Cloning and Expression inEscherichia coliof the Glutamate Racemase Gene fromPediococcus pentosaceus

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Abstract
The glutamate racemase (EC 5.1.1.3) gene of a lactic acid bacterium, Pediococcus pentosaceus, was cloned into Escherichia coli C600 with a vector plasmid, pBR322. The requirement of l-glutamate for the growth of E. coli in the minimum medium containing d-glutamate and the formation of a red pigment in a coupled enzyme reaction mixture were used to select clones expressing glutamate racemase activity. Glutamate racemase overproduced as 0.3— 2.0% of the total soluble proteins in a clone carrying the plasmid pICR221, 10.3 kb of DNA, was purified from cell extracts about 130-fold to homogeneity. The purified enzyme has a molecular weight of about 40,000 and is a single polypeptide chain. Glutamate is the sole substrate for the enzyme. Unlike many other amino acid racemases, glutamate racemase is devoid of cofactors: there is no evidence for pyridoxal 5’-phosphate or FAD in the ultraviolet spectrum of the purified enzyme, and the enzyme is not inactivated by carbonyl reagents such as hydroxylamine and sodium borohydride.