Cloning and Expression in Escherichia coli of the Glutamate Racemase Gene from Pediococcus pentosaceus

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.