Cloning and Expression of the Glutamate Racemase Gene of Bacillus pumilus

• Published in: Journal of biochemistry (Tokyo) Vol. 121; no. 6; pp. 1155 - 1161
• Main Authors: Liu, Lidong, Yoshimura, Tohru, Endo, Keiji, Esaki, Nobuyoshi, Soda, Kenji
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.06.1997
• Subjects: Amino Acid Isomerases - genetics; Amino Acid Sequence; Bacillus - genetics; Bacillus pumilus; Bacillus subtilis; Base Sequence; Cloning, Molecular; Codon; Escherichia coli; Gene Expression Regulation, Bacterial - physiology; Gene Expression Regulation, Enzymologic - physiology; glutamate racemase; Molecular Sequence Data; murl; Mutation; Peptide Chain Initiation, Translational - genetics; purification; Recombinant Fusion Proteins - biosynthesis; sequencing
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a021709

A glutamate racemase gene (murI) was found in Bacillus pumilus cells and cloned into Escherichia coli WM335, a D-glutamate auxotroph, by means of a genetic complement method. Murl of B. pumilus encodes a 272-amino acid protein with an unusual initiation codon, TTG. The deduced amino acid sequence shows significant similarity with those of glutamate racemases from E. coli (ratio of identical residues, 28%), Pediococcus pentosaceus (44%), and Staphylococcus haemolyticus (49%). B. pumilus Murl was expressed as a fusion protein connected to the N-terminal 12 residues of β-galactosidase; the fusion protein showed glutamate racemase activity, and resembled the enzyme of P. pentosaceus in physicochemical and enzymological properties.