Characterization of a glutamate decarboxylase (GAD) gene from Lactobacillus zymae.
Lactic acid bacteria (LAB) were isolated from Kimchi, a Korean traditional fermented vegetable food. LAB accumulating GABA (γ-aminobutyric acid) in the culture media were screened by TLC analysis. One isolate, GU240, produced the highest amount of GABA among the 3,000 isolates and identified as a Lactobacillus zymae strain. Glutamate decarboxylase (GAD) gene was cloned and over-expressed in E. coli BL21(DE3) using pET26b(+). The recombinant GAD was purified by using a Ni-NTA column. Its size was 53 kDa by SDS-PAGE. Maximum GAD activity was at pH 4.5 and 41 °C and the activity was dependent on pyridoxal 5'-phosphate. Km and Vmax of LzGAD were 1.7 mM and 0.01 mM/min, respectively, when glutamate was used as a substrate.
Division of Applied Life Science (BK21 Plus), Graduate School, Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, 660-701, Republic of Korea.
Ji Yeong
JY
Park
Seon-Ju
SJ
Jeong
Jeong Hwan
JH
Kim
D000595:Amino Acid Sequence; D001483:Base Sequence; D002855:Chromatography, Thin Layer; D003067:Coenzymes / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005516:Food Microbiology; D015870:Gene Expression; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D003067:Coenzymes; D011994:Recombinant Proteins; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid; D011732:Pyridoxal Phosphate; D005968:Glutamate Decarboxylase