Overexpression and Purification of Rhizobium etli Glutaminase A by Recombinant and Conventional Procedures

Huerta-Saquero, Alejandro; Calderón, Jorge; Arreguín-Espinosa, Roberto; Calderón-Flores, Arturo; Durán, Socorro

doi:10.1006/prep.2001.1394

Cited by 18 publications

(8 citation statements)

References 21 publications

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“…A01 toward its natural substrate L-glutamine. Glutaminases from Aspergillus oryzaezae AJ11728 3 , Streptomyces sp 35 , Rhizobium etli 36 have K m near to the rSAM. As shown in Supplementary Table S1 the changes in activation energy (ΔE * ), enthalpy (ΔH * ), entropy (ΔS * ) for rSAM thermo-activation according to the Arrhenius plot were respectively 4 kJ/mol, 1.3 kJ/mol and −0.17 J/mol°K while for L-glutaminase from E. coli were respectively 13.2 kcal/mol (55.44 kJ/mol), 8.2 kcal/mol (34.44 kJ/mol) and −14.1 e.u (59 J/mol°K).…”

Section: Discussionmentioning

confidence: 99%

Novel halo- and thermo-tolerant Cohnella sp. A01 L-glutaminase: heterologous expression and biochemical characterization

Mosallatpour

Aminzadeh

Shamsara

et al. 2019

Sci Rep

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L-glutaminase importance to use in the food industry and medicine has attracted much attention. Enzymes stability has always been a challenge while working with them. We heterologously expressed and characterized a novel stable L-glutaminase from an extremophile bacterium (Cohnella sp. A01, PTCC No: 1921). Km, Vmax, catalytic efficiency and specific activity of rSAM were respectively 1.8 mM, 49 µmol/min, 1851 1/(S.mM) and 9.2 IU/mg. Activation energy for substrate to product conversion and irreversible thermo-inactivation were respectively 4 kJ/mol and 105 kJ/mol from the linear Arrhenius plot. rSAM had the highest activity at temperature 50 °C, pH 8 and was resistant to a wide range of temperature and pH. In compare to the other characterized glutaminases, rSAM was the most resistant to NaCl. Mg2+, glycerol, DTT, and BME enhanced the enzyme activity and iodoacetate and iodoacetamide inhibited it. rSAM had only been partially digested by some proteases. According to the Fluorimetry and Circular dichroism analysis, rSAM in pH range from 4 to 11 and temperatures up to 60 °C had structural stability. A cysteine residue in the enzyme active site and a thiol bond were predicted upon the modeled tertiary structure of rSAM. Present structural studies also confirmed the presence of a thiol bond in its structure.

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Section: Discussionmentioning

confidence: 99%

Novel halo- and thermo-tolerant Cohnella sp. A01 L-glutaminase: heterologous expression and biochemical characterization

Mosallatpour

Aminzadeh

Shamsara

et al. 2019

Sci Rep

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“…The effect of pH on L-asparaginase activity was determined at different values ranging from 6.0 to 11.0 at 37 o C using buffers as described elsewhere [28]. The temperature of maximum activity was evaluated by incubating the enzyme for 2 min in a standard reaction mixture containing 30 mM KH…”

Section: Effects Of Ph and Temperaturementioning

confidence: 99%

Biochemical Characterization of Recombinant L-Asparaginase (AnsA) from Rhizobium etli, a Member of an Increasing Rhizobial-Type Family of L-Asparaginases

Moreno-Enríquez

Evangelista-Martínez²,

González-Mondragón³

et al. 2012

J. Microbiol. Biotechnol.

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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 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 o C, but the optimal temperature of activity was 37 o 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 × 10 4 M-1 s-1 , respectively. The L-asparaginase activity was reduced in the presence of Mn 2+ , Zn 2+ , Ca 2+ , and Mg 2+ metal ions for about 52% to 31%. In addition, we found that + NH 4 , 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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“…1). The enzyme obtained from Streptomyces canarius has a molecular weight of 44 kDa, 8 that obtained from Rhizobium etli has a molecular weight of 26.9 kDa, 34 and that obtained from Pseudomonas nitroreducens has a molecular weight of 40 kDa. 35 The recombinant enzyme has shown optimum activity at 70 C and pH 9.…”

Section: Discussionmentioning

confidence: 99%

Recombinantl-glutaminase obtained fromGeobacillus thermodenitrificansDSM-465: characterization andin silicoelucidation of conserved structural domains

et al. 2019

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Overexpression and Purification of Rhizobium etli Glutaminase A by Recombinant and Conventional Procedures

Cited by 18 publications

References 21 publications

Novel halo- and thermo-tolerant Cohnella sp. A01 L-glutaminase: heterologous expression and biochemical characterization

Novel halo- and thermo-tolerant Cohnella sp. A01 L-glutaminase: heterologous expression and biochemical characterization

Biochemical Characterization of Recombinant L-Asparaginase (AnsA) from Rhizobium etli, a Member of an Increasing Rhizobial-Type Family of L-Asparaginases

Recombinantl-glutaminase obtained fromGeobacillus thermodenitrificansDSM-465: characterization andin silicoelucidation of conserved structural domains

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