Cloning, purification, and properties of a cofactor-independent glutamate racemase from Lactobacillus brevis ATCC 8287.
A glutamate racemase gene of Lactobacillus brevis ATCC 8287 was cloned into Escherichia coli TM93 by the phenotypic complementation of a phosphoenolpyruvate carboxylase deficiency on minimum agar medium containing D-glutamate. The gene was localized to a 1.4-kb HindIII-EcoRI DNA fragment and the total nucleotide sequence of the fragment was analyzed. The gene has typical promoter and SD sequences which appeared to function in E. coli. The deduced amino acid sequence of the enzyme had 276 amino acids and the molecular weight was calculated as 29,426. Two cysteine residues and their surrounding regions of the enzyme are homologous to those of other cofactor-independent racemases. The glutamate racemase was purified from recombinant E. coli to homogeneity and characterized. The enzyme required no cofactors for the activity, and retained its activity even in 2 M (300 g/l) L-glutamate.
Yagasaki
M
M
Technical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Yamaguchi, Japan.
Iwata
K
K
Ishino
S
S
Azuma
M
M
Ozaki
A
A
D000591:Amino Acid Isomerases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism*; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007778:Lactobacillus / Q000201:enzymology*; D008969:Molecular Sequence Data
D003067:Coenzymes; D004269:DNA, Bacterial; D000591:Amino Acid Isomerases; C062292:glutamate racemase