Molecular identification of monomeric aspartate racemase from Bifidobacterium bifidum

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Abstract

Bifidobacterium bifidum is a useful probiotic agent exhibiting health-promoting properties and contains D-aspartate as an essential component of the cross-linker moiety in the peptidoglycan. To help understand D-aspartate biosynthesis in B. bifidum NBRC 14252, aspartate racemase, which catalyzes the racemization of D- and L-aspartate, was purified to homogeneity and characterized. The enzyme was a monomer with a molecular mass of 27 kDa. This is the first report showing the presence of a monomeric aspartate racemase. Its enzymologic properties, such as its lack of cofactor requirement and susceptibility to thiol-modifying reagents in catalysis, were similar to those of the dimeric aspartate racemase from Streptococcus thermophilus. The monomeric enzyme, however, showed a novel characteristic, namely, that its thermal stability significantly increased in the presence of aspartate, especially the D-enantiomer. The gene encoding the monomeric aspartate racemase was cloned and overexpressed in Escherichia coli cells. The nucleotide sequence of the aspartate racemase gene encoded a peptide containing 241 amino acids with a calculated molecular mass of 26 784 Da. The recombinant enzyme was purified to homogeneity and its properties were almost the same as those of the B. bifidum enzyme.

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Yamashita, T., Ashiuchi, M., Ohnishi, K., Kato, S., Nagata, S., & Misono, H. (2004). Molecular identification of monomeric aspartate racemase from Bifidobacterium bifidum. European Journal of Biochemistry, 271(23–24), 4798–4803. https://doi.org/10.1111/j.1432-1033.2004.04445.x

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