l-Theanine Synthesis Using Î³-Glutamyl Transpeptidase from Bacillus

Article pubs.acs.org/JAFC

L‑Theanine

Synthesis Using γ‑Glutamyl Transpeptidase from Bacillus licheniformis ER-15

Shruti Bindal and Rani Gupta* Department of Microbiology, University of Delhi, South Campus, New Delhi 110021, India S Supporting Information *

ABSTRACT: Recombinant γ-glutamyl transpeptidase (rBLGGT) from Bacillus licheniformis ER-15 was purified to homogeneity by ion-exchange chromatography. Molecular masses of large and small subunits were 42 and 22 kDa, respectively. The enzyme was optimally active at pH 9.0 and 60 °C and was alkali stable. Km and Vmax for γ-glutamyl-p-nitroanilide hydrochloride were 45 μM and 0.34 mM/min, respectively. L-Theanine synthesis was standardized using a one variable at a time approach followed by response surface methodology, which resulted in approximately 85−87% conversion of L-glutamine to L-theanine within 4 h. The standardized reaction contained 80 mM L-glutamine, 600 mM ethylamine, and 1.0 U/mL rBLGGTin 50 mM Tris-Cl (pH 9.0) at 37 °C. Similar conversions were also obtained with the enzyme immobilized in calcium alginate. Using immobilized enzyme, 35.2 g of L-theanine was obtained in three cycles of 1 L each. The product was purified by Dowex 50W X 8 hydrogen form resin and was confirmed by HPLC and proton NMR spectroscopy. KEYWORDS: theanine, γ-glutamyl transpeptidase (GGT), Bacillus, process optimization, RSM, immobilization

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INTRODUCTION L-Theanine, chemically named 2-amino-4-(ethylcarbamoyl) butyric acid, is a FDA-approved food supplement and a popular additive in health drinks. It is known for its various physiological and therapeutic benefits on human health,1 as in cases of cancer2,3 and Alzheimer’s disease.4 Hence, the demand for Ltheanine is expected to rise in the future. Theanine is largely extracted from the leaves of Camellia sinensis (green tea) and also synthesized chemically5,6 to meet the current demand. However, chemically synthesized theanine is often not accepted as a food additive as it is a racemic mixture of L- and D-forms and also its synthesis is more expensive. Hence, investigations were further shifted to enzymatic synthesis as an alternative for stereoselective production of L-theanine.1 Enzymes that are able to transfer a γ-glutamyl moiety to ethylamine find application in the synthesis of theanine. Three enzymes have shown potential, namely, glutamine synthetases (EC 6.3.1.2), glutaminases (EC 3.5.1.2), and γ-glutamyl transpeptidases (GGT; EC 2.3.2.2). The use of glutamine synthetases in industries is limited due to ATP requirement, the low reactivity of ethylamine with the enzyme, and difficulty in maintaining reaction conditions.7 Glutaminases could have been the second preference, but their low conversion rates due to poor transpeptidase to hydrolysis ratio8−10 make the process a costly affair. These limitations in the biosynthesis of L-theanine have been overcome to a major extent by the use of GGT. GGTs have been reported to give higher conversion rates of 60−95% using Lglutamine or L-glutamic acid as γ-glutamyl donors.11−15 However, optimization of the process parameters, maximizing the yield and minimizing the production cost, are the major constraints that have to be looked into to make the biosynthesis of theanine a feasible process for industrial-scale production. Here we present a statistical process optimization for the biosynthesis of L-theanine using recombinant GGT from Bacillus licheniformis ER-15 (rBLGGT). Effects of various process © XXXX American Chemical Society

parameters, such as concentration of substrates and enzyme and incubation time, on theanine synthesis were studied. Furthermore, the process was made cost-effective by enzyme recovery through immobilization along with better conversion rate.

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MATERIALS AND METHODS

Materials. Luria−Bertani (LB) medium for the cultivation of Escherichia coli was acquired from Difco Laboratories (USA). QSepharose resin, Dowex 50W X 8 hydrogen form resin, L-theanine, and glutathione (oxidized) were purchased from Sigma-Aldrich (Bangalore, India), L-Glutamine was from SRL (Mumbai, India), and ethylamine was acquired from Loba Chemie (Mumbai, India). All other chemicals used in the study were commercial products of either chemical or biological grade. Escherichia coli BL21 harboring a pET 51b vector containing ggt gene (GI: 162708169) from B. licheniformis ER-15 was procured from the laboratory. The construct was designed such that only the N-terminus was tagged with Strep-tag (see details in Figure S1 in the Supporting Information). Wild GGT from B. licheniformis ER-15 was produced by cultivating the cells in basal medium (Supporting Information Table S1) and purified using Q-Sepharose anion-exchange resin. rBLGGT Production and Purification. rBLGGT was produced by cultivating the recombinant E. coli BL21 cells in 50 mL of LB medium containing 100 μg/mL ampicillin at 37 °C and 200 rpm to an absorbance of 0.5 OD600. Isopropyl-β-D-thiogalactopyranoside (IPTG) was then added to a final concentration of 0.1 mM, and incubation was continued under the same conditions for 3 h. E. coli cells were then harvested by centrifugation at 7441g for 10 min at 4 °C and were resuspended in 10 mL of 10 mM Tris-HCl buffer (pH 9.0). rBLGGT was extracted by disrupting the cells by sonication (VCX 750, Sonics and Received: January 7, 2014 Revised: August 14, 2014 Accepted: August 22, 2014

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dx.doi.org/10.1021/jf5022913 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

Substrate Specificity for γ-Glutamyl Acceptors. Substrate specificity of rBLGGT toward various amino acids as γ-glutamyl acceptors, namely, alanine, threonine, leucine, methionine, phenylalanine, glycine, asparagine, isoleucine, tryptophan, glutamine, glutamic acid, aspartic acid, serine, histidine, valine, arginine, and lysine, was studied considering activity with glycylglycine, as an acceptor, as 100%. Effect of Metal Ions and Inhibitors. The effect of various metal ions on enzyme activity was studied by pre-incubating the enzyme in the respective metal ion solution (pH 9.0) for 5 min and then performing the assay as mentioned. Enzyme was investigated for nine different metal ions, namely, Co2+, Cu2+, Ca2+, Cd2+, Mn2+, Mg2+, Ni2+, Zn2+, and Pb2+, at a final concentration of 5 mM in the reaction. The influence of various inhibitors, for example, phenylmethanesulfonyl fluoride (PMSF), Nbromosuccinamide (NBS), bromoacetic acid, ethylenediaminetetraacetic acid (EDTA), β-mercaptoethanol, idoacetamide, sodium azide, iodoacetic acid, dithiothreitol, and 1,10-phenanthroline, on GGT activity was also investigated along with some specific inhibitors of GGT such as 6-diazo-5-oxo-L-norleucine (DON) and azaserine. Theanine Biosynthesis: Optimization of Reaction Parameters. Initially, theanine synthesis was determined in a 5 mL reaction containing 20 mM L-glutamine (donor) and 200 mM ethylamine (acceptor) at pH 9.0 and 37 °C using 0.4 U/mL rBLGGT for a period of 6 h to select an appropriate time for further optimization. Reactions were stopped by an equal volume of 0.5% (v/v) trifluoroacetic acid (TFA) and were then filtered through a 0.2 μm filter. The products were analyzed with a Shimadzu high-performance liquid chromatograph (HPLC) equipped with a C18 reverse phase column (Luna 5 μm C18 (2); dimensions, 250 × 4.6 mm). L-Theanine was detected according to the method of Zhang19 with a few modifications in the mobile phase: 0.05% (v/v) TFA in water; flow rate, 0.5 mL/min; oven temperature, 40 °C. UV−vis absorbance detection was done at 203 nm without any derivatization, and the L-theanine peak was identified by running commercial standards. However, a measurable amount of L-theanine was obtained within 1 h of the reaction; thus, further parameters, such as the concentrations of donor, acceptor, and enzyme, were optimized using a one variable at a time approach for a 1 h reaction at pH 9.0 and 37 °C. One Variable at a Time Approach. Ethylamine concentration was varied from 40 to 400 mM in a reaction containing 20 mM L-glutamine and 0.4 U/mL rBLGGT in the above specified conditions. Similarly, Lglutamine (10−100 mM) was varied at a constant concentration of ethylamine (200 mM) and rBLGGT (0.4 U/mL). The effect of enzyme concentration was also studied in reactions containing 40 mM Lglutamine and 200 mM ethylamine at pH 9.0 and 37 °C by varying its concentration from 0.1 to 1.0 U/mL. All of the reactions were done in triplicate, and theanine yield was determined after 1 h of incubation according to the method described above. Statistical Approach: Response Surface Methodology (RSM). The interaction pattern of three main components of L-theanine synthesis reaction, namely, L-glutamine (X1), ethylamine (X2), and rBLGGT (X3), was studied by employing RSM strategy. The experiment was designed using the Box−Behnken model following Design Expert software (version 6.0.10). Each of the independent variables was studied at three different coded levels of −1, 0, and +1, resulting in 17 sets of experiments (5 replications of the central run). The actual and coded forms with respect to the full experimental plan of all three variables are listed in Table 1. L-Theanine yield (Y1) and percent conversion (Y2) of L-glutamine to L-theanine were taken as the two responses for this study. All of the reactions were performed in 50 mM Tris-HCl buffer (pH 9.0) and were incubated at 37 °C for 4 h. A second-

Materials Inc., Newtown, CT, USA; 750 W power output with frequency of 20 kHz ± 50 Hz) using a sonication probe of 13 mm tip diameter for 5 min with on/off pulse of 5 s. The cell lysate was then clarified by centrifugation at 11627g for 20 min. The processing temperature was maintained at 4 °C throughout. Subsequently, the crude extract was concentrated by acetone precipitation (1:1, v/v) and purified by Q-Sepharose anion-exchange chromatography (column dimensions, 8 cm × 1.5 cm; flow rate, 3 mL/min) using 10 mM TrisHCl buffer (pH 9.0). Protein was eluted with a 0.1−3 M NaCl gradient. Five milliliter fractions were collected, and their protein content (at 280 nm) and GGT activity (at 410 nm) were determined. GGT Assay. GGT assay was performed according to the method described by Ikeda.16 One milliliter of reaction volume contained 1 mM γ-glutamyl-p-nitroanilide hydrochloride (γ-GpNA), 10 mM glycylglycine (excluded in hydrolytic reactions), and appropriately diluted enzyme. Unless otherwise indicated, GGT assay was performed in 50 mM Tris-HCl buffer (pH 9.0) at 60 °C for 5 min. Reaction was stopped by adding 100 μL of 3 M acetic acid, and the activity was monitored by measuring hydrolyzed γ-GpNA at 410 nm. One enzyme unit was defined as the amount of enzyme required to release 1 μmol of pnitroaniline per minute under standard assay conditions. End product inhibition was studied by adding L-theanine (10−40 mM) to the reaction while the GGT assay was performed and monitoring the change in transpeptidase activity. Electrophoresis, Western Blotting, and Activity Staining. SDS-PAGE of purified rBLGGT along with the crude enzyme was carried out according to the procedure of Laemmli17 to determine the molecular mass and subunit composition of the enzyme. A 12% (v/v) resolving gel stacked with a 5% (v/v) stacking gel was run at 20 mA for approximately 1 h using a Bio-Rad power pac. Gels were then stained with 0.25% (w/v) Coomassie Brilliant blue R-250 at mild shaking and destained thereafter. Western blot analysis was done by transferring the protein to a PVDF membrane (Bio-Rad). The membrane was then blocked for 1 h in 3% (w/v) BSA prepared in 1× PBS and thereafter incubated with 1:5000 streptactin antibody conjugated with horseradish peroxidase (HRP) for 1 h on mild shaking at room temperature. Subsequently, the membrane was washed with 0.05% (v/v) Tween-20 in 1× PBS. The protein band was then visualized using diaminobenzidine (DAB, 0.1% w/v) solution prepared in hydrogen peroxide. Native PAGE (10% v/v) was prepared, and purified rBLGGT was loaded in two wells of the gel along with native molecular markers and was run at 20 mA for 1 h. Thereafter, one of the lanes containing rBLGGT sample was sliced using a scalpel and subjected to activity staining, whereas the remaining gel was stained with 0.25% (w/v) Coomassie Brilliant blue R-250 to analyze the molecular mass of the native protein. For acitivity staining, the gel piece was first equilibrated at 50 mM Tris-HCl buffer (pH 9.0) for 10 min and then placed over an agar plate containing the substrates (γ-GpNA and glycylglycine). Gel was incubated at 45 °C until the yellow color of p-nitroaniline appeared. To intensify the stain, gel was further treated with N-(1-naphthyl)ethylenediamine employing the method described previously.18 Biochemical Characterization of rBLGGT. Effect of pH and Temperature. GGT assay was performed at various pH values (5.0− 12.0), using universal pH buffer (Britton−Robinson buffer) to determine the effect of pH on transpeptidase activity. pH stability was studied by preincubating the enzyme at pH 5.0−12.0 (10 mM) for 1 h and then performing the assay under standard conditions. The effect of temperature on enzyme activity and stability was determined by incubating the reaction at various temperatures ranging from 40 to 80 °C. All of the experiments were performed in triplicate. Relative and residual enzyme activities were plotted, and the activity obtained under standard assay conditions was regarded as 100%. Steady-state kinetics of purified rBLGGT was performed with various concentrations of γ-GpNA ranging from 0.016 to 0.2 mM at its optimal pH and temperature. The Km and Vmax values were calculated using a Lineweaver−Burk plot (graph was plotted using Systat Software, Inc., SigmaPlot for Windows).

Table 1. Experimental Ranges and Levels of the Three Independent Variables Used in RSM in Terms of Actual and Coded Factors variable L-glutamine

(mM) ethylamine (mM) rBLGGT (U/mL) B

actual

coded

20 200 0.5

−1 −1 −1

actual

coded

actual

coded

80 400 1.0

0 0 0

140 600 1.5

+1 +1 +1



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Figure 1. (A) Elution profile of rBLGGT from Q-Sepharose fast flow column pre-equilibrated with 10 mM, pH 9.0, Tris-Cl buffer (fractions 21, 22, and 23 were pooled together for biochemical characterization). (B) Native-PAGE (lane 5) and zymogram of purified rBLGGT (native protein markers used; lanes: 1, catalase, 240 kDa; 2, bovine albumin, 67 kDa; 3, egg albumin, 43 kDa; 4, trypsin soybean inhibitor, 20.1 kDa). (C) SDS-PAGE and Strep-tag Western blot of rBLGGT (lanes: 1, SDS protein marker; 2, crude cell lysate; 3, acetone precipitated fraction; 4, purified rBLGGT). degree polynomial equation was generated using a modified quadratic model

were calculated by monitoring the decrease in substrate concentration with time, and thus units were defined as micromoles of GSSG utilized per minute under standard assay conditions. GSSG peak was analyzed on HPLC using the same protocol as employed for theanine detection. Assuming equal distribution of the enzyme in the beads, theanine synthesis reaction was carried out by adding calcium alginate beads equivalent to 1.0 U/mL in 50 mL of reaction volume and was kept at 37 °C and 100 rpm for 4 h. Reaction was repeated five times by harvesting the beads from the previous reaction. Subsequently, the reaction was scaled up to 1 L. Purification of L-Theanine. A 50 mL reaction under optimized conditions was set up and terminated by lowering its pH to 3.0 by adding 3% (v/v) HCl solution. Batch purification was done by adding 10 g of Dowex 50W X 8 hydrogen form resin to the reaction vessel and kept at mild shaking (50 rpm) for 10 min at room temperature. The vessel was then allowed to stand for 10 min so that the resin settled at the bottom and was separated by decantation. L-Theanine was then eluted with 5 volumes of ammonia−water (pH 11.6; pH was adjusted by diluting 30% (v/v) ammonium hydroxide solution with ultrapure water), five times. Each fraction was analyzed on HPLC. Resin was regenerated using 2% (v/v) sulfuric acid and ultrapure water. The fraction containing purified theanine was then lyophilized and compared with the commercial standards on HPLC. 1H NMR analysis was done by dissolving 10 mg of the sample in 500 μL of D2O followed by analysis on a Bruker Avance 400 MHz spectrophotometer at the University Science Instrumentation Centre (USIC), University of Delhi, with TMS as internal standard. 1H NMR was done as single pulse (1D) at 23.7 °C, and acquisition duration and relaxation delay were 1.37 and 4 s per scan, respectively, with a pulse angle of 45°.

Y1or2 = β0 + β1X1 + β2X 2 + β3X3 + β11X12 + β22X 2 2 + β33X32 + β12X1X 2 where β0 is the constant coefficient, β1, β2, and β3 are linear, β11, β22, and β33 are quadratic, and β12 is the interaction coefficient. Statistical Analyses. RSM results were analyzed by performing an analysis of variance (ANOVA) test. F value and Prob > F generated by ANOVA were used to determine the statistical significance of the regression coefficients. Also, the model applicability was assessed by observing the values of R2, adjusted R2, and the coefficient of variance. Subsequently, the model was validated by setting up 10 random experiments (9 within the design space and 1 beyond), and the results thus obtained were checked against their predicted values. Enzyme Immobilization. rBLGGT was immobilized by entrapping the enzyme in calcium alginate beads. Acetone-precipitated enzyme (9.0 U/mL) was added to an equal volume of 6% (w/v) sodium alginate and mixed thoroughly, resulting in a 3% (w/v) sodium alginate enzyme mixture. Beads were prepared by adding the above mixture dropwise to a 0.2 M CaCl2 solution at 4 °C. Beads were left in the CaCl2 solution for 3 h at 4 °C for hardening, subsequently washed with ultrapure water, and air-dried. GGT activity retained in the beads as compared to the free enzyme was estimated by hydrolytic assay using oxidized glutathione (GSSG) as substrate. A 10 mM stock solution of GSSG was prepared in 50 mM Tris-HCl buffer (pH 9.0). GGT assay of free as well as immobilized enzyme was done using GSSG as substrate for hydrolytic reaction. Enzyme units C



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Article

RESULTS AND DISCUSSION Purification and Characterization of rBLGGT. rBLGGT was purified by Q-Sepharose ion-exchange chromatography, and the active fraction was eluted in 0.3 M NaCl (Figure 1A) with a yield of 28.8% and a specific activity of 4.58 U/mg (Table 2). The

respectively. The molecular masses of GGTs from other microbial sources range from 50 to 63 kDa, with variable sizes of large subunits, whereas the molecular masses of small subunits were almost constant.20,21 Biochemical Characterization. rBLGGT exhibited maximum activity at pH 9.0 and a temperature of 60 °C, similar to wild enzyme (Figure 2A,B). However, wild GGT showed a steep fall in activity when the pH was increased to 10.0, whereas rBLGGT retained around 60% of its activity until pH 11.0. GGTs from Bacillus sp. have been reported to work at alkaline pH ranging from 8.0 to 10.0, but there is a wide range (37−62 °C) with respect to temperature optima.21 rBLGGT was stable in a broad pH range of 6.0−12.0, following almost the same pattern as wild type (Figure 2C). During temperature stability experiments it was observed that the enzyme was stable at 40 °C up to 3 h, whereas at 50 °C enzyme activity decreased gradually with a t1/2 of 56 min. On the contrary, wild GGT was found to be more stable with a t1/2 of 192 min at 50 °C (Figure 2D and Figure S2 in the Supporting Information). rBLGGT was found to be stable for 3 months at 4 °C and for 1 week at 37 °C. Steady state kinetics of rBLGGT revealed a Km value of 45 μM against γ-GpNA, whereas that of wild GGT was 84 μM (Supporting Information Figure S3). This difference in Km values could be attributed to some conformational changes incurred by the recombinant protein while attaining its tertiary

Table 2. Purification Summary of rBLGGT step cell lysate acetone precipitated Q-Sepharose ionexchange purified (0.3 M fraction)a

total protein (mg)

total activity (U)

specific activity (U/mg)

yield (%)

purification fold

125 7.8 3.5

55.75 29.64 16.06

0.446 3.80 4.588

100 53.1 28.8

1.0 8.5 10.2

a

Acetone-precipitated fraction was purified in two lots from QSepharose fast flow column.

purity of the enzyme was checked with native-PAGE gel, which showed a single band of around 64 kDa. Activity staining was done to check the enzyme activity of the corresponding band (Figure 1B). rBLGGT is a heterodimeric protein, which was confirmed by SDS-PAGE gel (Figure 1C). The apparent molecular masses of large and small subunits were approximately 42 and 22 kDa,

Figure 2. (A) Effect of pH on rBLGGT activity. For pH optimization GGT assay was performed in different pH buffers (5.0−12.0) at 60 °C. rBLGGT activity of 0.53 U/mL at 60 °C was taken as 100%. All reactions were done in triplicate. (B) Effect of temperature on BLGGT activity. GGT assay was performed by incubating the reaction at specific temperatures (40−80 °C) for 5 min. (C) rBLGGT stability in different pH buffers. Enzyme was preincubated in different pH buffers (5−12; 10 mM) for 1 h, and then GGT assay was performed at pH 9.0 and 60 °C. (D) Determination of temperature stability of rBLGGT. Enzyme was preincubated at different temperatures, and then GGT assay was performed under standard conditions after every 30 min interval. At 60 °C enzyme stability was checked for 5 min intervals. rBLGGT was also compared with wild GGT form B. licheniformis ER-15 for pH and temperature optima and stability (0.37 U/mL of wild GGT under standard conditions was taken as 100%). D



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Figure 3. (A) Time profile for L-theanine synthesis (reaction containing 20 mM L-glutamine, 200 mM ethylamine, and 0.4 U/mL BLGGT in Tris-Cl, pH 9.0, buffer (50 mM), kept at 37 °C). (B) Optimization of acceptor concentration by a one variable at time approach (L-glutamine was kept constant at 20 mM. (C) Effect of L-glutamine concentration (ethylamine concentration was kept constant at 200 mM). (D) Optimization curve of enzyme concentration for theanine synthesis (5 mL reaction containing 40 mM L-glutamine and 200 mM ethylamine). All of the above reactions were done in triplicate and were carried out at 37 °C and pH 9.0 for 1 h.

structure. The affinity of rBLGGT for γ-GpNA as substrate is around 2 times that of B. licheniformis ATCC 27811 GGT,20 whereas the Km of B. subtilis SK11.004 GGT has been reported to be 7500 μM.21 Inhibition studies of rBLGGT and the wild type revealed that the two amino acids, namely, serine and tryptophan, are of utmost importance with respect to its activity as the enzyme is strongly inhibited by PMSF and NBS, which correlates with the previously reported results of Inoue et al.,22 wherein it has been shown that N-terminal threonine residue is crucial for both autocatalysis and activity of the enzyme. Although inhibition with NBS supports the hypothesis of Shuai et al.,21 complete inhibition of both the enzymes by GGT specific inhibitors such as DON and azaserine confirmed the nature of the enzymes as γglutamyl transferases (Supporting Information Figure S4). Metal ion inhibition studies demonstrated some contrasting features between the recombinant and wild enzymes. rBLGGT inhibition was observed with Zn2+, Mn2+, Ni2+, and Pb2+ metal ions, whereas there was apparently no change in GGT activity in the presence of Mg2+, Ca2+ Cu2+, and Cd2+ ions. On the other hand, wild-type GGT showed more sensitivity toward all of the metal ions used. Moreover, complete inhibition of the wild type was observed in the presence of Co2+, whereas rBLGGT retained

around 80% of the activity (Supporting Information Figure S5). rBLGGT was also inhibited by ethylene glycol tetraacetic acid (EGTA) with 37% residual activity, which was completely regained in the presence of Ca2+ or Mg2+ but not with Cd2+ metal ions; similar observations were also reported previously in the case of GGT from B. licheniformis.20 Inhibition with EGTA was not observed in case of wild- type. Substrate specificity of rBLGGT with various amino acids as γglutamyl acceptors was analyzed, and it showed >50% transpeptidase activity with most of the amino acid acceptors, whereas serine, alanine, and aspartic acid were weak acceptors. With respect to substrate specificity, rBLGGT showed a behavior highly similar to that of its wild counterpart Supporting Information Figure S6). High substrate specificity of rBLGGT makes the enzyme a good choice for synthesizing γ-glutamyl compounds. There are a very few reports of GGTs from Bacillus sp. having >50% transpeptidase activity with respect to monomers.23,24 Theanine Synthesis. Various parameters affecting the conversion of L-theanine from L-glutamine were investigated. LTheanine was quantified using HPLC, where theanine eluted in a linear fashion in the range from 0.25 to 2 mM between 10.8 and 11.4 min using 0.05% (v/v) TFA as a mobile phase. This is the E



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Table 3. Box−Behnken Design and the Results along with the Variance Analysis for the Selected Quadratic Modela (A) theanine yield (mM) run 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

L-glutamine

(mM)

0 −1 0 +1 0 +1 0 −1 +1 0 +1 0 −1 0 −1 0 0

% conversion

ethylamine (mM)

rBLGGT (U/mL)

actual

predicted

actual

predicted

−1 0 −1 −1 +1 0 0 +1 +1 0 0 0 −1 +1 0 0 0

+1 −1 −1 0 +1 −1 0 0 0 0 +1 0 0 −1 +1 0 0 (B)

17.09 2.45 12.31 10.41 68.88 31.02 65.63 17.18 56.27 62.51 26.37 58.42 1.95 54.97 13.36 67.12 63.41

21.99 4.04 15.75 5.49 60.88 26.32 63.42 22.1 59.7 63.42 32.56 63.42 −1.47 54.64 10.28 63.42 63.42

21.36 12.24 15.38 7.43 86.1 22.16 82.04 85.92 40.19 78.13 18.83 73.03 9.77 68.71 66.82 83.9 79.26

28.84 31.45 10.19 7.54 85.59 9.92 79.27 85.82 42.59 79.27 28.57 79.27 7.38 66.93 50.11 79.27 79.27

response

a

ANOVA values

theanine yield

% conversion

F value P>F mean R2 adjusted R2 coefficient of variance adequate precision

45.38 6 h29 except for GGT from B. subtilis14 where 94% conversion has been reported in 4 h. Statistical optimization was employed using Box−Behnken design. The actual and predicted values for both of the responses, L-theanine yield (mM) and percent conversion of L-glutamine to L-theanine, are listed in Table 3A. ANOVA studies revealed the model to be significant with F values of 45.38 (P > F < 0.0001) and 18.48 (P > F = 0.0001) for the former and the latter, F



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Figure 4. 3D plot for L-theanine yield (mM) and percent conversion of L-glutamine to L-theanine: effect of L-glutamine and ethylamine concentrations at a constant concentration of rBLGGT (1 U/mL).

600 mM ethylamine, and 1 U/mL rBLGGT at pH 9.0 and 37 °C within 4 h, which is nearly 4.25-fold the initial yield. rBLGGT enzyme concentration used in the current study is higher than in earlier studies14 because the enzyme has a temperature optimum of 60 °C and retains only 30% of its activity at 37 °C (Figure 2B). Furthermore, to make the reaction cost-effective, rBLGGT was immobilized by entrapment method using calcium alginate beads. Enzyme activity was retained at a level of 40−50% after entrapment as assessed by GGT assay using GSSG as substrate (retention time = 25 min). Immobilization had no effect on pH and temperature optima of the enzyme, whereas the thermal stability of the enzyme was enhanced with >80% activity at 60 °C until 15 min, in contrast to free rBLGGT that lost its activity at 60 °C within 5 min (Supporting Information Figure S9). Under optimized conditions 84% conversion was achieved using immobilized enzyme in 50 mL of reaction volume within 4 h, which was scaled up to 1 L with similar yields (Figure 5). Beads were reused for five times with >90% efficiency retained up to the third cycle. More than 90% of pure L-theanine was recovered by using the method of Zhu et al.30 with a few modifications. The purification was done in batch, and theanine was eluted using ammonia− water at pH 11.6 instead of pH 11.3. The purified fractions were then subjected to lyophilization, and finally a white theanine powder was obtained. The purity of the final product was assessed by HPLC (see Supporting Information Figure S10), and it was >95% pure. LTheanine was further identified by 1H NMR analysis (see Supporting Information Figure S10). Finally, through this process 35.2 g of theanine was obtained in three cycles of 1 L each. A brief comparison with the previously optimized methods for the synthesis of L-theanine using γglutamyl transpeptidase from various sources is listed in Table 4. This is the first report of the synthesis of L-theanine from B. licheniformis GGT. To date only E. coli and B. subtilis GGTs have been explored with 80−95% conversion efficiency. B. subtilis GGT has been reported to convert 94% L-glutamine to Ltheanine within 4 h14 in contrast to rBLGGT (84%), but the

respectively. Also, the adjusted R2 values were in good agreement with the predicted R2 for both responses (Table 3B). Thus, it can be concluded that the empirical model fits the experimental values very well. On the basis of the regression analyses the following equations were generated for Y1 and Y2: theanine yield (Y1) = 63.42 + 11.14X1 + 19.44X 2 + 3.12X3 − 30.99X12 − 10.97X 2 2 − 14.13X32 + 7.66X1X 2 % conversion (Y2) = 79.27 − 10.77X1 + 28.37X 2 + 9.33X3 − 30.66X12 − 12.78X 2 2 − 18.60X32 − 10.85X1X 2

X1, X2, and X3 are three independent variables included in the study. The interacting parameters, X13 and X23, were found to be insignificant and thus excluded from the model. From analysis of the interaction plots (Figure 4), it can be observed that on increasing the ethylamine concentration, yield as well as the percent conversion for L-theanine also increases at a constant concentration of L-glutamine and rBLGGT, whereas on increasing L-glutamine at a constant concentration of ethylamine, a decrease in percent conversion was observed. This could be attributed to either overcrowding of the active sites of rBLGGT or end product inhibition. However, with increasing enzyme, no benefit regarding percent conversion was encountered. Therefore, end product inhibition studies were carried out and a gradual decrease in enzyme activity was observed when the theanine concentration was increased from 10 to 40 mM during the GGT assay (Supporting Information Figure S8). The model was validated by conducting a set of 10 random experiments (9 within and 1 outside the design space), and the results were found to be in good agreement with the predicted values (Supporting Information Table S1). Thus, following RSM a maximum of 85−87% conversion of Lglutamine to L-theanine was obtained with the total theanine yield of 68−70 mM in a reaction containing 80 mM L-glutamine, G



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parameters that should be focused on to make the process more cost-effective.

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S9 and Tables S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(R.G.) Phone: +91-11-24111933. Fax: +91-11-24115270. Email: ranigupta15@rediffmail.com. Funding

We thank the Ministry of Food Processing Industries (MoFPI) for financial assistance. S.B. thanks DST-INSPIRE for a fellowship. Notes

Figure 5. Time profiling of theanine synthesis using free and immobilized enzyme. After all of the reaction conditions had been optimized, time was again standardized for 50 mL of reaction using free and immobilized enzyme.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the University of Delhi for facilities.

overall yield was less, as only 20 mM L-glutamine was used in that study. However, higher L-glutamine (or other γ-glutamyl donors) concentration (100−300 mM) in the case of E. coli GGTs resulted in higher yields, but accordingly high donor to acceptor ratios (1:7.5 or 1:10) and longer reaction times (8−24 h) were required to achieve >80% conversion. Thus, rBLGGT can be employed for the enzymatic synthesis of L-theanine with appreciable catalytic efficiency. Stereoselective synthesis of L-theanine with appreciable yields can thus be obtained from enzymatic reactions, as documented in Table 4. Also, Bacillus GGTs have been demonstrated to provide higher conversion rates as well as least reaction times. Thus, scaling up of the process, for L-theanine synthesis, at the industrial level could be achieved using B. licheniformis GGT. In contrast to natural extraction and chemical synthesis processes for L-theanine production, enzymatic synthesis has many advantages such as lower reaction time, high specificity, and higher yields. However, for such large-scale productions enzyme could be a limiting factor; therefore, now more emphasis should be given to increasing the enzyme production and immobilization. Reusability of the enzyme is one the most important

ABBREVIATIONS AND NOMENCLATURE rBLGGT:recombinant γ-glutamyl transpeptidase from Bacillus licheniformis ER-15 SDS-PAGE:sodium dodecyl sulfate−polyacrylamide gel electrophoresis Km:substrate concentration at which the reaction rate is half of Vmax Vmax:maximum rate achieved by the system, at maximum (saturating) substrate concentrations FDA:U.S. Food and Drug Administration L- and D-form:levorotatory and dextrorotatory ATP:adenosine triphosphate GGT:γ-glutamyl transpeptidase LB:Luria−Bertani SRL:Sisco Research Laboratory Pvt. Ltd. IPTG:isopropyl-β-D-thiogalactopyranoside NaCl:sodium chloride γ-GpNA:γ-glutamyl-p-nitroanilide hydrochloride PVDF:polyvinylidene difluoride BSA:bovine serum albumin PBS:phosphate buffer saline

Table 4. Comparative Analysis of the Methods Available for Enzymatic Synthesis of L-Theanine Using GGT from Various Sources donor concn (mM)

acceptor (ethylamine) concn (mM)

conditions

time (h)

80

600

pH 9.0, 37 °C

4

≥84

200

1500

pH 10.0, 37 °C

5

60

20

50

pH 10.0, 37 °C

4

94

recombinant E. coli GGT

267

2000

pH 10.5, 37 °C

24

80

immobilized E. coli cells

300

3000

pH 10.0, 50 °C

18

87.2 (after 6 uses)

E. coli cells

100

1000

pH 10.0, 45 °C

8

95

5

50

pH 9.0, 37 °C

6

93

γ-glutamyl donor used L-glutamine

enzyme recombinant B. licheniformis GGT (BLGGT) recombinant E. coli GGT Bacillus subtilis GGT

glutamic acid γ-methyl ester (GAME)

γGpNA

recombinant E. coli GGT

H

conversion (%)

ref current study Suzuki et al.11 Shuai et al.14 Wang et al.15 Zhang et al.13 Zhang et al.13 Zhang et al.29



Article

newly isolated strain of Bacillus subtilis, SK11.004. J. Sci. Food Agric. 2010, 90, 2563−2567. (15) Wang, Q.; Min, C.; Zhu, F.; Xin, Y.; Zhang, S.; Luo, L.; Yin, Z. Production of bioactive γ-glutamyl transpeptidase in Escherichia coli using SUMO fusion partner and application of the recombinant enzyme to L-theanine synthesis. Curr. Microbiol. 2011, 62, 1535−1541. (16) Ikeda, Y.; Taniguchi, N. γ-Glutamyl transpeptidase activity assay. Curr. Protoc. Toxicol. 2001, 5, 6.6.1−6.6.8. (17) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bateriophage T4. Nature 1970, 227, 680−685. (18) Rajput, R.; Verma, V. V.; Chaudhary, V.; Gupta, R. A hydrolytic γglutamyl transpeptidase from thermo-acidophilic archaeon Picrophilus torridus: binding pocket mutagenesis and transpeptidation. Extremophiles 2013, 17, 29−41. (19) Zhang, Y.; Chen, B.; Huang, Z.; Shi, Z. Preparative isolation and purification of L-theanine by HPLC. J. Liq. Chromatogr. Relat. Technol. 2005, 27, 875−884. (20) Lin, L. L.; Chou, P. R.; Hua, Y. W.; Hsu, W. H. Overexpression, one-step purification, and biochemical characterization of a recombinant γ-glutamyltranspeptidase from Bacillus licheniformis. Appl. Microbiol. Biotechnol. 2006, 73, 103−112. (21) Shuai, Y.; Zhang, T.; Mu, W.; Jiang, B. Purification and characterization of γ-glutamyltranspeptidase from Bacillus subtilis SK11.004. J. Agric. Food Chem. 2011, 59, 6233−6238. (22) Inoue, M.; Hiratake, J.; Suzuki, H.; Kumagai, H.; Sakata, K. Identification of catalytic nucleophile of Escherichia coli γ-glutamyltranspeptidase by γ-monofluorophosphono derivative of glutamic acid: N-terminal Thr-391 in small subunit is the nucleophile. Biochemistry 2000, 39, 7764−7771. (23) Hwang, S.-Y.; Ryang, J.-H.; Lim, W.-J.; Yoo, I.-D.; Oishi, K. Purification and properties of γ-glutamyl transpeptidase from Bacillus sp. KUN-17. J. Microbiol. Biotechnol. 1996, 6, 238−244. (24) Moallic, C.; Dabonne, S.; Colas, B.; Sine, J. P. Identification and characterization of a γ-glutamyl transpeptidase from a thermoalcalophile strain of Bacillus pumilus. Protein J. 2006, 25, 391−397. (25) Zhu, X.; Chen, B.; Ma, M.; Luo, X.; Zhang, F.; Yao, S.; Wan, Z.; Yang, D.; Hang, H. Simultaneous analysis of theanine, chlorogenic acid, purine alkaloids and catechins in tea samples with the help of multidimension information of on-line high performance liquid chromatography/electrospray-mass spectrometry. J. Pharm. Biomed. Anal. 2004, 34, 695−704. (26) Ying, Y.; Ho, J. W.; Chen, Z. Y.; Wang, J. Analysis of theanine in tea leaves by HPLC with fluorescence detection. J. Liq. Chromatogr. Relat. Technol. 2005, 28, 727−737. (27) Thippeswamy, R.; Gouda, K. G. M.; Rao, D. H.; Martin, A.; Gowda, L. R. Determination of theanine in commercial tea by liquid chromatography with fluorescence and diode array ultraviolet detection. J. Agric. Food Chem. 2006, 54, 7014−7019. (28) Tate, S. S.; Meister, A. γ-Glutamyl transpeptidase: catalytic, structural and functional aspects. Mol. Cell. Biochem. 1981, 39, 357−368. (29) Zhang, H.; Zhang, W.; Wang, Z.; Zhan, Y.; Xu, L.; Liu, J.; Liu, Q.; Jiao, Q. Enzymatic synthesis of theanine withEscherichia coli γglutamyltranspeptidase from a series of γ-glutamyl anilide substrate analogues. Biotechnol. Bioprocess Eng. 2013, 18, 358−364. (30) Zhu, S.; Wang, H. X.; Chen, S. W.; Dai, J. Study on adsorption and separation of theanine by ion exchange. Food Sci. 2007, 9, 148−153.

HRP:horseradish peroxidase DAB:diaminobenzidine PMSF:phenylmethylsulfonyl fluoride NBS:N-bromosuccinamide EDTA:ethylenediaminetetraacetic acid DON:6-diazo-5-oxo-L-norleucine HPLC:high-performance liquid chromatography TFA:trifluoroacetic acid UV−vis:ultraviolet−visible HCl:hydrochloric acid 1 H NMR:nuclear magnetic resonance with respect to hydrogen-1 nuclei D2O:heavy water TMS:tetramethylsilane kDa:kilodalton EGTA:ethylene glycol tetraacetic acid

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REFERENCES

(1) Voung, Q. V.; Bowyer, M. C.; Roach, P. D. L-Theanine: properties, synthesis and isolation from tea. J. Sci. Food Agric. 2011, 91, 1931−1939. (2) Huber, L. G. Green tea catechins and L-theanine in integrative cancer care: a review of the research. Altern. Complement. Ther. 2003, 9, 294−298. (3) Liu, Q.; Duan, H.; Luan, J.; Yagasaki, K.; Zhang, G. Effects of theanine on growth of human lung cancer and leukemia cells as well as migration and invasion of human lung cancer cells. Cytotechnology 2009, 59, 211−217. (4) Kim, T.; Lee, Y. K.; Park, S. G.; et al. L-Theanine, an amino acid in green tea, attenuates β-amyloid-induced cognitive dysfunction and neurotoxicity: reduction in oxidative damage and inactivation of ERK/ p38 kinase and NF-κB pathways. Free Radical Biol. Med. 2009, 47, 1601−1610. (5) Lichtenstein, N. Preparation of γ-alkylamides of glutamic acid. J. Am. Chem. Soc. 1942, 64, 1021−1022. (6) Gu, H.; Jiang, Y.; Wang, J. A practical synthesis of ethyl L-glutamine (L-theanine). Org. Prep. Proced. Int. 2004, 36, 182−185. (7) Tachiki, T.; Suzuki, H.; Wakisaka, S.; Yano, T.; Tochikura, T. Production of γ-glutamylmethylamide and γ-glutamylethylamide by coupling of baker’s yeast preparations and bacterial glutamine synthetase. J. Gen. Appl. Microbiol. 1986, 32, 545−548. (8) Tachiki, T.; Yamada, T.; Mizuno, K.; Ueda, M.; Shiode, J. I.; Fukami, H. γ -Glutamyl transfer reactions by glutaminase from Pseudomonas nitroreducens IFO 12 694 and their application for the syntheses of theanine and γ-glutamylmethylamide. Biosci., Biotechnol., Biochem. 1998, 62, 1279−1283. (9) Itoh, T.; Hoshikawa, Y.; Matsuura, S.; Mizuguchi, J.; Arafune, H.; Hanaoka, T.; Mizukami, F.; Hayashi, A.; Nishihara, H.; Kyotani, T. Production of L-theanine using glutaminase encapsulated in carboncoated mesoporous silica with high pH stability. Biochem. Eng. J. 2012, 68, 207−214. (10) Liu, B.; Li, P.; Zhang, C.; Wang, Y.; Zhao, Y. S. Theanine synthesized by immobilizing Pseudomonas nitroreducens LY in nanofibrous membranes. Process Biochem. 2010, 45, 1330−1333. (11) Suzuki, H.; Izuka, S.; Miyakawa, N.; Kumagai, H. Enzymatic production of theanine, an ‘umami’ component of tea, from glutamine and ethylamine with bacterial γ-glutamyltranspeptidase. Enzyme Microb. Technol. 2002, 31, 884−889. (12) Zhang, F.; Zheng, Q. Z.; Jiao, Q. C.; Liu, J. Z.; Zhao, G. H. Enzymatic synthesis of theanine from glutamic acid γ-methyl ester and ethylamine by immobilized Escherichia coli cells with γ-glutamyltranspeptidase activity. Amino Acids 2010, 39, 1177−1182. (13) Zhang, F.; Zheng, Q. Z.; Jiao, Q. C.; Liu, J. Z.; Zhao, G. H. Synthesis of theanine from glutamic acid γ-methyl ester and ethylamine catalyzed by Escherichia coli having γ-glutamyltranspeptidase activity. Biotechnol. Lett. 2010, 32, 1147−1150. (14) Shuai, Y.; Zhang, T.; Jiang, B.; Mu, W. Development of efficient enzymatic production of theanine by γ-glutamyltranspeptidase from a I


l-Theanine Synthesis Using Î³-Glutamyl Transpeptidase from Bacillus

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