Permeabilizing Escherichia coli for whole cell biocatalyst with enhanced biotransformation ability from l -glutamate to GABA

Journal of Molecular Catalysis B: Enzymatic 107 (2014) 39–46

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Permeabilizing Escherichia coli for whole cell biocatalyst with enhanced biotransformation ability from l-glutamate to GABA Wei-rui Zhao a , Jun Huang b , Chun-long Peng a , Sheng Hu c , Pi-yu Ke a , Le-he Mei a,c,∗ , San-jin Yao a a

Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, PR China c School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, PR China b

a r t i c l e

i n f o

Article history: Received 1 December 2013 Received in revised form 15 May 2014 Accepted 17 May 2014 Available online 27 May 2014 Keywords: Glutamate decarboxylase Escherichia coli Permeabilization Organic solvent Glutamte-GABA antiporter

a b s t r a c t ␥-Aminobutyric acid (GABA) is widely used as a pharmaceutical, nutraceutical, and as a precursor for synthesizing materials for industrial use. Bacterial cells can be exploited for use as biocatalysts with the potential to synthesize compounds such as GABA with greater efficiency, safety, and economy. However, efforts to use biocatalysts must overcome the permeability barrier of the cell envelope. Therefore, to produce a whole-cell biocatalyst with enhanced cell-associated glutamate decarboxylase (GAD) activity, we overexpressed the Glutamate (Glu)-GABA antiporter (GadC) in Escherichia coli (BL21(DE3)-pET28agadB). The cell-associated GAD activity of the transformants was higher by a factor of 2.6 in comparison; however, expression of GadC inhibited growth. Therefore, we permeabilized the cells using either organic solvents, surfactants, or heat. Permeabilization with organic solvents increased cell-associated GAD activity as a function of their hydrophobicity, and hexane was the most effective, increasing cell-associated GAD activity by a factor of 9.65 (6.72 U/mg). The surfactants Triton X-100, CHAPS, NP-40, OGP, and Brij-35 enhanced cell-associated GAD activity, and Triton X-100 was the most effective, increasing cell-associated GAD activity by a factor of 10.8 (7.53 U/mg). Heating BL21(DE3)-pET28a-gadB at 70 ◦ C for 30 min increased cell-associated GAD activity by a factor of 13.1. GAD did not leak from the permeabilized cells under optimum conditions. When the heat-permeabilized cells were immobilized on Ca-alginate gel beads, the biotransformation ability of beads maintained over 60% of their initial ability after 20 consecutive batches, and the beads retained 90% of their initial activity after 30 days of storage. These approaches for improving cell permeability to enhance cell-associated GAD activity show great promise for decreasing the cost of industrial production of GABA. © 2014 Elsevier B.V. All rights reserved.

1. Introduction ␥-Aminobutyric acid (GABA) is a four-carbon non-protein amino acid that acts as the major inhibitory neurotransmitter in the brain and directly affects personality and response to stress [1]. GABA acts as a hypotensive agent, diuretic, and tranquilizer and can prevent diabetes [2–5]. Furthermore, GABA elevates the concentrations of plasma growth hormone and the rate of protein synthesis in the brain [6,7], enhances the treatment of certain neurological disorders [8], and inhibits the proliferation of cancer cells [9,10].

∗ Corresponding author at: Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China. Tel.: +86 571 87953161; fax: +86 571 87951982. E-mail address: [email protected] (L.-h. Mei). http://dx.doi.org/10.1016/j.molcatb.2014.05.011 1381-1177/© 2014 Elsevier B.V. All rights reserved.

Moreover, GABA is used as a precursor in the environmentally safe synthesis of a variety of nitrogen-containing industrial chemicals such as N-methylpyrrolidone [11] and bioplastics such as polyamide 4 [12,13]. Because of the significance of GABA in the food, pharmaceutical, and chemical industries, numerous chemical and biological methods have been developed for synthesizing GABA. Biological methods to synthesize GABA from glutamate offer advantages, because synthesis is achieved using simple and mild reaction conditions that do not adversely affect the environment [14]. Because glutamate decarboxylase (GAD) is the only enzyme known to catalyze the irreversible ␣-decarboxylation of l-glutamate (Glu) or its salts to GABA [15], it is the focus of these efforts. Using whole cells as biocatalysts instead of isolated enzymes is preferred, because they eliminate the tedious and expensive procedures required to isolate and purify enzymes [16]. Therefore,

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much attention has been centered on developing whole-cell biocatalysts with high cell-associated GAD activity for commercial use. For this purpose, recombinant GADs have been expressed in various bacterial species to improve GABA production [17–20]. Although overexpression is an effective technique to produce cell preparations with high enzyme activity, the rates and yields of synthesis using whole-cell biocatalysts is strongly compromised because of the permeability barrier imposed by the cell envelope [16]. For example, the reaction rate achieved using a whole-cell biocatalyst is lower compared with enzymes by factors of 10–100 [16]. Therefore, it is important to develop effective methods to overcome cell envelope barrier to optimize the synthetic capability of whole-cell biocatalysts. Bacteria exchange substrates and products using an antiporter system [21]. For example, the glutamate (Glu)-GABA antiporter provides a specific channel for transporting GABA and Glu under acidic conditions [22,23]. In Escherichia and other bacterial genera, GAD and the Glu-GABA antiporter (called the GAD system) prevent acidification of the cytosol [22] by expelling intracellular protons through the decarboxylation of the acidic substrate (Glu) in the cytoplasm. This reaction converts Glu to the neutral compound GABA and exchanges GABA with extracellular Glu. GAD and the Glu-GABA antiporter catalyze the former and latter reactions, respectively [22,23]. Therefore, we reasoned that overexpression of the Glu-GABA antiporter in Escherichia coli might enhance the intracellular interaction of GAD with its substrate to increase the reaction rate and consequently overcome the permeability barrier imposed by the cell envelope. In addition, overcoming cell envelope permeability barrier can also been done by cell permeabilization. Certain chemicals increase cell permeability without lysing the cells and include detergents such as CTAB [24], TritionX-100 [25], and Tween [26], also chelating agents [27] and organic solvents such as toluene [25,28,29]. Furthermore, mechanical methods such as sonication, homogenization, and freeze–thawing [30,31] was also reported to increase cell permeability. However, few reports are available that describes the use of permeabilization techniques for preparing whole-cell biocatalysts with enhanced biotransformation ability from Glu to GABA. The E. coli strain BL21(DE3)-pET28a-gadB overexpresses GAD from Lactobacillus brevis CGMCC 1306 and therefore represents a potential candidate to produce GABA [32]. In the present study, we transformed BL21(DE3)-pET28a-gadB with the Glu-GABA antiporter (GadC). Because expression of GadC inhibited growth, we determined whether permeabilization with organic solvents, surfactants, and heat would enhance GAD activity.

2.2. Strains and culture conditions E. coli strains BL21(DE3)-pET28a-gadB and BL21(DE3) were kept in our laboratory and cultured at 37 ◦ C in 100 ml Luria–Bertani (LB) medium with 50 mg/ml of kanamycin in a 500 ml Erlenmeyer flask shaken at 200 rpm. When the optical density of the culture at 600 nm reached 0.7, the expression of GAD was induced by adding IPTG to a final concentration of 0.5 mM. The culture was then incubated at 30 ◦ C for 6 h with rotation reduced to 150 rpm. 2.3. Plasmid construction The gene (gadC) encoding the Glu-GABA antiporter was expressed in BL21(DE3)-pET28a-gadB to determine whether the cells produced higher levels of GAD activity. The primer pair (P1F/P1R), 5 -GAAGCTTAAAGGAGGTTCAAATATGGCTACATCAGTACAGAC-3 and 5 -TGCGGCCGCTTAGTGTTTCTTGTCATTCATC-3 was used to amplify gadC from BL21(DE3) genomic DNA. The primer sequences were derived from the United States National Center for Biotechnology Information reference sequence locus NC 012947.1. The amplified gadC fragment was subcloned into the Hind III/Not I sites of pET28a-gadB [32] to generate pET28agadBC. A ribosome-binding site (underlined in the sequence above) was included upstream of the ATG codon to enhance translation of the Glu-GABA antiporter. To express the Glu-GABA antiporter alone, gadC was amplified using the primers P2F (5 -AGCCATGGCTACATCAGTACAGACAG-3 )/P1R. The PCR product was digested with NcoI and NotI and cloned into the cognate sites of pET28a, yielding pET28a-gadC. Shanghai Sangon Co., Ltd. determined the nucleotide sequences of the plasmids. The plasmid DNAs were used to transform BL21(DE3), and the transformants were designated BL21(DE3)-pET28a-gadBC and BL21(DE3)-pET28a-gadC. 2.4. Permeabilization of BL21(DE3)-pET28a-gadB BL21(DE3)-pET28a-gadB was harvested from the culture medium by centrifugation at 10,000 × g for 1 min at 4 ◦ C, and the cell pellets were washed with sodium acetate buffer (0.2 M, pH 4.8). Cells treated with organic solvents or surfactants were incubated at room temperature, centrifuged at 10,000 × g, washed once with 0.2 M sodium acetate buffer (pH 4.8), and analyzed for GAD activity. For heat treatment, cell pellets were resuspended in sodium acetate buffer (0.2 M, pH 4.8). Permeabilization was carried out in triplicate. 2.5. Cell-associated GAD activity assay

2. Materials and methods 2.1. Chemicals The GABA standard was purchased from Acros Organics (Geel, Belgium). Dansyl chloride (Dns-cl) was obtained from Sigma-Aldrich (St. Louis, MO). Hexane, benzene, toluene, xylene, chloroform, ethyl acetate, acetone, methanol, polypropylene glycols 400 and 1000 (PPG-400, PEG-1000), and Tween 80 were purchased from China Medicine Co. Ltd. (Shanghai, China). Kanamycin, 5 -pyridoxal phosphate (PLP), isopropyl-␤-d-thiogalactoside (IPTG), Triton X-100, polyoxyethylene lauryl ether (Brij-35), nonyl phenoxypoly (ethyleneoxy) ethanol (NP-40), 3-((3-Cholamidopropyl)dimethylammonium)-1propanesulfonate (CHAPS), N-octyl-␤-d-glucopyranoside (OGP), peptone and yeast extracts were acquired from Sangon Inc. (Shanghai, China). Unless specified, all other chemicals were of analytical reagent grade or higher quality.

Cell-associated GAD activity was determined by measuring the amount of GABA formed at 37 ◦ C in a reaction mixture containing 0.25 mg (dry cell weight)/mL cell biomass, 0.2 M sodium acetate buffer (pH 4.8), 75 mM l-monosodium glutamate (MSG), and 0.1 mM PLP. The concentration of GABA was determined using high-performance liquid chromatography (HPLC) according to Huang et al. [14]. One unit (U) of GAD activity was defined as the amount of catalyst that produced 1 ␮mol of GABA per minute under the above conditions. Specific activity was defined as U/mg dry cell weight (DCW) cells. Total cell-associated GAD activity of a 1 mL of culture medium (the total cell-associated GAD activity per volume of culture medium, U) = The cell-associated GAD activity per milligram DCW (U/mg) × dry cell weight (mg) in 1 mL culture. 2.6. Scanning electron microscopy (SEM) E. coli cultures treated with different permeabilization methods were centrifuged at 2000 × g for 10 min, and the pellets were

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2.7. Transmission electron microscopy (TEM)

4.5 4.0 3.5 3.0

OD600

collected and resuspended in glutaraldehyde (2.5%, m/V) at 4 ◦ C for 12 h. The samples were centrifuged, and the cell pellets were washed three times with phosphate buffer (0.1 M, pH 7.0) and resuspended in phosphate buffer containing 1% (w/v) osmium tetroxide for 1 h. This cell pellet was washed twice with distilled water, dehydrated using a series of ethanol washes (50%, 15 min; 70%, 15 min; 90%, 15 min and 100%, 15 min), and washed twice with isoamyl acetate (15 min). The samples were subjected to critical point drying, coated with gold/palladium alloy, and observed using a scanning electron microscope (Philips XL-30 ESEM).

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2.5 2.0 1.5 1.0 0.5

For transmission electron microscopy (TEM), cells were harvested, fixed, and dehydrated as described for SEM. The sample was embedded in Spurr’s resin, cut into ultrathin sections (70 nm) using an Ultracut Ultramicrotome (Reichart-Jung) with a diamond knife, and fixed onto grids. The sections were double-stained with uranyl acetate and lead citrate. The grids were observed using a JEM-1230 (JEOL, Japan) electron microscope operating at 80 kV. 2.8. Immobilization of permeabilized cells Immobilization of the heat-permeabilized cells was performed according to Huang et al. [14] with some modifications. Different concentrations of permeabilized cell suspensions (2.5 mL) were added to 7.5 mL of 2% (w/v) sodium alginate in sodium acetate buffer (0.2 M, pH 4.8). A syringe fitted with a 0.6 mm (I.D.) hypodermic needle was filled with the cell suspension, and 0.5 mL was added drop-wise at 0.32 mL/min using a syringe pump onto a CaCl2 solution (20 g/L) to produce a set of gelled beads that was stored overnight at 4 ◦ C. The cell densities of gelled beads were as follows: 1.55, 3.09, 6.19, 9.28, 12.38, and 15.47 mg/mL (DCW). The stability of the immobilized cells was determined by measuring the yields of GABA from 20 batches of sequential preparations with immobilized permeabilized BL21 (DE3)-pET28a-gadB. The activity of each batch was determined in sodium acetate buffer (0.2 M, pH 4.4) containing 100 mM MSG, 50 mM CaCl2 , and 0.1 mM PLP with shaking at 100 rpm at 50 ◦ C for 4 h. CaCl2 was added to the reaction mixture to keep the beads from dissolving during the assays [33,34]. The GAD stability assay was carried out in triplicate. 3. Results and discussion 3.1. Effect of overexpressing the Glu-GABA antiporter on cell-associated GAD activity To increase the yield of GABA by Glu and to transport GABA out of the cells, the Glu-GABA antiporter was expressed together with GAD in E. coli BL21(DE3)-pET28a (BL21(DE3)-pET28a-gadBC). The cell-associated GAD activity was increased by a factor of 2.6 (U/mg) compared with that of BL21(DE3)-pET28a-gadB; however, expression of the Glu-GABA antiporter severely inhibited growth (Fig. 1). Relative to cell density, the total cell-associated GAD activity of a 1 mL of BL21(DE3)-pET28a-gadBC culture was increased by only a factor of 1.39 compared with BL21(DE3)-pET28a-gadB (Table 1). To

0

1

2

3

4

5

6

Time (h) Fig. 1. Growth of BL21 (DE3)-pET28a-gadB (), BL21 (DE3)-pET28a-gadBC (), and BL21 (DE3)-pET28a-gadC () in LB after IPTG induction. Data represent the mean ± SD from three independent determinations.

determine whether growth was inhibited by the overexpression of the Glu-GABA antiporter, gadC was overexpressed in the absence of gadB. There was little growth in cultures of BL21(DE3)-pET28agadC (Fig. 1). Recently, the GAD and Glu-GABA antiporter were also over-expressed using the synthetic protein complex strategy in E. coli (two enzyme are tied up closely using protein–protein interaction domains and the specific ligands, so the two enzymes catalyzing successive reactions are co-localized in a specific space) and the productivity of GABA could be improved 30% by the introduction of the complex between GAD and Glu-GABA antiporte (5.65 g/L) compare with normal co-overexpression of GAD and the Glu-GABA antiporter (4.34 g/L) [35]. However, our results showed overexpression of gadC could not only increase cell-associated GAD activity, but inhibit E. coli cell growth severely, which was negative to produce larger amount of whole cell catalyst with enhanced GAD activity. Overexpression of other membrane proteins inhibits cell growth [36,37], and this is attributed to limitations in the ability of the cells to synthesize phospholipids that mediate insertion of proteins into the membrane [36,37]. Overexpressed membrane proteins aggregate, and this impairs the functions of enzymes embedded in the membrane, particularly those involved in aerobic respiration [36,37]. Furthermore, the levels of respiratory-chain complexes in the cytoplasmic membrane are reduced to levels that produce insufficient levels of ATP [36]. Moreover, the results further convinced the presence of a permeability barrier that prevented the transport of Glu and GABA through the cell envelope [16]. Therefore, we explored the possibility that cell-associated GAD activity could be stimulated by permeabilizing the cells. 3.2. Permeabilization of BL21 (DE3)-pET28a-gadB with organic solvents Organic solvents increase cell permeability by destroying the lipid structure of the cell envelope, which increases membrane

Table 1 Cell-associated GAD activity of recombinant E. coli strains. Strains

Cell-associated GAD activity of whole cell (U/mg)

Cell density (OD600 )

Total cell-associated GAD activity of cells in one milliliter culture (U)a

BL21(DE3)-pET28a-gadB BL21(DE3)-pET28a-gadBC BL21(DE3)-pET28a -gadC

0.64 ± 0.11 1.70 ± 0.26 0

4.10 ± 0.08 2.34 ± 0.07 1.00 ± 0.07

0.62 ± 0.07 0.86 ± 0.17 0

a

GAD activity (DCW/ml). Data represent the mean ± standard deviation (SD) from three independent determinations.

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Table 2 Cell-associated GAD activity of permeabilized BL21 (DE3)-pET28a-gadB using organic solvents. Solvents

Log P (–)

Hexane Benzene Toluene Xylene Chloroform Ethyl acetate Acetone Methanol Control

3.5 2 2.5 3.1 2 0.68 −0.23 −0.76

Cell-associated GAD activity enhanced times 10.12 ± 0.22 9.58 ± 0.15 9.69 ± 0.37 8.50 ± 0.21 8.32 ± 0.54 2.7 ± 0.44 1.22 ± 0.14 1.13 ± 0.10 1

Cells were permeabilized with 2% organic solvent for 30 min. Log P values taken from Ref. [29]. P is the partition coefficient for the solvent between 1-octanol and water. Cell-associated GAD activity of untreated BL21(DE3)-pET28a-gadB = 0.65 ± 0.07 U/mg. Data represent the mean value ± SD from three independent determinations.

Cell-associated GAD activity enhanced times

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10 8 6 4 2 0 5 S ij3 AP H Br C

40 NP

P 00 00 00 80 -1 10 OG PG4 en X e G P on PE Tw iti Tr

Fig. 3. Cell-associated GAD activity of BL21 (DE3)-pET28a-gadB cells permeabilized for 30 min with 2% solutions of surfactants.

A 14 Cell-associated GAD activity enhanced times

fluidity [38,39]. We assayed the cell-associated GAD activities of BL21 (DE3)-pET28a-gadB cultures treated with 2% (v/v) solutions of eight organic solvents for 30 min (Table 2). Hexane, chloroform, and aromatic solvents such as toluene, benzene, and xylene enhanced cell-associated GAD activity by more than a factor of 8, and hexane was the most effective. Moreover, cell-associated GAD activity correlated with solvent hydrophobicity (Fig. 2) expressed as log P, where P is the octanol–water partition coefficient of the solvent. Less polar solvents (i.e., those with the highest log P values) significantly increased cell-associated GAD activity. Highly hydrophobic solvents (log P > 2) such as hexane, benzene, toluene, xylene, and chloroform are easier to incorporate into the cell membrane [40] and damage membranes, which is attributed to their higher membrane-buffer partition coefficients [38,40]. Therefore, when certain low solvent concentrations are used, the organic solvents with higher P values are more readily incorporated into the cell envelope and damaged cell envelope. We determined that treating cells with 0.5% (v/v) hexane for 5 min was optimal for stimulating cell-associated GAD activity (Supplementary Fig. S1) (6.27 U/mg). There was no detectable activity in supernatants prepared from the treated cells, indicating that the enzyme did not leak from the cells during permeabilization.

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Cell-associated GAD activity enhanced times

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Ethyl acetate

Methnol Acetone

0 -1

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Fig. 2. Correlation between hydrophobicity (log P) values of organic solvents and cell-associated GAD activity of BL21 (DE3)-pET28a-gadB exposed for 30 min to 2% solutions of organic solvents.

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Fig. 4. (a) Cell-associated GAD activity of BL21 (DE3)-pET28a-gadB permeabilized at different temperatures. Permeabilization conditions were as follows: 0.1 g dry cells were mixed within 1 mL sodium acetate buffer (0.2 M, pH 4.8) and incubated from 50 to 90 ◦ C for 30 min. (b) Effect of permeabilization time on cell-associated GAD activity of BL21 (DE3)-pET28a-gadB heated at 70 ◦ C. Data represent the mean ± SD from three independent determinations.

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3.3. Permeabilization of BL21 (DE3)-pET28a-gadB using surfactants Surfactants improve cell permeability by disrupting lipid–lipid and lipid–protein interactions. The type of surfactant is crucial for cell permeabilization [30]. E. coli cultures were treated with 2% (v/v) solutions of eight surfactants for 30 min (Fig. 3). Cell-associated GAD activity was increased using Triton X-100, CHAPS, NP-40, OGP, and Brij-35. In contrast, Tween-80 and PEG-100 had no detectable effect. Similar results were obtained using 4% (v/v) surfactant solutions (data not shown). Effective surfactants disrupt hydrophobic interactions between cell envelope components [41]. However, we were unable to correlate the changes in cell-associated GAD activity changes with the properties of surfactants, such as critical micelle concentration and hydrophilic–lipophilic balance. Among the surfactants tested here, Triton X-100 was the most effective (6.96 U/mg, 1% (v/v), 5 min, no leakage, Supplementary Fig. S2), consistent with its effects on other bacterial species and yeasts. For example, treatment of Pichia anomala with 5% Triton X-100 for 30 min enhances phytase activity by 15% [25]. Permeabilization of E. coli O44 K74 with 2% Triton X-100 for 10 min increases crotonobetaine synthesis by 75% [27], and treatment of Yarrowia lipolytica with 1% Triton X-100 enhances citric acid production by a factor of approximately 1.8 [42]. 3.4. Permeabilization of BL21 (DE3)-pET28a-gadB using heat Heating bacteria at certain temperatures denatures membrane proteins and causes the membrane phospholipids to become more

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fluid, leading to an increase in permeability. Recombinant GAD produced by BL21 (DE3)-pET28a-gadB retains significant activity above 60 ◦ C [25]. Therefore, we determined whether heating BL21(DE3)-pET28a-gadB increased cell-associated GAD activity. Cell-associated GAD activity was maximally elevated by a factor of 13.1 (8.38 U/mg, no leakage) compared with the control when the cells were heated at 70 ◦ C for 30 min (Fig. 4a and b). The increased reaction rate observed below 70 ◦ C was likely because of enhanced substrate availability, while activity decreased drastically at higher temperatures because of denaturation of the enzyme.

3.5. Morphology of permeabilized cells We next observed the morphology of cells treated with 0.5% hexane, 1% Triton X-100, and heat. SEM observations revealed nonevident exterior features difference exists between the control and treated cells (Fig. 5), but TME images showed micromorphological differences between control and treated cells (Fig. 6). The membranes of untreated cells were intact with uniformly distributed cytochylema and intracellular electron density. In contrast, ruffled outer membranes and concentrated protoplasts were observed in cells treated with hexane and Triton X-100 (Fig. 6). Heated cells also exhibited concentrated cytoplasms and transparent cell envelopes. The ultrastructural changes of the cell envelope were consistent with increased cell permeability. Moreover, TEM images of treated cells showed neither distinct pores in the membranes nor extracellular leakage of the cytoplasm.

Fig. 5. SEM images of BL21 (DE3)-pET28a-gadB. (a) Untreated or exposed to (b) 0.5% hexane for 5 min, (c) 1% Triton X-100 for 5 min, or (d) 70 ◦ C for 30 min.

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Fig. 6. TEM images of BL21 (DE3)-pET28a-gadB (a) without treatment or exposure to (b) 0.5% hexane for 5 min, (c) 1% Triton X-100 for 5 min, or (d) 70 ◦ C for 30 min.

3.6. Comparison of different permeabilization methods The expression of the Glu-GABA antiporter by E. coli BL21(DE3)pET28a-gadBC and permeabilization of cells using organic solvents, surfactants, and heating facilitated access of GAD to its substrate. Although expression of the Glu-GABA antiporter enhanced cell-associated GAD activity by a factor of 2.6, cell growth was significantly inhibited. We assumed initially that overexpression of the Glu-GABA antiporter would make it possible to avoid permeabilizing the cells, which may be undesirable for processing large-scale cultures [16]. We found that permeabilization of BL21(DE3)-pET28a-gadB with hexane and Triton X-100 for a short time elevated cellassociated GAD activity by more than a factor of 9.5. Hexane is relatively inexpensive and its volatility facilitates its removal from the reaction mixture, and Triton X-100 is also not expensive and relatively safe. Thus, permeabilizing BL21 (DE3)-pET28agadB cells should make scale-up feasible. Nevertheless, using permeabilizing agents, particularly surfactants, may complicate downstream processing and operating safety when potentially hazardous organic solvent are used [16]. We were able to successfully address these concerns by showing that heating BL21 (DE3)-pET28a-gadB at 70 ◦ C for 30 min was the optimum method for increasing cell-associated GAD activity (13.1-fold increase). Moreover, this method is more cost-effective and convenient to perform.

3.7. Effect of the concentration of immobilized heat-permeabilized cells on GAD activity We next determined the GAD activity of different concentrations of heat-permeabilized BL21 (DE3)-pET28a-gadB that were immobilized on Ca-alginate beads. The initial GAD activity of the beads increased as a function of cell density and then plateaued (Fig. 7). GAD activity could not be enhanced effectively when the cell concentration exceeded 9.28 mg/mL. And the special GAD activity of the immobilized cells and immobilized efficiency decreased as a function of cell density (Fig. 7). These findings may be accounted for by exacerbating the obstructive effect of increased tortuosity of the beads, because single or aggregated cells occupied the originally empty pores in the beads [43,44]. Thus, increased cell density likely limited diffusion through the beads and prevented a further increase in GABA synthesis rate and decreased the activity recovery rate of catalysts. Lower cell density with higher activity recovery of catalysts would increase the volume of immobilized beads and the cost of carrier materials. Therefore, we selected a cell concentration of 9.28 mg/mL to prepare the beads. 3.8. Optimization of pH and temperature We next determined the effects of pH and temperature on the GAD activity of immobilized heat-permeabilized BL21(DE3)pET28a-gadB. GAD activity was the highest at pH 4.4–4.8 (Fig. 8a)

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V0 (mM/min)

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60 50 40 30 20 10

with 0.1 mM PLP

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Immobilization efficiency (%)

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Special activity of immobilized cells (U/mg)

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without PLP

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Recycled number

Cell concerctration (mg/mL) Fig. 7. Effect of cell concentration on initial reaction rate (V0 ) of immobilized, permeabilized BL21(DE3)-pET28a-gadB (), specific activity of the immobilized cells (䊉) and immobilization efficiency(). Reactions were performed at 37 ◦ C in sodium acetate buffer (0.2 M, pH 4.8) using one set of beads (0.78–7.74 mg DCW), 75 mM MSG, 0.1 mM PLP with shaking at 100 rpm. Specific activity of the immobilized beads = V0 /the dry weight of immobilized cells. Immobilization efficiency = specific activity of the immobilized cells/cell-associated GAD activity of permeabilized cells.

A 110

and decreased when the pH exceeded 4.8, which is consistent with previous reports showing that GABA biosynthesis is strictly pHdependent [32,33]. Treatment at temperatures from 20 ◦ C to 50 ◦ C (optimum 50 ◦ C) resulted in higher GABA yields, which decreased at higher temperatures (Fig. 8b). These results are similar to those of another study showing that the optimum temperature for the activity of recombinant GAD purified from BL21(DE3)-pET28a-gadB is 48 ◦ C [32].

100

Realtive activity (%)

90 80 70 60 50

3.9. Recycling immobilized heat-permeabilized cells

40 30 20

3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 pH

B 100

Relative activity (%)

Fig. 9. GABA production of 20 sequential batches with immobilized heatpermeabilized BL21(DE3)-pET28a-gadB. Reactions were performed at 50 ◦ C for 4 h in sodium acetate buffer (0.2 M, pH 4.4), one set of beads (4.64 mg DCW), 100 mM L-MSG, 0.1 mM PLP with shaking at 100 rpm. The data represent the mean of three replicates, and the error bars represent the standard deviations.

80 60 40 20

GAD is a PLP-dependent decarboxylase that substitutes a carbonyl group of PLP with an amine group from GABA to produce pyridoxamine-5-phosphate (PMP) and succinic semialdehyde. PMP dissociates from the active site, and causes GAD to become conformationally less stable [44]. The stability of GAD is greatly increased by adding PLP to the reaction mixture [45]. Therefore, we determined the GAD activities of consecutive batches of Ca-alginate gel beads with and without PLP. GAD activity was rapidly lost without adding PLP to the beads (Fig. 9). We attribute this to the stoichiometric transamination of PLP that was covalently bound to the active site of GAD. In the presence of 0.1 mM PLP, 20 batches of beads retained over 60% of their original GAD activity over 80 h under optimum conditions (Fig. 9). Another study shows that 10 batches of sodium alginate and carrageenan gel beads containing GAD retain the ability to produce 50% of the initial level of GABA within 80 h [46]. We conclude that our present data indicate that immobilized permeabilized BL21(DE3)-pET28a-gadB was sufficiently stable and produced GABA at a level suitable for multibatch processing.

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o

T ( C) Fig. 8. (a) Effect of pH on GAD activity of immobilized heat-permeabilized BL21(DE3)-pET28a-gadB. Reactions were performed at 37 ◦ C in sodium acetate buffers (0.2 M, pH 4.0–5.6) using one set of beads (4.64 mg DCW), 75 mM MSG, 0.1 mM PLP, with shaking at 100 rpm. (b) Effect of temperature on the GAD activity of immobilized heat-permeabilized cells. Reactions were performed in sodium acetate buffer (0.2 M, pH 4.4), 75 mM MSG, one set of beads (4.64 mg DCW), 0.1 mM PLP, 25–80 ◦ C with shaking at 100 rpm.

3.10. Stability during storage of immobilized heat-permeabilized cells Stability during storage is a crucial factor for the utility of biocatalysts. In the present study, the immobilized cells were stored at 4 ◦ C, and the GAD activity was tested at 5-day interval for one month. We found that GAD activity remained greater than 90% during 30 days of testing (Fig. 10), indicating that immobilized permeabilized cells were sufficiently stable to allow scale-up.

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References

Relative activity (%)

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5

10

15

20

25

30

Storage days Fig. 10. Stability of GAD activity during storage of immobilized heat-permeabilized BL21(DE3)-pET28a-gadB. The immobilized cells were stored in sterile water at 4 ◦ C, and the GAD activity was tested at intervals of 5 days for 1 month.

4. Conclusions In the present study, we determined whether the expression of the Glu-GABA antiporter (GadC) would increase the cell-associated GAD activity of E. coli BL21(DE3)-pET28a-gadB by enhancing the access of GAD to its substrate. When the Glu-GABA antiporter was expressed in BL21(DE3)-pET28a-gadB, cell-associated GAD activity was elevated by a factor of 2.60; however, cell growth was markedly inhibited. Therefore, we used an alternative approach that involved permeabilizing the cells with organic solvents, surfactants, and heat. Highly hydrophobic (log P > 2) organic solvents increased cell-associated GAD activity. Hexane was the most effective and enhanced cell-associated GAD activity by a factor of 9.65. Triton X-100 was the most effective surfactant and increased cellassociated GAD activity by a factor of 10.8. Heating the cells at 70 ◦ C for 30 min increased cell-associated GAD activity by a factor of 13.1. To avoid the use of organic solvents or surfactants, we immobilized heat-permeabilized cells and found that the GAD activity of these preparations was high and stable during storage. These approaches for improving cell permeability to enhance cellassociated GAD activity show promise for decreasing the cost of producing GAD for use as an industrial catalyst and for producing GABA for pharmaceutical and nutritional purposes. Acknowledgements This work was supported by the grants from the National Natural Science Foundation of China (Nos. 21176220 and 31240054), Zhejiang Provincial Natural Science Foundation (No. Z13B060008), and the Key Technology Research and Development Project of Ningbo (No. 2011C11023). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.molcatb. 2014.05.011.

[1] K. Krnjevic, Physiol. Rev. 54 (1974) 418–540. [2] K. Inoue, T. Shirai, H. Ochiai, M. Kasao, K. Hayakawa, M. Kimura, H. Sansawa, Eur. J. Clin. Nutr. 57 (2003) 490–495. [3] C. Jakobs, J. Jaeken, K.M. Gibson, J. Inherit. Metab. Dis. 16 (1993) 704–715. [4] T. Okada, T. Sugishita, T. Murakami, H. Murai, T. Saikusa, T. Horino, A. Onoda, O. Kajimoto, R. Takahashi, T. Takahashi, J. Jpn. Soc. Food Sci. 47 (2000) 596–603. [5] H. Hagiwara, T. Seki, T. Ariga, Biosci. Biotechnol. Biochem. 68 (2004) 444–447. [6] K. Tujioka, M. Ohsumi, K. Horie, M. Kim, K. Hayase, H. Yokogoshi, J. Nutr. Sci. Vitaminol. 55 (2009) 75–80. [7] K. Tujioka, S. Okuyama, H. Yokogoshi, Y. Fukaya, K. Hayase, K. Horie, M. Kim, Amino Acids 32 (2007) 255–260. [8] C. Wong, T. Bottiglieri, O.C. Snead, Ann. Neurol. 54 (2003) S3–S12. [9] C.H. Oh, S.H. Oh, J. Med. Food 7 (2004) 19–23. [10] H.M. Schuller, H. Al-Wadei, M. Majidi, Carcinogenesis 29 (2008) 1979–1985. [11] T.M. Lammens, M. Franssen, E.L. Scott, Green Chem. 12 (2010) 1430–1436. [12] I. Saskiawan, Microbiol. Indones 2 (2008) 119–123. [13] C. Takahashi, J. Shirakawa, T. Tsuchidate, N. Okai, K. Hatada, H. Nakayama, T. Tateno, C. Ogino, A. Kondo, Enzyme Microb. Technol. 51 (2012) 171–176. [14] J. Huang, M. Le-He, H. Wu, D.Q. Lin, World J. Microbiol. Biotechnol. 23 (2007) 865–871. [15] H. Ueno, J. Mol. Catal. B 10 (2000) 67–79. [16] R.R. Chen, Appl. Microbiol. Biotechnol. 74 (2007) 730–738. [17] M. Kook, M. Seo, C. Cheigh, S. Lee, Y. Pyun, H. Park, J. Korean Soc. Appl. Biol. Chem. 53 (2010) 816–820. [18] K. Park, S. Oh, Biotechnol. Lett. 28 (2006) 1459–1463. [19] S.J. Park, E.Y. Kim, W. Noh, Y.H. Oh, H.Y. Kim, B.K. Song, K.M. Cho, S.H. Hong, S.H. Lee, J. Jegal, Bioprocess Biosyst. Eng. 36 (2013) 885–892. [20] A.Y. Plokhov, M.M. Gusyatiner, T.A. Yampolskaya, V.E. Kaluzhsky, B.S. Sukhareva, A.A. Schulga, Appl. Biochem. Biotechnol. 88 (2000) 257–265. [21] H. Jung, M. Buchholz, J. Clausen, M. Nietschke, A. Revermann, R. Schmid, K. Jung, J. Biol. Chem. 277 (2002) 39251–39258. [22] D. Ma, P.L. Lu, C.Y. Yan, C. Fan, P. Yin, J.W. Wang, Y.G. Shi, Nature 483 (2012), 632-U161. [23] P. Small, S.R. Waterman, Trends Microbiol. 6 (1998) 214–216. [24] Y. Ni, B. Zhang, Z. Sun, Chin. J. Catal. 33 (2012) 681–687. [25] P. Kaur, T. Satyanarayana, J. Appl. Microbiol. 108 (2010) 2041–2049. [26] D. Zehentgruber, C.A. Dragan, M. Bureik, S. Lutz, J. Biotechnol. 146 (2010) 179–185. [27] M. Canovas, T. Torroglosa, J.L. Iborra, Enzyme Microb. Technol. 37 (2005) 300–308. [28] K.O. Choi, S.H. Song, Y.J. Yoo, Biotechnol. Bioprocess Eng. 3 (2004) 147–150. [29] A. Kumar, A. Pundle, J. Mol. Catal. B 57 (2009) 67–71. [30] D.V. Cortez, I.C. Roberto, New Biotechnol. 29 (2012) 192–198. [31] M. Puri, S. Gupta, P. Pahuja, A. Kaur, J.R. Kanwar, J.F. Kennedy, Appl. Biochem. Biotechnol. 160 (2010) 98–108. [32] E.Y. Fan, J. Huang, S. Hu, L.H. Mei, K. Yu, Ann. Microbiol. 62 (2012) 689–698. [33] T.M. Lammens, D. De Biase, M. Franssen, E.L. Scott, J. Sanders, Green Chem. 11 (2009) 1562–1567. [34] H. Birgisson, J.O. Wheat, G.O. Hreggvidsson, J.K. Kristjansson, B. Mattiasson, Enzyme Microb. Technol. 40 (2007) 1181–1187. [35] T.D.L. Vo, J. Ko, S.J. Park, S.H. Lee, S.H. Hong, J. Ind. Microbiol. Biotechnol. 40 (2013) 927–933. [36] S. Wagner, M.M. Klepsch, S. Schlegel, A. Appel, R. Draheim, M. Tarry, M. Hogbom, K.J. van Wijk, D.J. Slotboom, J.O. Persson, J.W. de Gier, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 14371–14376. [37] S. Wagner, L. Baars, A.J. Ytterberg, A. Klussmeier, C.S. Wagner, O. Nord, P. Nygren, K.J. van Wijk, J.W. de Gier, Mol. Cell. Proteomics 6 (2007) 1527–1550. [38] S.J. Osborne, J. Leaver, M.K. Turner, P. Dunnill, Enzyme Microb. Technol. 12 (1990) 281–291. [39] M.V. Flores, C.E. Voget, R. Ertola, Enzyme Microb. Technol. 16 (1994) 340–346. [40] J. Sikkema, J. Debont, B. Poolman, J. Biol. Chem. 269 (1994) 8022–8028. [41] http://www.piercenet.com/ [42] M. Mirbagheri, I. Nahvi, G. Emtiazi, F. Darvishi, Appl. Biochem. Biotechnol. 165 (2011) 1068–1074. [43] K.C. Chen, J.Y. Wu, W.B. Yang, S. Hwang, Biotechnol. Bioeng. 83 (2003) 821–832. [44] J. Oyaas, I. Storro, H. Svendsen, D.W. Levine, Biotechnol. Bioeng. 47 (1995) 492–500. [45] P.L. Grant, J.M. Basford, R.A. John, Biochem. J. 241 (1987) 699–704. [46] Q. Wang, Y.Q. Xin, F. Zhang, Z.Y. Feng, J. Fu, L. Luo, Z.M. Yin, World J. Microb. 27 (2011) 693–700.