Lactococcus lactis contains only one glutamate decarboxylase gene.
Glutamate decarboxylase, which is associated with a glutamate-dependent acid-resistance mechanism, was purified from Lactococcus lactis subsp. lactis by a three-step procedure. The specific activity was increased about 114-fold with a yield of 16%. The N-terminal amino acid sequence of the enzyme was determined. The gene encoding this enzyme was cloned in Escherichia coli, and its nucleotide sequence was determined. The deduced amino acid sequence suggests that the enzyme is produced as a mature form (466 amino acid residues), not as a precursor protein. The subunit molecular mass of L. lactis glutamate decarboxylase was calculated to be 53 926 Da. The enzyme was maximally active at pH 4.7 and reacted only with L-glutamate among 20 alpha-amino acids. The apparent Km value was calculated to be 0.51 mM. The activity was stable at acidic pH values; there was no activity in the neutral pH range. At pH 4.1 the enzyme activity was retained at temperatures up to 70 degrees C in 10 min incubations. L. lactis glutamate decarboxylase behaved as a single protein when the enzyme was purified. A single band corresponding to the glutamate decarboxylase gene was detected on Southern blot analysis. These data suggest that there is one glutamate decarboxylase gene in L. lactis.
Nomura
Masaru
M
Department of Animal Products, National Institute of Animal Industry, Norindanchi, Tsukuba, Ibaraki 305--0901, Japan.
Nakajima
Ikuyo
I
Department of Animal Products, National Institute of Animal Industry, Norindanchi, Tsukuba, Ibaraki 305--0901, Japan.
Fujita
Yasuhita
Y
Department of Animal Products, National Institute of Animal Industry, Norindanchi, Tsukuba, Ibaraki 305--0901, Japan.
Kobayashi
Miho
M
Department of Animal Products, National Institute of Animal Industry, Norindanchi, Tsukuba, Ibaraki 305--0901, Japan.
Kimoto
Hiromi
H
Department of Animal Products, National Institute of Animal Industry, Norindanchi, Tsukuba, Ibaraki 305--0901, Japan.
Suzuki
Ichirou
I
Department of Animal Products, National Institute of Animal Industry, Norindanchi, Tsukuba, Ibaraki 305--0901, Japan.
Aso
Hisashi
H
Department of Animal Physiology, National Institute of Animal Industry, Norindanchi, Tsukuba, Ibaraki 305--0901, Japan.
D000595:Amino Acid Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D005798:Genes, Bacterial; D005968:Glutamate Decarboxylase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D005968:Glutamate Decarboxylase