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℃ 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℃ and the activity was dependent on pyridoxal 5'-phosphate. The K_m and V_max of GAD were 0.045 mM and 0.011 mM/min, respectively, when glutamate was used as the substrate.

Introduction

γ-Aminobutyric acid (GABA) is a non-protein amino acid distributed widely in microorganisms, plants, and animals [19]. Several GABA-containing functional foods and pharmaceuticals have been developed, since GABA possesses some physiological functions such as hypotensive, anti-anxiety, tranquilizing, analgesic, and diuretic effects [14,21]. GABA also has an improving effect for visual function of old animals [9]. GABA is produced from L-glutamate by the action of glutamate decarboxylase (GAD, E.C. 4.1.1.15), a product of gadB. GAD activity is widely distributed among lactic acid bacteria (LAB), since GAD production is a stress response when cells are exposed to acidic environments [11]. GABA-producing ability varies widely among the species of LAB, and Lactobacillus brevis strains are so far the most outstanding GABA producers [20]. GABA production has been reported for Lactobacillus paracasei [8], Lactobacillus plantarum [13], and Lactobacillus zymae [12]. GAD from Lb. brevis IFO 12005 was purified and its properties were examined [20]. GAD from Lactococcus lactis ssp. lactis 01-7 was purified. Its molecular mass was determined to be 54 kDa and the optimum pH was 4.7 [11]. In this study, a new GABA-producing Lactobacillus sakei was isolated from jeot-gal, a Korean fermented seafood. The gadB gene from Lb. sakei A156 was cloned and overexpressed in Escherichia coli BL21(DE3). Recombinant GAD was purified and the properties of GAD were reported.

 

Materials and Methods

Isolation of GABA-Producing LAB from jeot-gals

Various jeot-gal products were purchased at local markets in Jinju area (Gyeongnam, South Korea) during 2013. jeot-gals were homogenized with 0.1% sterile peptone water by using a Stomacher 80 (Seward, Worthing, UK). After being serially diluted with 0.1% sterile peptone water, 100 µl aliquots were spread on MRS agar plates with 1% CaCO3 and 0.006% bromocresol purple. The plates were incubated for 48 h at 30℃ and yellow colonies with clear zones were picked up [6]. Each putative LAB strain was cultured in MRS broth overnight and stored at –70℃. GABA-producing strains were screened by thin-layer chromatography (TLC) as shown below. One milliliter of MRS broth with monosodium glutamate (MSG, 3% (w/v)) was inoculated with five isolates (each isolate 10 µl) and incubated for 48 h at 30℃. The culture was centrifuged (12,000 ×g, 4℃ for 5 min) and 1 µl of culture supernatant was spotted onto an activated silica gel plate (Silica gel 60 F254; Merck Co., Darmstadt, Germany). After separated in n-butanol:acetic acid:water (4:1:1 (v/v/v)), the plate was treated with 2% ninhydrin solution and developed at 70℃ for 10 min. For samples showing GABA production, each isolate was individually examined for the GABA production ability by the same way.

Identification of GABA-Producing LAB

GABA-producing isolates were identified by using an API 50 CHL kit (bioMerieux, Lyon, France) and 16S rRNA gene sequencing [15]. 16S rRNA gene was amplified by PCR from genomic DNA as the template and using the universal primer pairs 27F (5’-AGAGTTTGATCMTGGCT CAG-3’) and 1492R (5’-TACGGYTACCTTGTTACGACTT-3’). Amplification reaction was performed in a total volume of 50 µl consisting of 5 µl of DNA (100 ng), 0.5 µl (1 U) of Taq DNA polymerase (Sigma, St. Louis, MO, USA), 5 µl of 10× buffer, 5 µl of dNTP mixture (2.5 mM each), 10 pmol of each primer, and 34 µl of nuclease-free water. The amplification condition was as follows: one cycle of 94℃ for 5 min, followed by 30 cycles of 94℃ for 30 sec, 58℃ for 30 sec, 72℃ for 2 min, and a final extension at 72℃ for 4 min. The PCR product was sequenced at Cosmogenetech (Seoul, Korea) and homology search was done using the Basic Local Alignment Search Tool (BLAST) and the GenBank data library (http://blast.ncbi.nlm.nih.gov).

Growth Characteristics of Lb. sakei A156

Lb. sakei A156 was grown in MRS broth for 18 h at 30℃ and the culture was used to inoculate 100 ml of MRS broth (1% (v/v)). Inoculated culture was incubated under different conditions and growth was monitored by measuring the OD600 value at time intervals.

Lb. sakei A156 was grown at different temperatures (4℃, 10℃, 25℃, 30℃, 37℃, and 45℃) for 60 h and on MRS broth with different initial pH values (pH 4-10) at 37℃. Growth of Lb. sakei A156 on MRS broth with NaCl (1%, 3%, 5%, 7%, 10%, 12%, and 15% (w/v) was also examined during 60 h incubation at 30℃ (pH 7).

Cloning of gadB and gadC Genes

The gadB gene of Lb. sakei A156 was amplified by using a primer pair: primers F (5‘-GGGCATATGAATAAAAACGATCAGG–3’, NdeI site underlined) and R (5’-GGGCTCGAGACTTCGAACGGTGGT-3’ XhoI site underlined). The reaction volume of 50 µl consisted of 5 µl of 10× buffer, 5 µl of dNTP (10 mM), 1 µl of each primer (10 mM), 36.5 µl of sterile water, 0.5 µl (1 U) of Taq polymerase (Sigma), and 1 µl of template DNA. The PCR procedure consisted of an initial denaturation of 94℃ for 5 min, followed by 30 cycles of 94℃ for 30 sec, 60℃ for 30 sec, 72℃ for 2 min, and the final extension step at 72℃ for 10 min. The PCR products were electrophoresed on a 1% agarose gel and stained with ethidium bromide. The PCR product with the expected size was eluted from the gel and cloned into the pET-26b (+) vector after being digested with NdeI and XhoI restriction enzymes. The ligation mixture was introduced into E. coli BL21 (DE3). The gadC gene was amplified from the genomic DNA by using a primer set based on gadC from Lb. brevis ATCC 367 (CP000416): gadCF (5’-TCGGCCGAATAATGAGTTCCC-3’) and gadCR (5’-AACGGAGCCTGTGTACGTAA-3’).

RT-PCR Experiment

RNA was isolated from Lb. sakei A156 by the Trizol/bead method [12]. RT-PCR was done after DNase (RQ1 RNase-free DNase; Promega, Madison, WI, USA) treatment. M-MLV Reverse Transcriptase (Enzynomics, Daejeon, Korea) was used and the reaction mixture consisted of 2 µl of 10× buffer, 2 µl of dNTP mixture (2 mM each), 0.5 µl of forward primer, 0.5 µl of reverse primer, 0.5 µl of reverse transcriptase, 2 µl of RNA (1 µg), 0.5 µl of RNase inhibitor, and 12 µl of water. The reaction was started by 30 min incubation at 37℃, followed by initial PCR activation for 15 min at 95℃. PCR cycles consisted of denaturation at 95℃ for 0.5 min, annealing at 58℃ for 0.5 min, and extension at 72℃ for 1min. A total of 29 cycles were repeated, and the final extension was done at 72℃ for 10 min.

Overexpression of gadB in E. coli BL21 (DE3) and Purification of GAD

E. coli BL21 (DE3) cells harboring pETA156 were grown in LB broth (50 ml) containing kanamycin (60 µg/ml) at 37℃ until the OD600 value reached 0.6. Then isopropyl β-D-1-thiogalactopyranoside (IPTG, 1 mM) was added and growth continued for 8 h at 20℃. Cells were harvested by centrifugation at 12,000 ×g for 10 min at 4℃, washed 3 times with phosphate-buffered saline (PBS, pH 7.4), and resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole, pH 7.0). Cells were disrupted by using a sonicator (Bandelin Sonopuls HD2070, Berlin, Germany) and centrifuged at 12,000 ×g for 15 min at 4℃. The resulting supernatant was used as the source of enzyme and loaded onto a Ni–NTA column (GE Healthcare, Stockholm, Sweden). Bound recombinant GAD was eluted by imidazole (40–500 mM). Protein concentration was determined by using a Bio-Rad protein assay kit [1]. GAD activity was determined by using the GABase method as described previously [12]. SDS-PAGE was done using a 12% (w/v) acrylamide gel [17].

Enzyme Assay of Recombinant GAD

TLC was used as the rapid method for the screening of GABA-producing LAB [5]. Enzyme solution (4 µg GAD in 0.1ml of lysis buffer) was mixed with 0.1 ml of 4 M ammonium sulfate. After 30 min of pre-incubation at room temperature, the enzyme solution was mixed with 1.3 ml of substrate (20 mM MSG, 0.2 mM pyridoxal 5’-phosphate (PLP), 0.2 M pyridine-HCl, pH 4.5) and incubated at 37℃. At time intervals (1, 3, 5, and 24 h), the reaction was terminated by immersing the sample tube in boiling water for 5 min. The GABA produced was visualized by TLC. The GABase assay was done for the quantitative analysis of GABA [12]. One milliliter of assay mixture consisted of 50 µl of sample, 700 µl of 0.5 M potassium pyrophosphate buffer (pH 8.6), 150 µl of 4 mM NADP+, 50 µl of GABase (1unit/ml; Sigma), and 50 µl of 20 mM α-ketoglutarate. The initial absorbance was read at 340 nm before adding α-ketoglutarate, and the final absorbance was read after 60 min at 25℃. The difference in OD340 values was used to calculate the GABA content in the sample. The equation of the GABA standard curve was OD340 = 0.0907X + 0.0551 (R2= 0.9929), where X = concentration of GABA in mM. One unit of GAD activity (U) is defined as the amount of enzyme producing 1 µmol GABA per minute.

Properties of Purified GAD

Purified GAD (4 µg) was incubated in broth at different pH (3 to 10) at 37℃ for 4 h and then the remaining GAD activity was measured by GABase assay. Purified GAD (4 µg) was incubated at temperatures ranging from 10℃ to 90℃ for 4 h (pH 5) and the remaining GAD activity was measured by GABase assay. The effect of PLP concentration (0–1.8 mM) and chemicals (2 mM) on the GAD activity were also determined by incubating GAD (4 µg) at 55℃ (pH 5) for 4 h as described above.

 

Results and Discussion

Isolation and Identification of GABA-Producing LAB from jeot-gals

LAB were isolated from various jeot-gals including myeolchi-jeot (anchovy), agami-jeot (gills), and saeu-jeot (shrimp). A total of 3,600 putative LAB were isolated by using MRS agar plates with CaCO3 and bromocresol purple. GABA production by isolates was examined by TLC and several isolates producing GABA were obtained (Fig. 1). The intensities of GABA spots were variable depending upon each isolate. Lb. zymae GU240 was included as the positive control (Fig. 1, lanes 3 and 9). Lb. zymae GU240 was previously isolated from kimchi and is one of the most efficient strains converting MSG into GABA [12]. The TLC result showed that A156 produced a large amount of GABA as comparable to Lb. zymae GU240 (Fig. 1A lane 5, 1B lane 8). A156 was isolated from myeolchi-jeot (anchovy). A156 was a non-spore-forming, grampositive, and short rod-type organism. The API kit test result indicated that A156 was Lb. brevis. To confirm the result, the 16S rRNA gene was amplified from the genomic DNA of A156 and the nucleotide sequence was determined. BLAST search for the determined 1,428 nucleotide sequence showed that the sequence was 99% identical with those of Lb. sakei LHL1-3, Lb. sakei PS49, and Lb. sakei TUB/2013/2-3 (data not shown). From the results, A156 was identified as Lb. sakei and named Lb. sakei A156. The 16S rRNA gene sequence of A156 was deposited into GenBank under the accession number KM982735.

Fig. 1.Thin-layer chromatogram showing GABA production by some LAB. Each isolate was incubated in MRS broth with 3% MSG for 48 h at 30℃. The culture was centrifuged (12,000 ×g, 4℃ for 5 min) and 1 µl of culture supernatant was analyzed by TLC as described in the text. (A) 1, 1 µl of 100 µM MSG; 2, 1 µl of 100 µM GABA; 3, Lb. zymae GU240 (+ control); 4, E104; 5, A156; 6, A157. (B) 1, 1 µl of 100 µM MSG; 2, 1 µl of 100 µM GABA; 3, negative control (MRS broth with 3% MSG); 4, C36; 5, C37; 6, C38; 7, C74; 8, A156; 9, Lb. zymae GU240; 10, Leu. citreum (negative control).

Lb. sakei A156 produced 15.81 ± 0.98 mg GABA per ml culture supernatant as determined by GABase assay. The value was much higher than that of Leu. mesenteroides ATCC10830 (0.05 ± 0.01 mg GABA), a negative control. Meanwhile, Lb. zymae GU240 produced 16.94 ± 1.14 mg GABA per ml culture supernatant. The conversion yield of L-glutamate into GABA was 86.5% for Lb. sakei A156 and 92.5% for Lb. zymae GU240. Lb. sakei A156 produced a significant amount of GABA from MSG and thus can be used as a starter for various fermented foods where GABA is one of the desirable components.

Growth Characteristics of Lb. sakei A156

Lb. sakei A156 was grown in MRS broth up to 192 h at different temperatures (Fig. 2A). Lb. sakei A156 grew rapidly at 25-45℃ and the OD600 values reached 1.5-1.6 at 24 h. At 20℃, growth was delayed until 12 h and then increased gradually, reaching OD600 values of 1.5-1.6 at 96 h. Lb. sakei A156 grew very slowly at 10℃ and did not grow at 4℃ in 192 h. The effect of initial pH (4.0–10.0) of MRS broth on the growth of Lb. sakei A156 was examined for 60 h incubation at 30℃. Lb. sakei A156 grew rapidly at the initial pH of 5.0-7.0, reaching the OD600 value of 1.6 in 18 h (Fig. 2B). When the initial pH was 4.0 and 8.0, Lb. sakei A156 grew slowly and reached the OD600 value of 1.5 at 60 h. Lb. sakei A156 grew slowly at pH 9.0, reaching the OD600 value of 1.2 at 60 h but did not grow at pH 10.0. Lb. sakei A156 grew well at 3% (w/v) NaCl, reaching the OD600 values of 1.3-1.5 in 18 h (Fig. 2C). Lb. sakei A156 grew slowly at 7% NaCl but did not grow at 10% NaCl. Thus, Lb. sakei A156 can be used as a starter for low-salt jeot-gals or other fermented foods where the NaCl concentration is below 10%.

Fig. 2.Growth of Lb. sakei A156 on MRS broth under different conditions. The absorbance of each culture was measured at 600 nm and each value represents the mean value from three independent measurements. (A) Temperature: ● , 4℃; ○ , 10℃; ▼ , 20℃; △ , 25℃; ■ , 30℃; □ , 37℃; ◆ , 45℃. (B) pH: ● , pH 4; ○ , pH 5; ▼ , pH 6; △ , pH 7; ■ , pH 8; □ , pH 9; ◆ , pH 10. (C) NaCl concentration: ● , 0%; ○ , 1%; ▼ , 3%; △ , 5%; ■ , 7%; □ , 10%; ◆ , 12%, ◇ , 15%.

Cloning of gadB and gadC Genes

The gadB gene of Lb. sakei A156 was amplified by PCR from the genomic DNA of A156 as the template and using the primers, which were designed from the nucleotide sequence of the gadB gene from Lb. zymae GU240 [12]. A 1.57 kb fragment was amplified and the nucleotide sequence was determined. The gadB sequence was deposited into GenBank under the accession number KM982734. An ORF of 1,440 nucleotides was located, and the amino acids translated from the nucleotide sequence showed high homologies to GADs in the database. gadB could encode a protein of 479 amino acids with the calculated size of 53.54 kDa and isoelectric point (pI) of 4.91. The GAD from Lb. sakei A156 contained a highly conserved catalytic domain that belongs to the PLP-dependent decarboxylase superfamily [12]. The domain includes a lysine residue essential for PLP binding, designated as PLP lysine [14]. The conserved lysine residue (K288) was located in GAD from Lb. sakei A156 with the adjacent histidine residue (data not shown). The active site residues (T224 and D255), known to promote decarboxylation, were also present [12]. Moreover, sequence (HVDAAFGG) conserved among PLP-dependent decarboxylases was found (data not shown).

The amino acid sequence of GAD from Lb. sakei A156 was aligned with those of GADs from other LAB (Fig. 3). GAD from Lb. sakei A156 showed 99% identity with the GAD from Lb. zymae GU240 (AHF72525), Lb. brevis ATCC367 (ABJ63253), and Lb. brevis BH2 (AIC75915), differing only at one amino acid. The GAD from Lb. sakei A156 differed from that of Lb. brevis CGMCC1306 (AEY81112.1) at two amino acids and that of Lb. brevis IFO12005 (BAF99137.2) at three amino acids.

Fig. 3.Alignment of the amino acid sequences of selected GADs. The amino acid sequence of GAD from Lb. sakei A156 was aligned with those of other GADs; Lb. sakei A156, Lb. zymae GU240, Lb. brevis ATCC 367, Lb. brevis BH2, Lb. brevis CGMCC 1306, and Lb. brevis IFO12005.

gadC was amplified from the genome of Lb. sakei A156 and the nucleotide sequence was determined (KP310071). An ORF of 1,506 nucleotides was located, which could encode a protein of 501 amino acids with the calculated molecular mass of 55.11 kDa and isoelectric point (pI) of 9. It was found that gadC was located in the immediate upstream of gadB and both genes were in the same orientation. The intervening space between the stop codon of gadC and the start codon of gadB was only 55 nucleotides in length (data not shown). These strongly indicated the operon structure of gadCB in Lb. sakei A156 (Fig. 4A). To confirm the operon structure, RT-PCR experiments were done for RNA samples prepared from Lb. sakei A156. A transcript covering both gadC and gadB was detected (Fig. 4B), supporting the gadCB operon structure in Lb. sakei A156. The same operon structure was reported for Lb. brevis IFO12005 [4], Lactococcus lactis subsp. lactis 01-7 [11], and Lb. zymae GU240 [12]. However, except for Lb. zymae, the operon structure was not proven experimentally.

Fig. 4.The operon structure of gadB and gadC. (A) Operon structure of gadB and gadC genes in Lb. sakei A156. The arrows indicate the binding sites for primers used for RT-PCR. (B) Agarose gel electrophoresis result of RT-PCR. Lane M, GeneRuler 1 kb DNA ladder (Invitrogen, Carlsbad, CA, USA); 1-4, RNA preps; 5-8, RNA preps treated with DNase I. Lanes 1 and 5, RT-PCR using universal primers (27F and 1492R) for 16S rRNA; 2 and 6, RT-PCR using primers A and B; 3 and 7, RT-PCR using primers C and D; 4 and 8, RT-PCR using primers A and D.

Overproduction and Purification of Recombinant GAD

The gadB gene (1.44 kb) was cloned into pET26b(+) at the NdeI and XhoI restriction sites, resulting in pETA156 (6.7 kb). pETA156 was introduced into E. coli BL21(DE3) by electroporation. Expression of gadB was initiated by the addition of IPTG (1 mM). The cell extract was prepared from induced E. coli cells and analyzed by SDS-PAGE. The result confirmed that GAD was overproduced in E. coli (Fig. 5A). A band was observed from the insoluble and soluble fractions and the size matched well with the size of GAD calculated from the sequence. In E. coli, GAD was produced as a His-tagged fusion protein to facilitate its purification. The size of recombinant GAD was calculated to be 54.4 kDa when six histidines at the C-terminus were included. After sonication and centrifugation, the soluble fraction was obtained and loaded onto a Ni–NTA column. GAD was eluted by imidazole at 300 mM concentration (Fig. 5B). The size of eluted GAD matched well with the expected size of recombinant GAD (54.4 kDa).

Fig. 5.SDS-PAGE of recombinant GAD. (A) M, prestained SDS-PAGE standards (DokDo MARK; Elpisbiotech, Korea); 1, insoluble fraction; 2, soluble fraction; 3, purified GAD eluted from a HiTrap affinity column at 300 mM imidazole concentration. (B) M, prestained SDS-PAGE standards (Dokdo marker). Recombinant GAD eluted at 40 mM imidazole (1), 100 mM imidazole (2), 300 mM imidazole (3), and 500 mM imidazole (4) concentration.

Properties of Recombinant GAD

The effects of temperature, pH, and PLP concentration on the GAD activity were examined and the results are shown in Fig. 6. The optimum temperature and pH for the GAD activity were 55℃ (Fig. 6A) and pH 5 (Fig. 6B), respectively. The optimum pH is similar to GADs from other LAB [20]. Most GADs from LAB are active at pH 4–5.5 as reported for Lb. brevis 877G [16], Lb. brevis IFO12005 [20], Lb. brevis CGMCC 1306 [2], Lb. paracasei NFRI 7415 [7], and Lactococcus lactis [11] (Table 1). GAD is involved in the maintainence of the cellular pH near neutral values under the acidic environments and its role is especially important for LAB [12]. The GAD of Lb. sakei A156 depended on PLP for its activity (Fig. 6C) and the maximum activity was observed at 0.1 mM PLP. No significant increase was observed above 0.1 mM. The activity of GAD was increased by MnCl2 (152%), CoCl2 (118%), CaCl2 (113%), and ZnCl2 (115%), whereas it was decreased by (NH4)2SO4 (92%), MgCl2 (84%), and AgNO3 (79%) (Fig. 6D). The Km of GAD was 16.0 ± 0.05 mM and the Vmax was 0.011 ± 0.0001 mM/min when glutamate was used as the substrate.

Fig. 6.GAD activity changes under different conditions. (A) Temperature, (B) pH, (C) pyridoxal 5’-phosphate (PLP) concentration, and (D) chemicals.

Table 1.Characteristics of some selected glutamate decarboxylases.

A GABA-producing Lb. sakei A156 was isolated from jeot-gal and its gadB gene was cloned and overexpressed in E. coli. Lb. sakei A156 and its gadB gene might be useful when GABA-enriched foods are attempted to be produced, serving as a new source of strain and gene.