PMID:22450783

Citation	Moreno-Enriquez, A, Evangelista-Martinez, Z, Gonzalez-Mondragon, EG, Calderon-Flores, A, Arreguin, R, Perez-Rueda, E and Huerta-Saquero, A (2012) Biochemical characterization of recombinant L-asparaginase (AnsA) from Rhizobium etli, a member of an increasing rhizobial-type family of L-asparaginases. J. Microbiol. Biotechnol. 22:292-300
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.
Links	PubMed
Keywords	Amino Acid Sequence; Asparaginase/chemistry; Asparaginase/genetics; Asparaginase/isolation & purification; Asparaginase/metabolism; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/isolation & purification; Bacterial Proteins/metabolism; Enzyme Stability; Kinetics; Molecular Sequence Data; Molecular Weight; Phylogeny; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Rhizobium/classification; Rhizobium/enzymology; Rhizobium/genetics; Rhizobium etli/chemistry; Rhizobium etli/classification; Rhizobium etli/enzymology; Rhizobium etli/genetics; Substrate Specificity
edit table

Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
RHIET:Q9RFN5		GO:0004067: asparaginase activity	ECO:0000314:		F		Fig. 5. shows the Michaelis- Menten substrate dependence for R. etli L-asparaginase. The steady-state kinetic properties of the recombinant AnsA were investigated at various concentrations of the substrate, and Km, Vmax, and kcat for L-asparagine were determined.	complete

References

See Help:References for how to manage references in GONUTS.