Cloning and Expression inEscherichia coliof the Glutamate Racemase Gene fromPediococcus pentosaceus

Distribution of Glutamate Racemase in Lactic Acid Bacteria and Further Characterization of the Enzyme fromPediococcus pentosaceus

“…-5) Although intensive studies have been done on the reaction mechanisms of various pyridoxal 5' -phosphate-dependent racemases,6) those of the cofactor-independent enzymes are not well understood, has been reported to occur only in a very few bacterial species such as Lactobacillus fermenti, 9) The cofactor requirement for the enzyme activity has also been ambiguous.lO,ll) Recently, we cloned the glutamate racemase gene from a lactic acid bacterium, Pediococcus pentosaceus, into Escherichia coli, and established an efficient purification procedure for the enzyme on the basis of the high expression of the gene in E. coli. 12 ) We obtained no evidence for the presence of pyridoxal 5'-phosphate or FAD in the purified enzyme.…”

“…Thus, it seems to be of particular interest in our future studies to identify the essential cysteinyl residue in the entire primary sequence of glutamate racemase, the identification of which is now under way based on the DNA sequencing of the cloned gene. 12 ) …”

Enzymatic Racemisation and its Application to Synthetic Biotransformations

“…Among the PLP-independent amino acid racemases, only glutamate racemase (EC 5.1.1.14), [41] and proline racemase (EC 5.1.1.4) [42] are discussed in more detail. For other racemases of this group, such as aspartate racemase (EC 5.1.1.13) [43,44] and 4-hydroxyproline racemase (EC 5.1.1.8, more exactly an epimerase acting on diastereomers rather than enantiomers), only limited data are available.…”

Isomerazation

“…Aspartate racemase requires no cofactors and contains an essential cysteine residue in the same manner as glutamate racemase [80]. When L-or maspartate was incubated with aspartate racemase in tritiated water, tritium was incorporated preferentially into the product enantiomer.…”