title: Purification and characterization of urease from dehusked pigeonpea (Cajanus cajan L) seeds.
abstract: Urease has been purified from the dehusked seeds of pigeonpea (Cajanus cajan L.) to apparent electrophoretic homogeneity with approximately 200 fold purification, with a specific activity of 6.24 x10(3) U mg(-1) protein. The enzyme was purified by the sequence of steps, namely, first acetone fractionation, acid step, a second acetone fractionation followed by gel filtration and anion-exchange chromatographies. Single band was observed in both native- and SDS-PAGE. The molecular mass estimated for the native enzyme was 540 kDa whereas subunit values of 90 kDa were determined. Hence, urease is a hexamer of identical subunits. Nickel was observed in the purified enzyme from atomic absorption spectroscopy with approximately 2 nickel ions per enzyme subunit. Both jack bean and soybean ureases are serologically related to pigeonpea urease. The amino acid composition of pigeonpea urease shows high acidic amino acid content. The N-terminal sequence of pigeonpea urease, determined up to the 20th residue, was homologous to that of jack bean and soybean seed ureases. The optimum pH was 7.3 in the pH range 5.0-8.5. Pigeonpea urease shows K(m) for urea of 3.0+/-0.2 mM in 0.05 M Tris-acetate buffer, pH 7.3, at 37 degrees C. The turnover number, k(cat), was observed to be 6.2 x 10(4) s(-1) and k(cat)/K(m) was 2.1 x 10(7) M(-1) s(-1). Pigeonpea urease shows high specificity for its primary substrate urea.
authors.affiliation: School of Biotechnology, Faculty of Science, Banaras Hindu University, - 221 005, Varanasi, India.
authors.forename: Nilanjana
authors.initials: N
authors.lastname: Das
authors.forename: Arvind M
authors.initials: AM
authors.lastname: Kayastha
authors.forename: Punit K
authors.initials: PK
authors.lastname: Srivastava
mesh_terms: D000595:Amino Acid Sequence; D036561:Cajanus / Q000201:enzymology*; D012639:Seeds / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014510:Urease / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D014510:Urease