title: Biochemical characterization of recombinant L-asparaginase (AnsA) from Rhizobium etli, a member of an increasing rhizobial-type family of L-asparaginases.
abstract: We report the expression, purification, and characterization of L-asparaginase (AnsA) from Rhizobium etli. The enzyme was purified to homogeneity in a single-step procedure involving affinity chromatography, and the kinetic parameters K(m), V(max), and k(cat) for L-asparagine were determined. The enzymatic activity in the presence of a number of substrates and metal ions was investigated. The molecular mass of the enzyme was 47 kDa by SDS-PAGE. The enzyme showed a maximal activity at 50 degrees C, but the optimal temperature of activity was 37 degrees C. It also showed maximal and optimal activities at pH 9.0. The values of K(m), V(max), k(cat), and k(cat)/K(m) were 8.9 +/- 0.967 × 10⁻³ M, 128 +/- 2.8 U/mg protein, 106 +/- 2 s⁻¹, and 1.2 +/- 0.105 × 10⁴ M⁻¹s⁻¹, respectively. The L-asparaginase activity was reduced in the presence of Mn²⁺, Zn²⁺, Ca²⁺, and Mg²⁺ metal ions for about 52% to 31%. In addition, we found that NH₄⁺, L-Asp, D-Asn, and beta-aspartyl-hydroxamate in the reaction buffer reduced the activity of the enzyme, whereas L-Gln did not modify its enzymatic activity. This is the first report on the expression and characterization of the L-asparaginase (AnsA) from R. etli. Phylogenetic analysis of asparaginases reveals an increasing group of known sequences of the Rhizobialtype asparaginase II family.
authors.affiliation: Centro de Investigacion Cientifica de Yucatan, A.C-CICY, Merida Yucatan, Mexico.
authors.forename: Angelica
authors.initials: A
authors.lastname: Moreno-Enriquez
authors.forename: Zahaed
authors.initials: Z
authors.lastname: Evangelista-Martinez
authors.forename: Edith G
authors.initials: EG
authors.lastname: Gonzalez-Mondragon
authors.forename: Arturo
authors.initials: A
authors.lastname: Calderon-Flores
authors.forename: Roberto
authors.initials: R
authors.lastname: Arreguin
authors.forename: Ernesto
authors.initials: E
authors.lastname: Perez-Rueda
authors.forename: Alejandro
authors.initials: A
authors.lastname: Huerta-Saquero
mesh_terms: D000595:Amino Acid Sequence; D001215:Asparaginase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012231:Rhizobium / Q000145:classification / Q000201:enzymology / Q000235:genetics; D042163:Rhizobium etli / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001215:Asparaginase