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KMID : 0545120120220030292
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Journal of Microbiology and Biotechnology
2012 Volume.22 No. 3 p.292 ~ p.300
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Biochemical Characterization of Recombinant L-Asparaginase (AnsA) from Rhizobium etli, a Member of an Increasing Rhizobial-Type Family of L-Asparaginases
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Angelica Moreno-Enriquez
Zahaed Evangelista-Martinez
Edith G. Gonzalez-Mondragon
Arturo Calderon-Flores
Roberto Arreguin
Ernesto Perez-Rueda
Alejandro Huerta-Saquero
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Abstract
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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 Km, Vmax, and kcat 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 50oC, but the optimal temperature of activity was 37oC. It also showed maximal and optimal activities at pH 9.0. The values of Km, Vmax, kcat, and kcat/Km were 8.9 ± 0.967 × 10-3 M, 128 ± 2.8 U/mg protein, 106 ± 2 s-1, and 1.2 ± 0.105 × 104M-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 β-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.
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KEYWORD
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Type II asparaginase, Rhizobium etli, biochemical characterization
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