Purification and characterization of a methylene urea-hydrolyzing enzyme from Rhizobium radiobacter (Agrobacterium tumefaciens)

Marja E. Koivunen, Christophe Morisseau, John W. Newman, William R. Horwath, Bruce D. Hammock

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


Slow-release fertilizers are gaining acceptance to increase fertilizer use efficiency and reduce environmental impact. The release of nitrogen from methylene urea, a common slow release N fertilizer, is controlled by microbial decomposition. An enzyme hydrolyzing slow-release nitrogen fertilizer, methylene urea, was purified from Rhizobium radiobacter (Agrobacterium tumefaciens) to homogeneity using a four-step purification procedure with an overall yield of 3%. The active enzyme has a molecular mass of approximately 180 kDa determined by size exclusion chromatography, and the SDS page of the purified protein indicated three subunits of different sizes (62, 34 and 32 kDa). The N-terminal amino acid sequence of the 62 kDa fragment indicates identity with urease subunits from Mycobacterium tuberculosis (73%) and Helicobacter pylori (71%). However, for the internal amino acid sequences of the 62 kDa fragment no matches with known proteins were found. Some internal peptides in the smaller subunits (32 and 34 kDa) are homologous to urease subunits and unknown proteins in Agrobacterium tumefaciens. Based on the kinetic properties, substrate selectivity, and inhibition characteristics, the novel enzyme (MUase) is an intracellular enzyme complex with urease activity. The enzymatic mechanism of methylene urea breakdown was studied using a novel LC-MS method for MU analysis, which indicates that all cold-water soluble nitrogen forms of methylene urea are subjected to hydrolysis, and the hydrolysis proceeds via methylurea, urea and other yet unidentified hydrolysis-products, suggesting that the isolated enzyme complex performs a multistep hydrolysis. The microbiological and molecular data is useful in determining the soil factors affecting the efficacy of methylene urea as a slow release fertilizer in agricultural production systems.

Original languageEnglish (US)
Pages (from-to)1433-1442
Number of pages10
JournalSoil Biology and Biochemistry
Issue number11
StatePublished - Nov 1 2003


  • Agrobacterium tumefaciens
  • Methylene urea
  • Purification
  • Rhizobium radiobacter
  • Slow-release fertilizer
  • Soil enzymes
  • Ureaformaldehyde

ASJC Scopus subject areas

  • Soil Science
  • Biochemistry
  • Ecology


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