Heterologous expression, purification and biochemical characterization of a glutamate racemase (MurI) from Streptococcus mutans UA159.

Background

Glutamate racemase (MURI) is a cofactor-independent enzyme which is essential for bacterial peptidoglycan biosynthesis pathway and therefore has been considered an attractive target for antimicrobial drug development. While in our previous study of gene essentiality MURI indicated Streptococcus mutans, a metiologicalajor agent of human Purified Recombiant Proteins dental caries, the study of S. mutans Muri not provide definitive results. This study aims to produce and characterize the biochemical properties of S. mutans UA159 Muri of the genome.

method

Structural characterization and prediction of multiple sequence alignment performed by bioinformatics analysis. Ilis6-tagged recombinant S. mutans MURI was expressed in pColdII expression vector and further purified using Ni 2+ affinity chromatography. protein solubility, purity and the state of aggregation were analyzed by SDS-PAGE, Western blotting, native PAGE and SEC-HPLC. assay kinetic parameters assessed by circular dichroism (CD). The kinetic constants calculated based on a curve fit to the Michaelis-Menten equation. Effect of temperature and pH on enzymatic activity is determined by a series of coupled enzymatic reaction mixture.

result

Glutamate racemase gene of S. mutans UA159 was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3). 264-amino acid protein, as a mixture of dimeric and monomeric enzyme, purified to electrophoretic homogeneity.

In CD trials, S. mutans Muri displayed a unique kinetic parameters (Km, D-Glu-Glu → l = 0.3631 ± 0.3205 mM, Vmax, d-Glu-Glu → l = 0.1963 ± 0.0361 min-1 mM, kcat, d -Glu-Glu → l = 0.0306 ± 0.0065 s-1, kcat / Km, d-Glu-Glu → l = 0.0844 ± 0.0128 s-1 mM 1, with d-glutamic acid as substrate; Km, l-Glu → d -Glu = 0.8077 ± 0.5081 mM, Vmax, d → l-Glu-Glu = 0.2421 ± 0.0418 mM min-1, kcat, l-Glu-Glu → d = 0.0378 ± 0.0056 s-1, kcat / Km, l-Glu-Glu → d = 0.0468 ± 0.0176 s-1 mM-1, with l- glutamate as a substrate). S. mutans is owned Muri optimal assay temperature of 37.5 ° C and the optimum pH was 8.0.

Heterologous expression, purification and biochemical characterization of a glutamate racemase (MurI) from Streptococcus mutans UA159.

Conclusion

The study's findings provide insight into the structure and Viral Recombinant Proteins biochemical properties of glutamate racemase in S. mutans and provide guidance envisioned to employ glutamate racemase in anti-caries drug design.

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