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Research ArticleReceived: 27 August 2013 Revised: 5 October 2013 Accepted article published: 26 October 2013 Published online in Wiley Online Library: 25 November 2013

(wileyonlinelibrary.com) DOI 10.1002/jctb.4251

Salt-free production of γ -aminobutyric acidfrom glutamate using glutamatedecarboxylase separated from Escherichia coliThu Huong Dinh,a Ngoc Anh Thu Ho,a Taek Jin Kang,a Karen A. McDonaldb

and Keehoon Wona∗

Abstract

BACKGROUND: Gamma(γ )-aminobutyric acid (GABA) has been used extensively in pharmaceuticals and functional foods andis also a building block for bioplastics. GABA is produced from glutamate through decarboxylation catalyzed by glutamatedecarboxylase (GAD). The reaction medium should be kept acidic because a pH rise resulting from the reaction inactivates theenzyme catalyst, which is active only at acidic pH. The use of conventional buffers and acids inevitably accompanies salts, whichcause serious problems in separation and purification of GABA. In this work, we have applied heterogeneous solid acids for thefirst time.

RESULTS: The GAD-catalyzed reaction was conducted in 0.2 mol L−1 sodium acetate buffer (pH 4.6) with 1 mol L−1 monosodiumglutamate at 37 ◦C. When commercial cation-exchange resins as solid acids were simply added to the reaction medium, theconversion improved from 13% to 67% without salt formation. Even when water was used as the reaction medium, acidicion-exchange resins enhanced the reaction conversion significantly.

CONCLUSION: In a salt-free manner, acidic resins suppress the pH rise during the reaction so that they can enhance the reactionconversion. In addition, they can be recovered and reused easily after the reaction. Heterogeneous solid acids make the GABAproduction process more economical and eco-friendly.c© 2013 Society of Chemical Industry

Keywords: gamma-aminobutyric acid; glutamate; glutamate decarboxylase; solid acids; salt-free reaction system

INTRODUCTIONRecently there has been a tremendous demand for chemicals andmaterials derived from renewable resources, biomass.1 Gamma(γ )-aminobutyric acid (GABA) is a non-protein amino acid widely foundin nature and has been used extensively in pharmaceuticals andfunctional foods.2 GABA is also a building block for polyamide4, which is a novel biobased and biodegradable plastic with

excellent properties.3–5 Owing to increasing demand for GABA,

extensive research on its production has been undertaken.5–8

From an industrial and economical point of view, biologicalproduction of GABA from glutamate, a cheap renewable source, isa completely reasonable and valuable process. GABA is producedthrough CO2 release from the α-carboxyl group of glutamate,which is irreversibly catalyzed by pyridoxal phosphate-dependentglutamate decarboxylase (GAD, EC 4.1.1.15) widely distributed inliving cells of various creatures from bacteria to mammals.9

In bacteria, GAD not only satisfies metabolic demands for aminoacids but also plays a key role in glutamate-based acid resistancesystem. By consuming a proton through its incorporation intoglutamate, GAD protects bacteria from extracellular acid stress.10,11

As can be inferred from its role, most bacterial GAD exhibit thehighest activity only under acidic conditions and show little activityat neutral to alkaline pH values.9,11,12 This characteristic of GADmust be critical to the host to prevent the significant increase in the

cytosolic pH while keeping its general acid resistance. However,the pH-dependence is disadvantageous to employment of thisenzyme catalyst for GABA production because the pH increaseresulting from the enzymatic reaction will eventually inactivatethe enzyme, leading to low conversion. Buffer solutions are not agood choice, taking into consideration the synthesis of GABA onan industrial scale. In order to counteract the increase in pH, andthus to improve the productivity of GABA, addition of conventionalacids such as hydrochloric acid to the reaction medium during thereaction has widely been performed. For example, Plokhov et al.used hydrochloric acid in a whole cell reaction using E. coli cellsoverexpressing E. coli GAD.13 Similarly, hydrochloric acid was usedfor maintaining the optimal pH in their non-buffered reaction usingimmobilized E. coli GAD.14 However, this method brings aboutformation of a significant amount of salts, which can cause seriousproblems in separation and purification of GABA after the reaction.

∗ Correspondence to: Keehoon Won, Department of Chemical and BiochemicalEngineering, Dongguk University-Seoul, 30 Pildong-ro 1-gil, Jung-gu, Seoul100– 715, Republic of Korea. E-mail: keehoon@dongguk.edu

a Department of Chemical and Biochemical Engineering, Dongguk University-Seoul, 30 Pildong-ro 1-gil, Jung-gu, Seoul 100-715, Republic of Korea

b Department of Chemical Engineering and Materials Science, University ofCalifornia at Davis, 1 Shields Avenue, Davis, CA 95616, USA

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In this respect, it is essential to devise a simple and efficientmethod for suppressing the pH increase without salt formation.Solid acids have been mainly used as catalysts for variousreactions such as acetylation, alkylation, acylation, hydrolysis,and dehydration. Application of heterogeneous solid acids isan efficient method for environment-friendly processes becauseof its easy separation and recyclable nature.15,16 In this work, forthe first time we have employed heterogeneous acids instead ofconventional homogeneous acids in order to counteract the pHincrease during GABA production catalyzed by GAD. Commercialcation-exchange resins have been used as solid acids, and theireffects on the enzymatic GABA production from glutamate havebeen examined in the present study.

MATERIALS AND METHODSMaterialsE. coli recombinant strain (E. coli BL21(DE3): pET-28b-GAD)was constructed. L-monosodium glutamate (MSG), pyridoxal 5’-phosphate (PLP), DL-dithiothreitol (DTT), and the other chemicalswere purchased from Sigma-Aldrich (St. Louis, MO, USA) and wereused without further purification. Amberlyst 15 is a strongly acidiccation-exchange resin containing sulfonic acid (macroreticularbeads with total exchange capacity ≥ 4.7 meq g−1). AmberliteIRC86 is a weak acid cation exchange resin containing carboxylicacid groups (gel polyacrylic copolymer beads with total exchangecapacity ≥ 5.2 meq g−1).

Cloning, expression, and separation of GADThe E. coli chromosome contains two homologous GAD genes,gadA and gadB, the protein products of which are named GADα andGADβ , respectively.17,18 The DNA sequence that encodes GADβ

was PCR-amplified from E. coli DH5α and cloned into pET-28b. Aplasmid harbouring the gene of interest was used to transform E.coli BL21(DE3) for overexpression. From the streaking plate, oneloop of colony was inoculated into 5 mL of LB medium containing0.1 mg mL−1 of pyridoxine and 0.05 mg mL−1 of kanamycin, andincubated at 37 ◦C and 120 rpm for 18 h. Two milliliters of starterculture was then inoculated into 100 mL of the above LB medium.The recombinant cells were grown at 37 ◦C and 120 rpm for 2 h,and GAD expression was induced by adding IPTG at 0.1 mg mL−1

for 3 h. The cells collected by centrifugation (7000 rpm at 4 ◦C for30 min) were washed three times with phosphate buffered saline(PBS, pH 7.3) and then recovered by centrifugation (10 000 rpm at4 ◦C for 10 min). The washed cells (0.5 g) were suspended in 10 mLof PBS (pH 7.3) and disrupted by sonication (1 min × 5 times) onice. Cell debris was removed by centrifugation at 10 000 rpm,4 ◦C for 30 min. Proteins in the supernatant were fractionatedwith 20%, 40%, 60%, and 80% saturated ammonium sulfate.The precipitated protein collected between 40 and 60% of saltsaturation was redissolved in 10 mL of PBS (pH 7.3) and stored at4 ◦C.

GAD-catalyzed decarboxylation of glutamate into GABAFor activity assay of GAD separated from E. coli, the GADsolution was added to sodium acetate buffer (0.2 mol L−1,pH 4.6) containing 100 mmol L−1 MSG, 0.1 mmol L−1 PLP, and0.2 mmol L−1 DTT. Samples were withdrawn from the reactionmedium incubated at 37 ◦C and then analyzed using HPLC inorder to determine the initial rate. One unit (U) of GAD was defined

as the amount of enzyme required to produce 1 μmol of GABA permin under this condition.

To determine the effect of pH on GAD activity, the enzymaticdecarboxylation was carried out with 0.4 U of GAD for 5 h in1 mL of several buffers containing 20 mmol L−1 MSG, 0.1 mmol L−1

PLP, and 0.2 mmol L−1 DTT: 0.2 mol L−1 sodium citrate bufferfor pH values of 3 and 3.5; 0.2 mol L−1 sodium acetate bufferfor pH values of 4, 4.6, 5, and 5.5; 0.1 mol L−1 MOPS [3-(N-morpholino)propanesulfonic acid] buffer for pH values of 6 and7. For high substrate concentrations, it was conducted with 8U of GAD in 2 mL of sodium acetate buffer (0.2 mol L−1, pH 4.6)containing 1 mol L−1 MSG, 0.1 mmol L−1 PLP, and 0.2 mmol L−1

DTT without or with 0.2 g of cation-exchange resins for 24 h.All the reactions were carried out at 37 ◦C in a microtubeshaking incubator (Hangzhou Allsheng Instruments Co., China).At predetermined intervals, samples were withdrawn from thereaction mixture and diluted with NaOH solution (5 mol L−1). Allthe experiments were performed at least in triplicate, and the datawere presented as the mean and the standard deviation.

Analysis of substrate and productThe substrate and the product were determined withoutderivatization by a HPLC system (Waters 2960 series) equipped witha UV/Vis dual absorbance detector (Waters 2487). After removalof contaminants by centrifugation, the samples were injected intothe HPLC system through an autosampler. The stationary phasewas a Waters Atlantis T3 C18 column (4.6 mm × 250 mm, 5 μmparticle), which retains and separates highly polar compoundsover a wide pH range, and the column temperature was 30 ◦C.The mobile phase was phosphoric acid aqueous solution (pH 2.8),and the flow rate was 0.4 mL min−1. GABA and MSG were clearlyseparated and detected at 210 nm, and their retention times were6.6 min and 7.6 min, respectively.

RESULTS AND DISCUSSIONEffects of pH on GAD separated from E. coliGlutamate decarboxylase was prepared from cell cultivationand separation: recombinant strain (E. coli BL21(DE3)::pET-28b-GAD) was constructed and cultivated to produce GAD. Theoverexpressed enzyme was separated by cell disruption followedby ammonium sulfate fractionation. SDS-PAGE revealed that thefraction of the precipitated protein collected between 40 and60% of salt saturation contained most of GAD (data not shown).The pH effect on activity of the GAD separated from E. coli wasinvestigated changing pH from 3 to 7. In order to ensure that thepH does not change during the reaction, 20 mmol L−1 glutamatewas used – wild type GADβ was reported to have Km for glutamateof 2.32 mmol L−1 in sodium acetate buffer, pH 4.6.12 As shown inFig. 1(A), GAD was active in the range pH 3.5 to 5.5 with optimalpH at 4.6. It is well known that E. coli GAD shows the highestactivity between pH 4 and 5.9,12,13 At pH values above 6, GADwas found to be inactive. It was explained by conformationalchange of the hexameric enzyme at its N- and C-termini fromacidic to neutral pH.11,12,19 Especially, His465 at the C-terminusof the enzyme together with Glu89 was demonstrated to beinvolved in the conformational change in a cooperative manner.20

When the GAD-catalyzed reaction was carried out in sodiumacetate buffer (0.2 mol L−1, pH 4.6), 20 mmol L−1 glutamate wascompletely converted into GABA within 5 h (Fig. 1(B)).

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Figure 1. (A) Effect of pH on the activity of GAD separated from E. coli.(B) Time course of the reaction conversion at pH 4.6. The enzymaticreaction was carried out in 0.2 mol L−1 buffer containing 20 mmol L−1

MSG, 0.1 mmol L−1 PLP, and 0.2 mmol L−1 DTT.

GAD-catalyzed reaction with high substrate (glutamate)concentrationTo ensure a high level of productivity in industrial applications,as high a substrate concentration as possible is needed.GAD-catalyzed reaction was attempted with a high substrateconcentration (1 mol L−1 glutamate). The reaction was carried outin 0.2 mol L−1 acetate buffer (pH 4.6) at 37 ◦C. As shown in Fig. 2(A)(closed circle), the reaction conversion reached 13% after 3 hreaction and did not increase any more even though the reactionwas allowed to proceed to 24 h. The final conversion did notimprove even when the enzyme was used fourfold more; the initialrate increased (data not shown). The conversion of 13% is verylow compared with the final conversion of 100% with 20 mmol L−1

glutamate (Fig. 1(B)). Product inhibition is not responsible for thislow conversion because GABA did not decrease GAD activity up to5 mol L−1.14 This low conversion can be explained by an increasein pH of the medium during the reaction from acidic to neutral;GAD is hardly active at pH values above 6 (Fig. 1(A)).

In order to verify this explanation, the pH was monitored usinga pH meter during the reaction (closed square, Fig. 2(A)). The initialpH was found to be 5.3 although the buffer (0.2 mol L−1, pH 4.6)was used. This is due to 1 mol L−1 MSG in the reaction medium,which is a neutral compound: the pH of 1 mol L−1 MSG in purewater was about 6.9. A proton is consumed as a co-substrate at astoichiometric ratio as the enzymatic decarboxylation reaction

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Figure 2. (A) Time course of GAD-catalyzed reaction with 1 mol L−1

glutamate. Symbols: conversion ( ) and pH ( ). (B) Effects of substrateconcentration on the final conversion ( ) and the initial pH ( ). Thereaction was conducted in sodium acetate buffer (0.2 mol L−1, pH 4.6)containing 0.1 mmol L−1 PLP and 0.2 mmol L−1 DTT for 24 h.

progresses. It is obvious that the 0.2 mol L−1 buffer used inthis experiment cannot supply enough protons to keep the pHunchanged. As a result, the pH of the reaction mixture increasedbeyond the range for GAD activity, leading to the low conversion.

The effects of substrate concentration on the reactionconversion were also investigated: the MSG concentration wasincreased from 0.5 mol L−1 to 3 mol L−1. It is known that GAD doesnot suffer from substrate inhibition.13 With increasing substrateconcentration, the conversion decreased, whereas the measuredinitial pH rose (Fig. 2(B)). Because the concentrations used exceedthe buffering capacity (0.2 mol L−1), the addition of neutral MSGto the acidic buffer (pH 4.6) increases the initial pH. The greaterthe initial pH above 4.6, the narrower the pH range over which theGAD can function; as a result, the conversion becomes lower.

INTRODUCTION OF CATION-EXCHANGERESINS TO THE ENZYMATIC GABAPRODUCTIONIt is essential to maintain acidic pH for GAD activity throughoutthe reaction in order to achieve higher conversion. For thispurpose, hydrochloric acid has been widely used, but formsa significant amount of salts, which result in separation andpurification problems. We have introduced solid acids to enzymaticGABA production and employed cation-exchange resins as aheterogeneous source of acid. It is expected that they can supplyprotons to the reaction without salt formation by replacing Na+

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Figure 3. GAD-catalyzed decarboxylation of 1 mol L−1 glutamate in sodiumacetate buffer (0.2 mol L−1, pH 4.6) in the presence of 0.2 g of acidic cation-exchange resins, (A) Amberlyst 15 and (B) Amberlite IRC86. Symbols:conversion ( ) and pH ( ).

from MSG in the reaction medium with H+ on the cation-exchangeresin. First a strong acid cation-exchange resin (Amberlyst 15) wasadded to 0.2 mol L−1 acetate buffer (pH 4.6) containing 1 mol L−1

MSG, 0.1 mmol L−1 PLP, and 0.2 mmol L−1 DTT used in Fig. 2(A).Figure 3(A) reveals that the reaction conversion was successfullyenhanced from 13% to 67%. This is because GAD remainedactive for a longer time than without the solid acid; the pHwas kept below 6 for about 12 h. After the pH exceeded 6, thereaction conversion hardly increased. Similarly, the conversion alsoimproved significantly to 67% when a weak acid cation-exchangeresin (Amberlite IRC86) was used (Fig. 3(B)). The final conversionmay be influenced by the cation-exchange capacity of the resins.Though these two cation-exchange resins have different functionalgroups, their effects were similar perhaps because they had similarion-exchange capacity.

One of the most important things considered for industrialapplications is separation and purification of products after reac-tion. Even though the use of solid acids prevents salt formation,salts still exist in the reaction medium owing to buffers. In thisrespect, a buffer-free reaction system is highly advantageous;other merits of a buffer-free system would be to eliminateproblems such as incompatibility with the coenzyme PLP andto reduce process operation costs by using fewer chemicals.14

GAD-catalyzed reaction was conducted in water instead of sodiumacetate buffer as the reaction medium; Amberlite IRC86 (0.6 g)was added to water (HPLC grade) containing 1 mol L−1 MSG,0.1 mmol L−1 PLP, and 0.2 mmol L−1 DTT. Changes in conversionand pH during the enzymatic reaction are shown in Fig. 4. The

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Figure 4. GAD-catalyzed decarboxylation of 1 mol L−1 glutamate in purewater in the presence of Amberlite IRC86 (0.6 g). Symbols: conversion ( )and pH ( ).

conversion was almost 100% after 24 h (for reference, when 0.2 gof the resin was used, the final conversion was 60%), which isbecause GAD remained active throughout the reaction; the pHvalue remained less than 6 during the reaction. Thus, glutamatewas completely converted into GABA in the salt-free medium.

CONCLUSIONSSolid acids have been applied to counteract the pH rise duringdecarboxylation of glutamate into GABA catalyzed by GAD.The reaction conversion was significantly improved without saltformation by using cation-exchange resins as a solid acid. Evenwhen water was used instead of buffer solutions as a reactionsolvent, acidic ion-exchange resins enhanced the conversion of theGABA synthesis reaction in a salt-free manner. After the reaction,acidic resins in the reaction medium can be recovered and reusedeasily. Solid acids will make the GABA production process moreeconomical and eco-friendly by lowering process operating costsand by simplifying separation and purification of GABA.

ACKNOWLEDGEMENTSThis work was supported by the Research and DevelopmentProgram of the Ministry of Trade, Industry and Energy/theKorea Evaluation Institute of Industrial Technology (10033199);the Agriculture Research Center Program of the Ministry ofAgriculture, Food and Rural Affairs (ARC-710003-03-4-SB120); theConverging Research Center Program (NRF-2009-0082276) andthe Brain Research Program (NRF-2008-2006261) through theNational Research Foundation of Korea (NRF) funded by theMinistry of Science, ICT and Future Planning; the Research Programof Dongguk University (2012).

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