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World J Microbiol Biotechnol (2011) 27:693–700 DOI 10.1007/s11274-010-0508-2

ORIGINAL PAPER

Enhanced c-aminobutyric acid-forming activity of recombinant glutamate decarboxylase (gadA) from Escherichia coli Qi Wang • Yinqiang Xin • Feng Zhang • Zhiyong Feng • Jin Fu • Lan Luo • Zhimin Yin

Received: 21 April 2010 / Accepted: 7 July 2010 / Published online: 20 July 2010 Ó Springer Science+Business Media B.V. 2010

Abstract c-aminobutyric acid (GABA) is an important bioactive regulator, and its biosynthesis is primarily through the a-decarboxylation of glutamate by glutamate decarboxylase (GAD). The procedures to obtain GABA by bioconvertion with high activity recombinant Escherichia coli GAD have been seldom understood. In this study, Escherichia coli GAD (gadA) was highly expressed (about 70–75% of total protein) as soluble protein in Escherichia coli BL21(DE3) containing pET28a-gadA, which was induced by 0.4 mM IPTG in LB medium, and maximal GABA-forming activity of the recombinant GAD was 40 U/mL at a concentration (0.15 mM) of pyridoxal phosphate (PLP) and a concentration (0.6 mM) of Ca2? at optimal pH of 3.8. The optimal concentration (7.5 mM) of Mn2? can also improve the activity of recombinant enzyme, but the co-effect of Ca2? and Mn2? exhibited antagonism effect when added simultaneously. LB and 0.1% (w/v) lactose were selected as culture medium and inducer, respectively. The relative activity was markedly higher activated by Ca2? (174%), Mn2? (164%) than that by other seven bivalent cations. Finally, the yield of GABA was high of 94 g/L detected by paper chromatography or HPLC in 1 L reaction system with 30 mL crude GAD (12 U/mL). By

Q. Wang Y. Xin F. Zhang Z. Feng J. Fu Z. Yin (&) Jiangsu Province Key Laboratory for Molecular and Medicine Biotechnology, College of Life Science, Nanjing Normal University, No. 1 Wenyuan Road, Nanjing 210046, Jiangsu, People’s Republic of China e-mail: [email protected] L. Luo (&) State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, People’s Republic of China e-mail: [email protected]

entrapping Escherichia coli glutamate decarboxylase into sodium alginate and carrageenan gel beads, the activity of immobilized GAD (IGAD) remained 85% during the initial five batches and the activity still remained 50% at the tenth batch, these results indicated that the recombinant Escherichia coli GAD was feasible for the future industrial production of GABA. Keywords Escherichia coli c-Aminobutyric acid-forming activity Glutamate decarboxylase Ca2? Pyridoxal phosphate Immobilized glutamate decarboxylase

Introduction GABA, a four-carbon non-protein amino acid, acts as a major inhibitory neurotransmitter in the central nervous system (Krnjevic 1974). GABA has several physiological functions such as neurotransmission, induction of hypotensive effects, diuretic effects, treatment of epilepsy and tranquilizer effects (Jakobs et al. 1993; Cohen et al. 2002; Komatsuzaki et al. 2005). Recent studies showed that GABA was also a strong secretagogue of insulin from the pancreas effectively preventing diabetes (Adeghate and Ponery 2002; Hagiwara et al. 2004). Some reports indicate that GABA is, in microorganisms, functionally involved in the germination of the Bacillus megaterium spore (Foester and Foester 1973). It has also been reported that GABA production confers resistance to an acidic pH in Escherichia coli and Lactococcus lactis (Castanie-Cornet et al. 1999; Sanders et al. 1998; Warnecke and Gill 2005). To date, GABA has been used extensively in pharmaceuticals and functional foods such as gammalone, cheese, gabaron tea, and shochu (Nomura et al. 1998; Sawai et al. 2001; Yokoyama et al. 2002).

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Glutamate decarboxylase (GAD: EC 4.1.1.15) is a pyridoxal 50 -phosphate (PLP)-dependent enzyme, which catalyses the irreversible a-decarboxylation of L-glutamate to c-aminobutyric acid (GABA), and is widely distributed amongst eukaryotes and prokaryotes (Ueno 2000). The Escherichia coli chromosome contains distinct genes encoding two biochemically identical isoforms of glutamic acid decarboxylase, GadA and GadB (De Biase et al. 1999), and the crystal structure and functional analysis for GAD in E. coli has been studied already (Capitani et al. 2003). Some researchers developed some GAD from various microorganisms to produce GABA, but the recovery of GABA from such complex fermentation broth is generally difficult and expensive to perform (Komatsuzaki et al. 2005; Choi et al. 2006). Low activity of cell preparations was the main restriction for commercial enzymatic synthesis until now. Therefore, the study about how to obtain the recombinant enzyme with high GABA-forming activity and low cost is very significant. Due to the increasing commercial demand for GABA, various chemical and biological synthesis methods for GABA have been studied (Plokhov et al. 2000; Komatsuzaki et al. 2005; Choi et al. 2006). Biosynthesis of GABA may be a much more promising method due to simple reaction procedure, high catalytic efficiency, mild reaction conditions and environmental compatibility. Glutamate decarboxylase is the unique enzyme to catalyse the conversion of L-glutamate or its salts to GABA through the single-step a-decarboxylation (Ueno 2000; Battaglioli et al. 2003). Immobilized enzyme technology is a useful method for preparation of metabolic products in the pharmaceutical, food, and other industries. The applications of immobilized enzymes in analytical chemistry have been stimulated because of their well known advantages (Wen et al. 1997; Wu et al. 1995; Xie et al. 1995; Marko-Varga and Dominguez 1991; Bartlett and Cooper 1993; O’Hara et al. 1994; Simonian et al. 1994). It can be advantageous to use immobilized microbial recombinant enzyme with GAD activity, thus avoiding separation and purification of the enzyme, simplifying the product purification process, and enhancing long-term operation stability (Huang et al. 2006). Although the Escherichia coli GAD and GABA synthesis have been profoundly investigated, how to further obtain the recombinant GAD with high GABA-forming activity remains poorly understood. Our study here provided an efficient method to produce recombinant Escherichia coli GAD by using pET system. Our results showed that factors such as pH, bivalent cations and PLP, could play an important roles in enhancing GABA-forming activity of the recombinant Escherichia coli GAD. Immobilized Escherichia coli glutamate decarboxylase (IGAD) provided a good sight for future industrial production of GABA.

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Materials and methods Materials The Escherichia coli K-12, DH5a and BL21 (DE3) were stored in this laboratory at -70 °C. PITC, c-GABA, PLP were obtained from Sigma Chemical Co. L-glutamic acid and L-glutamate sodium were purchased from China Medicine Co. Ltd (Shanghai, China). National 717 anion exchange resin were obtained from Forestry and Biochemistry Institute of Nanjing Forestry University. Carrageenan, Sodium Alginate were the products of Sinopharm chemical reagent Co. Isopropyl-b-D-thiogalactoside (IPTG) was from Amersco. Lactose was from Xilong Pharceutical Co (Guangxi, China). Peptone, Yeast extract were produced by England OXOID LTD. Restriction endonucleases were purchased from New England Biolabs and T4 DNA ligase from Fermentas. Pfu polymerase and SDS–PAGE protein marker were obtained from Takara. The pET28a, Ni–NTA-his resin and Anti-His6 monoclonal antibody were products of Novagen. Mouse IgG secondary antibodies was obtained from Calbiochem. Coomassie brilliant blue R-250 was from Bio-Rad. The other reagents were of the highest grade commercially available. Plasmid construction, heterologous expression and purification of recombinant protein Escherichia coli glutamate decarboxylase gene (gadA) was amplified by PCR using primers FW: 50 - TCAGGA CATATG ATG GAC CAG AAG CTG TTA ACG-30 and RV: 50 - ACTGAG GGATCC TCA GGT GTG TTT AAA GCT GTT-30 from E. coli K-12 according to sequences reported in EMBL data bank under accession NO. EG50009. The amplified products were digested by Nde I and BamH I and then ligated into the corresponding sites of the expression plasmid pET28a, which produced an open frame encoding 19 amino acids containing hexahistidine tag at the end of N-terminal of recombinant GAD. The recombinant plasmid was identified by PCR and restriction endonuclease (Nde I and BamH I) digestion, and the sequence of gadA gene inserted into pET28a was proved to be the same as that reported in the data bank by DNA sequencing (by Shanghai invitrogen bio.co.Ltd). BL21(DE3)-pET28a-gadA strain was obtained by transforming the recombinant plasmid into BL21 (DE3) strain to express His6-tagged GAD. BL21(DE3)-pET28a-gadA strains were cultured in a sterilized Luria–Bertani (LB) medium at pH 7.0. When the cultures reached an OD600 of *0.5, 0.4 mM IPTG or 0.1% (m/v) lactose was added to induce the recombinant GAD expression. Cultures were collected by centrifugation at 11,627 g, at 4 °C for 10 min after growing at 37 °C for induction of 8 h. The cell pellet from 200 mL E. coli cell


culture was washed twice and suspended in chilled potassium phosphate buffer (PBS, pH 7.0) to reach cell concentration at 30 mg/mL. The suspended solution was disrupted with ultrasonication at 20 kHz for 20 min. Debris were removed by centrifugation at 16,743 g, at 4 °C for 15 min. The supernatants (crude GAD) were subjected to IDA-Ni2? affinity column (Novagen) according to the manufacture’s instruction. Purified GAD was dialyzed against 2 L of PBS buffer (pH 7.0). Unless otherwise indicated, all purification procedures were carried out either at 4 °C or on ice. SDS–PAGE, western blotting analysis SDS–PAGE (Bio-RAD, USA) was used for the recombinant GAD expression analysis of BL21-pET28a-gadA according to Laemmli. The protein samples were mixed with equivalent 29 sample buffer (125 mM Tris–HCl, pH 6.8, 20% glycerol, 0.005% bromophenol blue) and were denatured by SDS, electrophoresed on a 12% SDS–polyacrylamide gel at 4 °C, 135 V for 1.5 h, and the proteins were detected by Coomassie brilliant blue R-250 staining. The proteins were also transferred to a PVDF membrane for Western blotting analysis, which was carried out as the method literature (Sambrook et al. 1989). Determination of GABA-forming activity of GAD GABA-forming activity of recombinant GAD was measured by estimating of the amount of GABA which was determined by HPLC. One unit (U) of GABA-forming activity was defined as the amount of enzyme that liberates 1 lmol GABA per minute under the following activity assay mixture. To start the reaction, an aliquot of the enzyme solution was added into the reaction mixture in a total volume of 1.0 mL, contained 31 g/L sodium glutamate, 200 mM HAc-NaAc buffer and 0.15 mM PLP or 0.6 mM Ca2? or 7.5 mM Mn2? or 7.5 mM other bivalent cations at pH 3.8. After being incubated at 37 °C for 30 min, the reaction was terminated by immersing the sample tube in boiling water for 3 min. The supernatant was obtained by centrifugation at 1,046 g, for 3 min at room temperature, which was submitted to HPLC assay procedure then. Protein concentration was determined by Bradford. Synthesis and purification of GABA products The 1 L reaction system (150 g/L glutamate, 0.6 mM Ca2?, 0.15 mM PLP, 200 mM HAc-NaAc buffer of pH 3.8 at 37 °C,30 mL crude GAD/12 U/mL) was tested the GABA production at 37 °C, finally. The crude GABA was obtained by vacuum concentration drying (60 °C, RE-3000A Rotary Evaporator, Shanghai. Yarong Instruments Company, China)

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of the reaction supernatants after ion exchange experiments, then by integrating alcohol-adding (two times volume of the crude GABA) with cooling (0–4 °C) and salting out to crystallize colorless columnar crystal of GABA. Immobilized glutamate decarboxylase (IGAD) and its application for determination of GABA The preparation of crude GAD followed the method above. The concentration of the GAD protein was measured by Bradford. Factors which affected the activity of IGAD and optimum reaction conditions of IGAD were investigated (data not shown). The mixed solution of sodium alginate (25 g/L) and carrageenan (10 g/L) were stirred and heated in magnetic stirring apparatus to melt absolutely, crude GAD were added to the mixed solution using injector when the solution cooled and mixed well then. The mixed solution was added drop by drop into calcium chloride solution (0.2 M). The gel beads thus formed through continuously stirring in calcium chloride solution for 30 min. Then the beads were collected by filtration, rinsed several times with acetate buffer solution (0.2 M, pH 3.8) and stored in the distilled water at 4 °C. The beads with a mean diameter (Dm) of about 2.2 mm were prepared without tails. IGAD activity was assayed according to enzyme reaction by measuring the amount of GABA synthesized from sodium glutamate as substrate. The reaction mixture contained IGAD, 0.2 M HAc-NaAc buffer at pH 3.8 and sodium glutamate solution with the concentration of 31 g/L. The reaction was performed at 37 °C, 130 rpm. Paper chromatography and HPLC assay of GABA Paper chromatography assay was performed according to normal procedure (Woolfolk et al. 1966). HPLC (Agilent 1100 series, USA) system equipped with a PicoTagTM Amino Acid column (300 9 3.9 mm, 5 lm, Waters, USA) was used for determining the GABA synthesized by GAD. The HPLC conditions were flow rate of 1.0 mL min-1, UV detection at 254 nm and ambient column temperature. A gradient elution program with A (methanol)/B (tetrahydrofuran/methanol/50 mM pH 6.2 sodium acetate, 5:75:420, by vol), as mobile phase was described as Chen et al. (1997). The retention time of GABA was 19.5 min. The samples from reaction mixture were operated as the procedure by Agilent. The derivatization product (PTC-GABA) was detected by a Diode Array Detector (Agilent, USA) as the absorbance at 254 nm. Statistical analysis A one-way analysis of variance was used to examine the statistical significance between the sample performances,

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using an Origin Pro 8.0 Program Package. Data from experiment assays were obtained from three replicates, and reported as the mean value ± standard deviation (SD).


Effect of divalent cations and pH on GABA-forming activity of the recombinant GAD

The recombinant GAD plasmid exhibited a high expression up to 70–75% of total cellular protein (densitometry was performed using Scion Image Analysis Software). The recombinant GAD was totally soluble in BL21 (DE3) after 8 h of induction with 0.4 mM IPTG or 0.1% (m/v) lactose by SDS–PAGE analysis. The calculated molecular weight of the recombinant GAD was about 53.5 kDa, which was consistent with the result of SDS–PAGE measurement (Fig. 1). Considering the high toxicity of IPTG, lactose was selected as the inducer. To further identify that the target band in SDS–polyacrylamide gel was the recombinant GAD, we detected it by Western blotting using His-Tag Monoclonal Antibody against hexahistidine-tagged GAD. According to the Western blot analysis, a band related to the predicted size of the protein, which was detected only in induced BL21 (DE3) transformed with pET28a-His6-gadA construct. The recombinant GAD protein started to express at 3 h and reached its maximum at 6 h after induction. The target proteins had not been degraded with time passing. The result of Western blot showed that the target band was indeed the recombinant GAD (data not shown).

A unique feature of plant and yeast Gad is the presence of a calmodulin (CaM)-binding domain in the C-terminal region (Coleman et al. 2001). In plants, Gad is thought to be a stress-adapter chaperonin sensing Ca2? signals. Removal of the CaM binding domain causes a deregulation of the activity, leading to severe developmental problems (Baum et al. 1996). Bacterial Gad has some features similar to those of the plant enzyme (Sukhareva et al. 1994). Besides, acknowledged as an essential trace element, Mn2? is generally accorded importance in the cellular physiology (David and Michael 2003). To know whether the GABAforming activity of the recombinant GAD from Escherichia coli activated by Ca2? or Mn2? was similar to GAD activity of plants and bacteria, we first investigated the optimal pH for the recombinant GAD activated by Ca2? or Mn2? at range of pH from 2.6 to 5.4 (Fig. 2). Activity of the recombinant GAD activated by Ca2? was markedly higher than that by Mn2? at almost whole range of pH from 2.6 to 5.4. The optimum pH for the recombinant Escherichia coli GAD activated by Ca2? was the same as that of the Mn2? (with the optimum at pH 3.8), the results were almost the same as those reported by Capitani et al. (2003) and Matsumoto et al. (1986), but the optimum pH of E. coli GAD was a little lower than those of GAD reported in plants and animals (Zhang et al. 2007; Matsumoto et al. 1986; Satyanarayan and Nair 1985). Thereby, it seems that some properties of the recombinant GAD in Escherichia coli is different from those of the GAD in plants and animals, although the recombinant GAD of bacterial bears

Fig. 1 SDS-PAGE analysis the heterologous expression of the recombinant GAD from LB (induced by 0.1% lactose) compared with that from LB (induced by 0.4 mM IPTG). M, protein MW marker; lines 1 and 2, lysates of BL21-pET28a, BL21-pET28a-gadA not induced grown in LB, respectively; line 3 lysates of BL21pET28a-gadA in LB and induced by 0.4 mM IPTG; line 6 lysates of BL21-pET28a-gadA in LB and induced by 0.1% lactose; lines 4 and 7, supernatants of lines 3 and 6 after sonication, respectively; lines 5 and 8, precipitations after sonication of lines 3 and 6, respectively; line 9 purified GAD. Arrow indicated the position of target protein

Fig. 2 Influence of pH on c-aminobutyric acid-forming activity of the recombinant GAD in the presence of 7.5 mM Mn2?(filled square) or 0.6 mM Ca2?(filled triangle). The assay mixture contained 31 g/L sodium glutamate, 7.5 mM MnCl2 or 0.6 mM CaCl2, 200 mM HAcNaAc buffer, pH 2.6–5.4 at 37 °C. Velocity is given in relative activity (set the maximal value tested as 100, the results are the mean values obtained from three replicates, and the error bars represent the standard deviations)

Results and discussion Heterologous expression analysis and identification of recombinant E. coil GAD

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greater sequence similarity to plant than to animal GAD (Baum et al. 1993). Seven other cations at 7.5 mM were also tested for the maintenance of the activity of the recombinant GAD at the optimum pH 3.8. The activity activated by seven cations were compared with that supported by Mn2? and Ca2? (Fig. 3). The result showed that most of the cations were less efficient in the maintenance of the recombinant GAD activity than Ca2? (44–50% of that for Ca2?) and Ni2?, Co2?, Ba2? showed some inhibitory effect on the activity of the recombinant enzyme compared with the control. The activity of GAD from Escherichia coli was known to depend upon the concentrations of Ca2? or Mn2?. It is considered that Ca2? plays a central role in intracellular signal transduction pathways. The response to various stimuli is mediated by calcium-binding proteins such as calmodulin (CaM) which translates a transient calcium signal into a variety of cellular processes (Weinstein and Mehler 1994; Crivici and Ikura 1995). Some reported that plants GAD could be stimulated in the presence of calcium/ calmodulin at its optimum pH, activity of purified recombinant plant GAD was essentially inactive in the absence of calcium and calmodulin, but it could be stimulated to high levels of activity by the addition of exogenous calmodulin in the presence of calcium (Baum et al. 1993; Zhang et al. 2007; Snedden et al. 1996). The yeast GAD also bounded to calmodulin as did the plant enzyme, suggesting a conservation of calcium regulation of this protein (Coleman et al. 2001). Ca2? showed positive effect on the activity of lactic acid bacterium GAD even when the concentration was under 100 lmol (Liu et al. 2005). In this study, we also found that purified recombinant Escherichia coli GAD activity was significantly affected by the Ca2?. The GAD activity exhibited a maximal value of its relative activity at

174% at the optimum pH after the addition of 0.6 mM of Ca2?(date not shown). The results of Ca2?-supported activity is almost the same as that reported from rice germ (Zhang et al. 2007). It has been reported that the importance of glutamate and GABA transport and regulation involved the brain GAD mRNA expression in the aged manganese-treated rats (Fitsanakis and Aschner 2005). To elucidate whether the Mn2? had the effect on the activity of purified recombinant Escherichia coli GAD, different concentrations of Mn2? have been chosen to investigate the GABA-forming activity of the purified recombinant GAD. The maximum of Mn2?-supported GAD activity (164%) was achieved at 7.5 mM and decreased sharply with the concentration of Mn2? increased, the optimal concentration of Mn2? was obviously higher than that of Ca2?(date not shown). Our results showed that the Escherichia coli GAD was a Ca2?-enzyme. Mn2? or Ca2? was known to be ion activator of Escherichia coli GAD on GABA-forming. Here, the co-effect of Ca2? and Mn2? on the recombinant GAD was further investigated when added simultaneously. As shown in the Fig. 4, the activation effect of Ca2? was obviously inhibited with the concentration of the Mn2? increased. It showed that the compound of Mn2?, Ca2? exhibited antagonism effect on Escherichia coli GAD activity, thus only Ca2? ion was selected as the activator in our experiment.

Fig. 3 Effect of bivalent cations on c-aminobutyric acid-forming activity of the recombinant GAD. The assay mixture contained 31 g/L sodium glutamate, 0.6 mM Ca2? or 7.5 mM other bivalent cations, 200 mM HAc-NaAc buffer (pH 3.8) at 37 °C. Velocity is given in relative activity (set the maximal value tested as 100, the control value was the GAD relative activity that Ca2? and Mn2? not added in the reaction system, the results are the mean values obtained from three replicates, and the error bars represent the standard deviations)

Fig. 4 Co-effect of Ca2? and Mn2? on the recombinant GAD when added simultaneously. The assay mixture contained 31 g/L sodium glutamate, 0–30 mM Mn2? when the concentration of Ca2? was fixed (0.6 mM), 200 mM HAc-NaAc buffer (pH 3.8) at 37 °C. Velocity is given in relative activity (set the GAD relative activity value of Ca2? and Mn2? not added in the reaction system as 100, the results are the mean values obtained from three replicates, and the error bars represent the standard deviations)

Effect of PLP on Escherichia coli GAD activity Glutamate decarboxylase from Escherichia coli requires pyridoxal-P for activity (Yamada and O’Leary 1977). To estimate the PLP effect on GAD biosynthesis, we

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Fig. 5 Effect of the PLP concentrations on the GAD reaction rate in the presence of a series concentration of PLP (0–0.3 mM). The assay mixture contained 31 g/L sodium glutamate, PLP (0–0.3 mM), 200 mM HAc-NaAc buffer (pH 3.8) at 37 °C. Velocity is given in relative activity (set the control value that PLP not added as 100, the results are the mean values obtained from three replicates, and the error bars represent the standard deviations)

investigated the optimal concentration effect of PLP on improving the activity of recombinant Escherichia coli GAD. As expected, PLP addition effectively enhanced GAD activity (Fig. 5). The maximum of PLP-supported GAD activity(198%)was achieved at 0.15 mM and gradually decreased when the concentration of PLP was above 0.15 mM. Previous studies showed that PLP plays an important role in stimulating GAD activity as a cofactor of the enzyme (Tong et al. 2002), and that addition of PLP to the culture medium might influence GABA production (Komatsuzaki et al. 2005). GAD activity in the spore of Aspergillus oryzae was increased 40-fold by addition of PLP (Noriyoshi and Katanori 2002). An increase of PLP concentration also favoured activity of GABA-transaminase, which was also dependent on PLP concentration and converted GABA (Satyanarayan and Nair 1990). The optimal concentration of PLP (50 lM) enhanced GAD activity in germinated millet by 32% compared with the control, whose activity is also decreased by high concentration (above 50 lM) of PLP (Bai et al. 2009). Cell growing in the presence of 0.02 mM pyridoxal phosphate (PLP) causes the 2- to 2.5-fold increase of total productivity of the gene-engineered GAD superproducer strain of Escherichia coli GADK10 cells (Plokhov et al. 2000). In our experiment, PLP also had an important effect on regulation of the Escherichia coli GAD activity, and the results demonstrated that the Escherichia coli GAD was a PLP-dependent enzyme. GABA synthesis with recombinant GAD To investigate whether it was feasible for future industrial production of GABA by the recombinant GAD, the 1 L

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Fig. 6 Paper chromatography analysis of c-aminobutyric acid synthesis by crude GAD from LB. The reaction system (1 L) containing 150 g/L glutamate, 0.6 mM Ca2?, 0.15 mM PLP, 200 mM HAcNaAc buffer of pH 3.8 and 0 or 30 mL crude GAD from BL21pET28a-gadA grown in LB was carried out at 37 °C, with shaking (200 rpm) for 24 h. 0, without GAD; 1,3, 5,7,10,13,16,24 h, reaction time last 1–24 h, respectively; L-Glu (glutamate), 0.15 M; c-GABA (aminobutyric acid), 0.1 M

reaction system (details are described in the legend) was tested. Glutamate was added each 2 h, GABA content was about 94 g/L after 24 h reaction, and the conversion of glutamate was about 90% (Fig. 6). GABA-producing ability of recombinant Escherichia coli GAD was apparently higher than those reported by Komatsuzaki et al. 2005 and Yokoyama et al. 2002. This result demonstrated that the recombinant GAD was applicable for large scale GABA production. Repeated batch process of IGAD The Escherichia coli GAD was immobilized according to the materials and methods. The half-life of the activity is 127 days when immobilized L-glutamic decarboxylase (GDC) was stored in the cold (4 °C) by using a new biosensor (Ling et al. 2000). The yield of GABA still remained 56% in the tenth batch by entrapping Lactobacillus brevis cells with higher glutamate decarboxylase (GAD) activity into Ca-alginate gel beads (Huang et al.

Fig. 7 IGAD activity of sequential 10 batches operation. Biotransformation of each batch was carried out with HAc-NaAc buffer (200 mM, pH 3.8), 31 g/L sodium glutamate, 0.6 mM Ca2?, 0.15 mM PLP and a 130 rpm min-1 shaking speed at 37 °C for 8 h (set the IGAD activity of first biotransformed as 100, the results are the mean values obtained from three replicates, and the error bars represent the standard deviations)


2006). In our results, the stability of IGAD was examined. The activity of IGAD reached about 85% during five cycles under the optimized reaction conditions and IGAD activity remained above 70% of original activity after eight times recycling, it dropped to 50% at the tenth repeat (Fig. 7). These data showed that the IGAD were stable and efficient for GABA production in multi-batch processes.

Conclusions Our study revealed the effect of Ca2?, Mn2? as well as other seven bivalent cations on the GABA-forming activity of the recombinant GAD from Escherichia coli. The results showed that the recombinant GAD was highly expressed (70–75%) and totally soluble. PLP was the coenzyme of the Escherichia coli GAD and played an important role in the activity of recombinant enzyme. Ca2? was much more efficient to activate GABA-forming of the recombinant protein than other bivalent cations and the optimum pH was at pH 3.8 for recombinant Escherichia coli GAD. The yield of GABA was high of 94 g/L in the 1 L reaction system with 30 mL crude GAD (12 U/mL) from LB and 150 g/L glutamate as substrate. The stability of IGAD has been examined, the use of IGAD by carrageenan and sodium alginate entrapment offered a promising means of GABA production for industrial application. Anyway, a procedure was established to get a recombinant GAD with high GABA-forming activity (40 U/mL) in LB medium, which eventually can be used for future large scale production of GABA. Acknowledgments This work was supported by the special funding from NJNU for talent faculty. We thank zhili Liu, associate professor, for performing the HPLC analysis.

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