1), 189-94. https://doi.org/10.1104/pp.70.1.189
Rajendran R, Pandi A, Ramchary A, Thiagarajan H, Panneerselvam J, Niraikulam A, Kuppuswami GM, Ramudu KN. (2019). Extracellular urease from Arthrobacter creatinolyticus MTCC 5604: scale up, purification and its cytotoxic effect thereof. Mol Biol Rep. 46(1):133-141. https://doi.org/10.1007/s11033-018-4453-8
Wassermann GE, Olivera-Severo D, Uberti AF, Carlini CR. (2010). Helicobacter pylori urease activates blood platelets through a lipoxygenase-mediated pathway. J Cell Mol Med. 14(7), 2025-34. https://doi.org/10.1111/j.1582-4934.2009.00901.x
Weber M & Jones M & Ulrich J. (2008). Optimisation of isolation and purification of the jack bean enzyme urease by extraction and subsequent crystallization. Food and Bioproducts Processing – FOOD BIOPROD PROCESS 86, 43-52. https://doi.org/10.1016/j.fbp.2007.10.005
Witte CP, Medina-Escobar N. (2001). In-gel detection of urease with nitroblue tetrazolium and quantification of the enzyme from different crop plants using the indophenol reaction. Anal Biochem. 290(1):102-7. https://doi.org/10.1006/abio.2000.4933
Witte CP, Tiller SA, Taylor MA, Davies HV. (2002). Leaf urea metabolism in potato. Urease activity profile and patterns of recovery and distribution of (15)N after foliar urea application in wild-type and urease-antisense transgenics. Plant Physiol. 128(3), 1129-36. https://doi.org/10.1104/pp.010506
Zusfahair, Ningsih D, Fatoni A & Pertiwi, D. (2018). Determination of Urease Biochemical Properties of Asparagus Bean (Vigna unguiculata ssp Sesquipedalis L.). IOP Conference Series: Materials Science and Engineering. 349. 012073. https://doi.org/10.1088/1757-899X/349/1/012073