Characterization of a Glutamate Decarboxylase (GAD) from Enterococcus avium M5 Isolated from Jeotgal, a Korean Fermented Seafood

Abstract

To develop starters for the production of functional foods or materials, lactic acid bacteria producing gamma-aminobutyric acid (GABA) were screened from jeotgals, Korean fermented seafoods. One isolate producing a high amount of GABA from monosodium L-glutamate (MSG) was identified as Enterococcus avium by 16S rRNA gene sequencing. E. avium M5 produced 18.47 +/- 1.26 mg/ml GABA when incubated for 48 h at 37 degrees C in MRS broth with MSG (3% (w/v)). A gadB gene encoding glutamate decarboxylase (GAD) was cloned and overexpressed in E. coli BL21 (DE3) using the pET26b (+) expression vector. Recombinant GAD was purified through a Ni-NTA column and the size was estimated to be 53 kDa by SDS-PAGE. Maximum GAD activity was observed at pH 4.5 and 55 degrees C and the activity was dependent on pyridoxal 5'-phosphate. The K-m and V-max values of GAD were 3.26 +/- 0.21 mM and 0.0120 +/- 0.0001 mM/min, respectively, when MSG was used as a substrate. Enterococcus avium M5 secretes a lot of GABA when grown on MRS with MSG, and the strain is useful for the production of fermented foods containing a high amount of GABA.