Microbial Production and Enzymatic Biosynthesis of γ-Aminobutyric Acid (GABA) Using Lactobacillus plantarum FNCC 260 Isolated from Indonesian Fermented Foods

Abstract

In the present study, we isolated and screened thirty strains of GABA (γ-aminobutyric acid)-producing lactic acid bacteria (LAB) from traditional Indonesian fermented foods. Two strains were able to convert monosodium glutamate (MSG) to GABA after 24 h of cultivation at 37 °C based on thin layer chromatography (TLC) screening. Proteomic identification and 16S rDNA sequencing using MALDI-TOF MS identified the strain as Lactobacillus plantarum designated as L. plantarum FNCC 260 and FNCC 343. The highest yield of GABA production obtained from the fermentation of L. plantarum FNCC 260 was 809.2 mg/L of culture medium after 60 h of cultivation. The supplementation of 0.6 mM pyridoxal 5’-phosphate (PLP) and 0.1 mM pyridoxine led to the increase in GABA production to 945.3 mg/L and 969.5 mg/L, respectively. The highest GABA production of 1226.5 mg/L of the culture medium was obtained with 100 mM initial concentration of MSG added in the cultivation medium. The open reading frame (ORF) of 1410 bp of the gadB gene from L. plantarum FNCC 260 encodes 469 amino acids with a calculated molecular mass of 53.57 kDa. The production of GABA via enzymatic conversion of monosodium glutamate (MSG) using purified recombinant glutamate decarboxylase (GAD) from L. plantarum FNCC 260 expressed in Escherichia coli was found to be more efficient (5-fold higher within 6 h) than the production obtained from fermentation. L. plantarum FNCC 260 could be of interest for the synthesis of GABA.

1. Introduction

γ-aminobutyric acid (GABA), which is a non-protein amino acid and plays a major role as a suppressive neurotransmitter, is widely present in plants, microorganisms, and the mammalian brain [1,2,3]. GABA has been extensively studied due to its physiological and pharmacological effects including anti-depressant, hypotensive activity, anti-diabetic in humans [4,5,6]. Recently, GABA administration in fluoride-exposed mice showed protective effects against hypothyroidism and maintained lipid and glucose levels in vivo [4]. Furthermore, GABA-enriched foods have been developed [5,6,7,8,9,10,11]. GABA-rich chlorella has been shown to significantly lower high blood pressure in hypertensive subjects [12]. A number of GABA-enriched foods such as soymilk [13], fermented milk [14], natto [15], green tea [16], and cheese [5] have been reported to suppress the elevation of blood pressure in spontaneously hypertensive rats (SHR) and hypertensive subjects.

GABA can be synthesized using chemical or biochemical means, of which the latter involves enzymatic conversion, whole-cell biocatalysts, or microbial fermentation. The chemical synthesis is considered hazardous due to corrosive nature of used reagents [17,18,19], hence the application in the food industry is limited. Moreover, the supplementation of synthetic GABA to food system is considered unnatural and unsafe [14]. Therefore, it is important to develop a natural and safe method to increase GABA in foods, since there are no side effects of natural GABA supplementation [16]. Recent studies showed that several strains of lactic acid bacteria (LAB) are promising candidates as GABA-producing bacteria [1,2,3,5] due to their GRAS (generally recognized as safe) status. A number of GABA-producing bacteria has been isolated from fermented foods such as L. brevis (from kimchi) [20], L. rhamnosus (from fermented pickles) [21], L. plantarum (from fermented dairy products) [22], L. helveticus (from koumis fermented milk) [23], L. buchneri (from kimchi) [24], L. otakiensis (from Pico cheese) [25], L. namurensis (from fermented green papaya) [11], and L. paracasei (from Italian cheese) [3]. These reports have shown that fermented foods are promising sources of GABA-producing bacteria. In addition, screening GABA-producing LAB from various fermented foods might open the possibilities to obtain newly isolated strains for the use as functional starter cultures in the food industry.

Several fermented foods from Indonesia namely gatot, growol, tape ubi, bekasam, and tempoyak are spontaneously fermented by LAB, which mainly involve the strains of the genera Lactobacillus, Pediococcus, and Streptococcus [26,27]. However, the potential of these LAB strains from Indonesian fermented foods to be used as GABA-producing bacteria as well as their relevant enzymes have not yet been studied. Therefore, the development of GABA-enriched foods using suitable LAB is a promising strategy, to bring new functional foods to the market. In addition, biosynthesis of GABA using LAB also provides advantageous effects including probiotic activity and extension of the shelf-life of food products [9].

The biosynthesis of GABA involves irreversible decarboxylation reaction of glutamate to GABA and carbon dioxide catalyzed by glutamate decarboxylase (GAD). GAD (EC 4.1.1.15) is a major enzyme for GABA synthesis and it requires pyridoxal 5’-phosphate (PLP) as a cofactor [28,29,30]. The GAD genes from various sources have been cloned, expressed and their biochemical properties have been characterized [20,31,32,33,34]. The use of purified GAD for the biosynthesis of GABA is also of interest because only simple downstream purification of GABA is required and yet, the process could overcome the limitation of microbial fermentation (i.e., GABA catabolism). In the present study, we describe the screening of GABA-producing LAB from Indonesian fermented foods (fermented soybeans, growol, gatot, tempeh, and bekasam) and GABA productions using microbial fermentation of the isolated strain and the purified GAD of this strain for the conversion of glutamate to GABA.

2. Materials and Methods

2.1. Screening of GABA-Producing LAB

Thirty isolates of Lactobacillus spp. were previously isolated from Indonesian fermented foods such as fermented soybeans, growol, gatot, tempeh, and bekasam (fermented fish) [35]. Lactobacillus spp. were the predominant genus according to cell morphology, Gram reactions and catalase tests. All strains were obtained and stored in the Food and Nutrition Culture Collection, Universitas Gadjah Mada (Yogyakarta, Indonesia). Prior to screening, all strains were grown in MRS broth containing 118 mM monosodium glutamate (MSG) (Ajinomoto, Tokyo, Japan) for 24–48 h at 37 °C under microaerophilic conditions. The cultures broth was then centrifuged at 8000× g for 5 min at 4 °C. GABA formation in the supernatant was analyzed using thin layer chromatography (TLC). Briefly, 0.5–1.0 µL of supernatants were spotted onto TLC plates Silica gel 60 F₂₅₄ (Merck, Darmstadt, Germany). The mobile phase consists of a mixture of 1-butanol: acetic acid: distilled water (5:2:2). Subsequently, the plates were sprayed with 0.5% ninhydrin and heated at 105 °C for 5 min to visualize the spots. GABA (Sigma Aldrich, St. Louis, MO, USA) was used as a standard, and the Rf values were calculated. LAB cultures showing the same Rf values as GABA standard were considered as positive GABA-producers. Positive GABA-producing strains were identified using proteomic and genotype techniques. Furthermore, the amount of GABA produced was determined by the GABase assay [36]. All chemicals were of the highest grade.

2.2. Identification of GABA-Producing LAB

Proteomic and genotype techniques were performed to identified GABA-producing LAB. Genomic DNA of GABA-producing LAB was extracted using peqGOLD Bacterial DNA Mini Kit (PeqLab, Erlangen, Germany) according to manufacturer’s instructions. The extracted DNA was used as a template for partial 16S rDNA amplification. The amplifications of 16S rDNA were performed using forward primer bak4 (5′-AGGAGGTCATCCARCCGCA-3′) and reverse primer bak11w (5′-AGTTTGATCMTGGCTCAG-3′) [37], The PCR reaction mixtures consisted of 10× PCR buffer (Dynazyme buffer 10× Thermo scientific, Waltham, MA, USA), 10 nmol/µL dNTP mix (GE Healthcare Buckinghamshire, UK), 2 U/µL DNA polymerase (Dynazime II, Thermo scientific) and high-quality sterile water to a total volume of 25 µL. The conditions for PCR amplification were as follows: initial denaturation at 95 °C for 3 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s, extension 72 °C for 2 min, and a final extension at 72 °C for 7 min. After PCR amplification, the amplified products were visualized by gel electrophoresis. The gel was stained with GelRed Nucleic Acid (Biotium, Hayward, CA, USA) and subsequently visualized with an ultraviolet transilluminator (BioRad, Hercules, CA, USA). The PCR products were purified using QIAquick PCR purification Kit (Qiagen, Venlo, The Netherlands) and sent for sequencing (Eurofins MWG Operon, Ebersberg, Germany). Subsequently, the partial 16S rDNA sequence was compared with the National Center for Biotechnology Information (NCBI) sequence database using Basic Local Alignment Search Tool (BLAST) program.

Proteomic identification was performed using matrix-assisted laser desorption/ionizing time-of-flight mass spectrometry (MALDI-TOF MS). GABA-producing bacteria were identified by the extended direct transfer method. A single colony was directly spread onto a MALDI target plate. The spot was overlayed with 1 μL of 70% formic acid and allowed to dry at room temperature. Furthermore, 1 μL of 10 mg/μL HCCA (α-cyano-4-hydroxycinnamic acid) solution was then added to the spot and allowed to dry at room temperature. The target plate was immediately applied to MALDI-TOF MS and analyzed using Microflex LT bench-top mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with the FlexControl 3.4 software. A mass spectrum was processed using BioTyper software (version 3.0, Bruker Daltonics, Bremen, Germany). MALDI-TOF MS profiles were obtained from bacteria isolates and matched with a database containing 8223 reference MALDI-TOF MS profiles.

2.3. Determination of GABA Production and GAD Assay

The GABAse method was performed to determine GABA concentration in culture supernatants. Briefly, the culture broth was centrifuged at 8000× g for 5 min at 4 °C. 10 µL of supernatants were mixed with 140 µL of 100 mM K₄P₂O₇ buffer (pH 8.6), 30 µL of 4 mM NADP⁺, 10 µL of 1 U/mL GABase (Sigma-Aldrich, St. Louis, MO, USA). The mixtures were dispensed into each well of 96-well plate. The initial absorbance was read at 340 nm in PerkinElmer plate reader (PerkinElmer, Buckinghamshire, UK). After the initial reading, 10 µL of 20 mM α-ketoglutarate were added and the mixtures were incubated for 1 h. The final absorbance was read after 1 h at the same wavelength. GABA concentrations were determined based on the difference of A₃₄₀ values and the standard curve of GABA.

The GAD assay was carried out using colorimetric method [38]. The reaction mixtures consist of 200 mM Na₂HPO₄-citric acid buffer (pH 5.0), 20 mM L-MSG, 0.2 mM PLP, and 20 µL of purified GAD. The mixtures were thoroughly mixed and incubated at 37 °C for 1 h and then deactivated by boiling for 5 min. The reaction mixtures were used to determined GAD activity using the Berthelot reaction method, which was composed of 100 µL of reaction sample, 250 µL of H₂O, 50 µL of 200 mM sodium borate (pH 9.0), 250 µL of 6% phenol and 200 µL of 5% (w/v) sodium hypochlorite. Subsequently, the reaction mixtures were thoroughly mixed and boiled for 10 min until the blue color developed, then immediately placed on ice for 15 min. The mixtures were analyzed colorimetric at 630 nm to determine the absorption value. One unit of GAD activity was defined as the amount of enzyme that liberates 1 µmol of GABA per minute under activity assay conditions.

The concentrations of GABA formed in supernatants and after enzymatic conversions were confirmed using Ultra Performance Liquid Chromatography (UPLC Acquity H- Class, Waters Corporation, Milford, MA, USA) equipped with a PDA detector and an AccQ. Tag Ultra C18 column (1.7 µm particle, 2.1 × 100 mm). The samples were hydrolyzed using 6 N HCL and followed by derivatization of the samples and the GABA standard using AccQ-Tag ultra-derivatization kit (Waters, Milford, MA, USA) according to the manufacturer’s instructions. For UPLC analysis, the derivatized samples were injected to Acquity UPLC H class [39]. The system was operated at a flow rate of 0.5 mL/min at 49 °C with a wavelength of 260 nm. The mobile phase used were AccQ. Tag Ultra Eluent A 100%; Accq. Tag Ultra Eluent B (Aquabides 90:10); Aquabides Eluent C; AccQ. Tag Ultra Eluent B 100%.

2.4. GABA Production

The MRS medium was inoculated with 5% inoculum of GABA-producing LAB and incubated at 37 °C for 108 h. The optical densities (OD₆₀₀) of the cultures were measured every 12 h. A concentration of MSG (25–100 mM) (Sigma Aldrich, St. Louis, MO, USA), pyridoxal 5-phosphate (PLP, 0.2 and 0.6 mM) and pyridoxine (vitamin B6, 0.1–0.3 mM) were added to the MRS medium and GABA production under these conditions was investigated subsequently.

2.5. Cloning of gadB Gene

The glutamate decarboxylase gene (gad) from L. plantarum FNCC260 was amplified using degenerated primers gad_FwdNdeI (5′-CATATGATGGCAATGTTRTAYGGTAAAC-3′) and gad_RevEcoRI (5′-GAATTCCAGTGTGTGAATMSGTATTTC-3′), which were designed based on the sequences of the gad genes of Lactobacillus spp. available in GenBank (Accession numbers JN248358.1, KU214639.1, AB986192.1, CP029349.1, AL935263.1, CP018209.1, CP028977.1, GU987102.1, JX545343.1).

The primers were supplied by VBC-Biotech Service (Vienna, Austria) and the appropriate endonuclease restriction sites were introduced in the forward and reverse primers (underlined sequences). The conditions for PCR reactions were as follows: initial denaturation at 98 °C for 20 s; 30 cycles of denaturation at 98 °C for 20 s, annealing at 58 °C for 20 s, extension at 72 °C for 1 min 45 s, and final extension at 72 °C for 2 min. The amplified PCR products were purified using the Monarch DNA Gel Extraction Kit (New England Biolabs, Ipswich, MA, USA), digested with NdeI and EcoRI and cloned into the pET 21(+a) vector (Novagen, Merck KGaA, Darmstadt, Germany) resulting in the plasmid pET21GAD. E. coli NEB5α was used as a host for obtaining the plasmids in sufficient amounts. The sequence of the insert was confirmed by DNA sequencing performed by a commercial provider (Microsynth, Vienna, Austria). The alignment tool (BLAST) from the National Center for Biotechnology Information BLAST website was used for the alignment of the nucleotide sequence of the gad gene from L. plantarum FNCC 260 with the available gad sequences from LAB. The comparison of glutamate decaroboxylases (GAD) from different LAB species was carried out using the program Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) [31,33].

2.6. Overexpression of GADLbFNCC260 in E. coli and Protein Purification

The expression vector of pET21GAD harboring the gad gene from L. plantarum FNCC 260 was transformed into E. coli T7 Express GRO carrying the plasmid pGRO7, which encodes the chaperones GroEL and GroES (Takara, Shiga, Japan). Subsequently, E. coli T7 Express GRO carrying the plasmid pET21GAD was cultivated in LB broth medium supplemented with 100 µg/mL ampicillin, 20 µg/mL chloramphenicol, and 1 mg/mL arabinose until OD_600nm of 0.6 was reached. Thereafter, the isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM for induction. The culture was further incubated at 18 °C for 20 h with shaking at 180 rpm. The cells were harvested, washed twice with 50 mM sodium phosphate buffer (pH 6.5), and resuspended in buffer A (50 mM NaH₂PO₄, 300 mM NaCl, and 10 mM imidazole, pH 7.0). The resuspended cells were disrupted using a French press (Aminco, Silver Spring, MD, USA) and centrifuged at 15,000× g for 20 min at 4 °C. The cell-free extracts were collected and loaded to a prepacked 1 mL HisTrap HP Ni-immobilized metal ion affinity chromatography (IMAC) column (GE Healthcare, Uppsala, Sweden) that was pre-equilibrated with buffer A (50 mM NaH₂PO₄, 300 mM NaCl, and 10 mM imidazole, pH 7.0). The His-tagged protein was eluted at a rate of 1 mL/min with a 15 mL linear gradient from 0 to 100% buffer B (20 mM NaH₂PO, 500 mM imidazole, 500 mM NaCl, pH 6.5). Active fractions were pooled, desalted, and concentrated by ultrafiltration using an Amicon Ultra centrifugal filter unit with a 30 kDa cut-off membrane (Millipore, Burlington, MA, USA). The purified enzyme was stored in 50 mM citrate-phosphate buffer (pH 5.0) for further characterization and enzymatic conversion. The molecular masses of purified GAD were determined by SDS-PAGE and Native PAGE. Protein bands were visualized by staining with Bio-safe Coomassie (Bio-Rad). The determination of protein mass was carried out using Unstained Precision plus Protein Standard (Bio-Rad, Hercules, CA, USA).

The size of the protein was also confirmed by LC-ESI-MS analysis. The proteins were S-alkylated with iodoacetamide and digested with Trypsin (Promega, Madison, WI, USA). The digested proteins were directly injected to LC-ESI-MS (LC: Dionex Ultimate 3000 LC). A gradient from 10 to 80% acetonitrile in 0.05% trifluoroacetic acid (using a Thermo ProSwiftTM RP-4H column (0.2 × 250 mm) at a flow rate of 8 µL/min was applied (30 min gradient time). Detection was performed with a Q-TOF instrument (Bruker maxis 4G, Billerica, MA, USA) equipped with standard ESI source in positive ion, MS mode (range: 400–3000 Da). Instrument calibration was performed using ESI calibration mixture (Agilent, Santa Clara, CA, USA). Data were processed using Data analysis 4.0 (Bruker) and the spectrum was deconvoluted by MaxEnt.

2.7. Enzymatic Synthesis of GABA

Batch conversion reactions were carried out in 2 mL scale with 0.64 U/mL purified GAD using 100 mM MSG in 50 mM citrate-phosphate buffer (pH 4.5) containing 0.2 mM of PLP as cofactor. Decarboxylation reactions were performed at 30 °C with 300 rpm agitation using a Thermomixer (Eppendorf, Hamburg, Germany). The samples were withdrawn at time intervals and the enzyme GAD was inactivated at 100 °C for 5 min. The samples were stored at −20 °C for subsequent analysis. GABA content in the reaction mixtures was determined using the GABase assay and confirmed with UPLC analysis as described in Section 2.3.

3. Results and Discussion

3.1. Screening and Identification of GABA-Producing LAB

Thirty isolates of Lactobacillus spp. from Indonesian fermented foods were screened for the formation of GABA in the culture medium using the TLC method and only two isolates showed clear spots on TLC plate (Figure 1), which have similar Rf value (0.78) as the GABA standard. These two isolates were FNCC 260 and FNCC 343 isolated from fermented cassava and fermented fish, respectively. The two strains FNCC 260 and FNCC 343 were cultivated in MRS broth containing 118 mM MSG for 48 h to determine the GABA production in the culture medium, which was analyzed to be 352 mg and 328 mg of GABA per liter of culture medium, respectively. Based on morphological observation, these two strains were Gram positive, rod-shape, microaerophilic, and catalase negative.

Subsequently, these two GABA-producing LAB were identified using 16S rDNA and MALDI-TOF MS (Bruker Biotyper). Proteomic identification by MALDI-TOF MS was performed since this technique is very effective in identifying species and subspecies of LAB. A number of species and subspecies of LAB have been successfully identified using proteomic-based identification technique [40,41,42,43]. Based on MALDI-TOF MS identification, both strains FNCC 260 (2.15 log score) and FNCC 343 (2.12 log score) were identified to be L. plantarum, which matches with the strain in a reference database. The log scores also indicate the accuracy and reliability of MALDI-TOF MS identification. A log score between 2.00 and 2.30 indicates accurate identification to the genus and species level [44,45]. Subsequently, GABA-producing LAB were subjected to 16S rDNA sequencing.

Based on partial 16S rDNA sequencing (~1400 bp), both strains belong to the species L. plantarum, with 99.81% sequence identity with L. plantarum strain CIP 103,151 (accession number, NR_104573.1) for the strain FNCC 260, and 99.81% sequence identity with L. plantarum strain NBRC 15,891 (accession number, NR_113338.1) for the strain FNCC 343, respectively. The results confirmed that both strains FNCC 260 and FNCC 343 are indeed L. plantarum.

3.2. Time-Course of GABA Production by L. plantarum FNCC 260

L. plantarum FNCC 260 was cultivated in MRS medium supplemented with 118 mM MSG at 37 °C. The time courses of GABA production, the pH value and the growth profile of L. plantarum FNCC 260 are shown in Figure 2. GABA production started when cell growth reached the stationary phase after 12 h of cultivation. A slightly higher GABA production was obtained after 48 h (450 mg/L) compared to the GABA production mentioned above in the screening experiment (352 mg/L). This was due to different MSG used in these two experiments (see Materials and Methods). The highest GABA production was 809.2 mg/L of cultivation medium after 60 h of cultivation, at which cell growth is still in the stationary phase. This observation agrees with previous reports in the literature. The maximum GABA production of L. brevis L-32 was observed between 36 to 72 h of cultivation and GABA was mainly produced during the stationary growth phase [46,47,48]. However, GABA production decreased when the cultivation time was prolonged further. This might be due to the activity of the enzyme GABA transaminase (GABA-T), which degrades GABA. This enzyme catalyzes GABA degradation to succinic semialdehyde by using either pyruvate or α-ketoglutarate as the amino acceptors and succinic semialdehyde is irreversibly oxidized to succinate by succinic semialdehyde dehydrogenase [46,49,50]. Interestingly, we observed that the cell growth did not show a decreasing trend when the cultivation time was extended up to 108 h since GABA is utilized as a nutrient during prolonged cultivation [49]. Ko et al. (2013) reported a similar observation of cell growth of L. brevis FPA 3709 during GABA synthesis when GABA production decreased [49].

During GABA production, a decrease in pH of MRS medium was observed. The pH of the cultivation medium rapidly decreased from the initial pH 6.5 to pH 4.1 after 12 h of cultivation. The decrease in pH was due to lactic acid and acetic acid formation during the cultivation of the organism [50]. GABA production involves cytoplasmic decarboxylation, which results in extracellular proton consumption after the uptake of glutamate by its specific transporter [28]. This may lead to the removal of hydrogen ions and an increase of pH in the cytoplasm [51]. Apparently, we observed that the decrease in pH of the cultivation medium (to below pH 4.0) correlated with the increase in GABA production during cultivation of L. plamtarum FNCC 260. Similarly, the maximum GABA production of L. buchnerii was achieved when the pH of the cultivation medium decreased to pH 5.0 [24]. In this study, although GABA production started to decrease after 60 h of cultivation, which might be due to the activity of the enzyme GABA transaminase, the pH of the cultivation medium kept decreasing until 84 h of cultivation indicating that decarboxylation of glutamate still occurred.

3.3. The Effect of Cofactors on GABA Production and Cell Growth

Glutamate decarboxylase is a pyridoxal 5’-phosphate (PLP) dependent enzyme. Theoretically, the addition of PLP to the medium could increase GAD activity and GABA production [2,21,52,53]. PLP and pyridoxine were added into the medium at various concentrations. As expected, the addition of 0.2 mM and 0.6 mM PLP led to the increase in GABA production, reaching 903.0 mg/L and 945.3 mg/L after 108 h of cultivation, respectively (Figure 3a). Furthermore, GABA was still produced when cultivation time was prolonged to 108 h. In contrast, GABA production in a medium without PLP rapidly decreased after 72 h. it was clear that the strain was able to utilize PLP to produced GABA. The addition of PLP to the medium did not inhibit cell growth during the cultivations (Figure 3b). Previous studies by Komatsuzaki et al. [2] and Yang et al. [53] reported that the addition of 0.1 mM and 0.02 mM PLP significantly enhanced GABA production and GAD activity in the culture media of L. paracasei NFRI 7451 and S. thermophillus Y2, respectively.

Pyridoxine (vitamin B6) is a water-soluble vitamin that is ubiquitously found in nature. Pyridoxine can be taken by the cells at the plasma membrane and is subsequently phosphorylated to form PLP within the cytoplasm [52]. The utilization of pyridoxine could be an alternative to replace PLP since PLP is significantly more expensive with low availability. Therefore, we hypothesized that the addition of pyridoxine would improve GABA production. It is shown that the addition of 0.1 mM pyridoxine had a better enhancement on GABA production compared to higher concentrations of pyridoxine tested. The addition of 0.1 mM pyridoxine enhanced GABA production reaching 839.5 mg/L and 969.5 mg/L after 60 h and 96 h of cultivation, respectively (Figure 4a), which are higher compared to the cultivation without pyridoxine (754 mg/L and 606 mg/L, respectively). This observation suggests that pyridoxine can be taken up by the cells and is phosphorylated to form PLP, which is essential for GAD activity. However, GABA production decreased to 878.8 mg/L after 108 h of cultivation (Figure 4a). This could be due to the degradation of pyridoxine during cultivation and hence it lost its activity as a cofactor [53]. The addition of pyridoxine did not have notable effects on cell growth during the cultivation of L. plantarum FNCC 260 (Figure 4b). In both cases of PLP and pyridoxine additions, we observed that the growth reached OD₆₀₀ ~ 8 within 12 h and then maintained at OD ~ 6–7 during the entire cultivation time up to 108 h. Li et al. reported that the addition of cofactor did not improve or inhibit the cell growth of L. brevis NCL912 [28].

3.4. The Effect of Monosodium Glutamate on GABA Production

The presence of MSG is a key factor in producing GABA. The optimal culture conditions for GABA production were determined by measuring the GABA content in the cultivation medium of L. plantarum FNCC 260 with different initial MSG concentrations in the MRS medium. As shown in Figure 5 increasing MSG concentrations increased GABA production (Figure 5a) and maintained cell viability (Figure 5b). The maximum GABA production was achieved at 1226 mg/L at 96 h in an MRS medium containing 100 mM MSG. It appears that a prolonged incubation time did not increase GABA productivity of the strain. A possible reason led to such circumstances was due to GABA catabolism, resulting from GABA transaminase activity. The activity of GABA transaminase could decrease GABA production by converting GABA to succinic semi-aldehyde (SSA).

Similarly, high concentrations of MSG resulted in decreased GABA production of the strains L brevis CRL 1942, S. thermophillus Y2 and L. paracasei NFRI 7415 [2,54,55]. It was suggested that high glutamate concentrations become more toxic to some strains of LAB and suppressed the expression of gadB genes [56]. In this study, we observed that MSG concentration up to 100 mM did not have effects on bacterial growth (Figure 5b) indicating that GABA is consumed by the cells to maintain its viability during the cultivation period. However, as it was shown in Figure 2, when the initial concentration of MSG in cultivation medium was 118 mM, the production of GABA was significantly lower compared to the production obtained with 100 mM MSG. This suggests that the observations from previous studies [2,28,53] about the negative effects of high glutamate concentrations on bacterial growth of some LAB strains and the expression of gadB genes could be an explanation for a similar observation in our study.

3.5. Cloning, Expression of Glutamate Decarboxylase from L. plantarum FNCC 260 in E. coli and Purification of the Enzyme

The gadB gene from L. plantarum FNCC 260 was cloned and its complete open reading frame consists of 1410 bp, encoding 469 amino acids. The predicted molecular mass of GAD is 53. 57 kDa and the theoretical isoelectric point (pI) is 5.62 as calculated using ExPASy program (www.expasy.org). The GadB sequence from L. plantarum FNCC 260 shared 98% homology with the GadB from L. futsaii CS3 (accession number AB839950), L. plantarum Taj-Apis362 (accession number AHG59384) and L. plantarum WCFS1(accession number CCC80401.1).

The gadB gene was cloned into the expression vector pET-21a(+) (Novagen, Merck KGaA, Darmstadt, Germany). The resulting vector pET21Gad was subsequently transformed into E. coli T7 Express carrying the plasmid pGRO 7 for the enhanced expression of the chaperones GroEL/GroES (E. coli T7 Express GRO). E. coli cells were cultivated in LB medium and induced with 0.5 mM IPTG as described in Section 2. The obtained expression yield was 1.38 kU/L fermentation medium with a specific activity of 0.24 U/mg. The recombinant GAD was purified with a single-step purification using the His-trap HP column and the specific activity of the purified enzyme was 1.12 U/mg with a purification factor of 4.5. The apparent molecular mass as judged by SDS-PAGE and native PAGE was estimated to be ~51 kDa and ~140 kDa, respectively (Figure 6a,b). The size of the protein was also confirmed by LC-ESI-MS and it was determined to be 51.79 kDa (data not shown). Several bands were found in native PAGE with the largest band was ~140 kDa. The LC-ESI-MS analysis revealed that these bands on native PAGE contain components of the subunit. The first band represent the intact dimeric enzyme and the other bands with lower molecular masses result from degradation of the intact protein. It also suggested that GadB from L. plantarum FNCC 260 is a homodimeric enzyme. GadB from L. plantarum ATCC 14917 was also reported as a homodimer [56].

The deduced amino acid sequence of L. plantarum FNCC 260 GAD contains a highly conserved catalytic domain that belongs to the PLP-dependent decarboxylase superfamily (Figure 7). A lysine residue (K280) is considered as the PLP-binding site for most bacterial GADs [51,56]. Lysine residue is also found in plant GADs since high homology between bacterial and plant GADs has been revealed [57,58]. In addition, the two residues T215 and D247 are crucial to promote decarboxylation [50]. Furthermore, the consensus sequence HVDAASGG is highly conserved in many bacterial GADs (Figure 7) and is also found in several GADs from Lactobacillus spp including L. futsaii CS3, L. brevis HYE1, L. zymae, and L. paracasei NFRI 7415 [19,34,36,51].

3.6. GABA Synthesis by Recombinant Glutamate Decarboxylase from L. plantarum FNCC 260

For enzymatic GABA synthesis, we performed the conversion of MSG using 0.64 U/mL purified recombinant GAD in a 2-mL scale of reaction mixtures. Most GADs from Lactobacillus spp. have optimum activities at acidic pH values [33,36,57,58], and the recombinant GAD from L. plantarum FNCC 260 showed an optimum pH at pH 4.5 as expected (data not shown). MSG (100 mM) in 50 mM citrate-phosphate buffer (pH 4.5) containing 0.2 mM PLP was used as substrate and the reaction was performed at 30 °C. GABA synthesis reached its highest yield at 6450 mg/L (63 mM) within 6 h of reaction (Figure 8), and the enzyme retained 73% of its initial activity after 6 h of reaction. It was clear that the use of purified GAD was more efficient in terms of both GABA production and conversion time. Enzymatic synthesis of GABA using purified recombinant GAD from L. plantarum FNCC 260 showed 5 to 7-fold higher product concentrations than microbial fermentations in a significantly shorter time. This suggests that the use of purified GAD is crucial to overcome the limitations in GABA production due to GABA-degrading enzymes in the cells, slow reaction rate, and low production yield [28,53,59,60]. Furthermore, UPLC analysis was performed to confirm GABA production in both microbial fermentation and enzymatic conversion with a retention time of GABA at 8.5 min (Figure 9).

4. Conclusions

In the present study, we compared GABA synthesis between microbial fermentation of L. plantarum FNCC 260, which was isolated from Indonesian fermented cassava, and enzymatic conversion of glutamate using recombinant glutamate decarboxylase (GAD) from L. plantarum FNCC 260 expressed in E. coli. MSG, PLP, and pyridoxine were shown to positively affect GABA production during the cultivations of L. plantarum FNCC 260. Enzymatic synthesis of GABA using purified recombinant GAD from L. plantarum FNCC 260 showed at least 5-fold higher GABA titres than microbial fermentations in a significantly shorter time. The newly isolated GABA-producing LAB is of great interest to extend the area of applications. L. plantarum FNCC 260 should be considered as a potential candidate for GABA production via both fermentation and enzymatic synthesis and can be also developed as functional starter culture.