Characterization of Glutamate Decarboxylase (GAD) from Lactobacillus sakei A156 Isolated from Jeot-gal

Abstract

A gamma-aminobutyric acid (GABA)-producing microorganism was isolated from jeot-gal (anchovy), a Korean fermented seafood. The isolate, A156, produced GABA profusely when incubated in MRS broth with monosodium glutamate (3% (w/v)) at 37°C for 48 h. A156 was identified as Lactobacillus sakei by 16S rRNA gene sequencing. The GABA conversion yield was 86% as determined by GABase enzyme assay. The gadB gene encoding glutamate decarboxylase (GAD) was cloned by PCR. gadC encoding a glutamate/GABA antiporter was located immediately upstream of gadB. The operon structure of gadCB was confirmed by RT-PCR. gadB was overexpressed in Escherichia coli BL21(DE3) and recombinant GAD was purified. The purified GAD was 54.4 kDa in size by SDS-PAGE. Maximum GAD activity was observed at pH 5.0 and 55°C and the activity was dependent on pyridoxal 5'-phosphate. The Km and Vmax of GAD were 0.045 mM and 0.011 mM/min, respectively, when glutamate was used as the substrate.

Screening of gamma-aminobutyric acid-producing lactic acid bacteria and its application in Monascus-fermented rice production

“…The enhancement in bioconversion ability appeared to be closely related to the increase in GAD activity. Previous studies showed that the activity of GAD could be increased by MnCl 2 (152%), CoCl 2 (118%), and ZnCl 2 (115%) (Sa et al, 2015), which was consistent with increased R GLu detected in this study (114%, 191%, and 416%, respectively). Besides, a positive effect of MgSO 4 (125%) was also observed, as described by Ueno et al (1997).…”

A Brief Review on Glutamate Decarboxylase From Lactic Acid Bacteria

“…Different ions can affect the stability and activity of GAD from different sources ( Table 2). GAD from E. avium M5 is activated in the presence of CaCl2 and MnCl2 but the activity is decreased by CuSO4 and AgNO3 [82]; comparable results were also obtained for GAD from other LAB sources, L. zymae [46] and L. sakei A156 [80].…”

“…As mentioned above, a number of LAB carry two GAD-encoding genes, gadA and gadB. Frequently, studies have focused on the purification and characterization of GadB, e.g., from E. raffinosus [75], L. plantarum [79], L. brevis [78], L. sakei [80], L. paracasei [18] since the expression levels of recombinant GadB are typically higher than those for GadA [55]. A recent study by Wu et al showed that gadA transcript was highly upregulated (55-fold) in strain L. brevis NPS-QW-145 at the stationary phase of growth.…”

Exploring the contributions of two glutamate decarboxylase isozymes in Lactobacillus brevis to acid resistance and γ-aminobutyric acid production

“…cremoris MG1363 and L. buchneri NRRL B-30929. However, there is no evidence that the gadR gene exists in other LAB species, except for strain L. sakei A156, the genome sequence of which has not yet been published [ 36 ]. In line with these differences, gadC and gadB of L. brevis and those genes of L. sakei share high sequence identity but are more distantly related to these genes in L. lactis , L. reuteri and L. plantarum .…”