Efficient Production of L-Theanine Using Immobilized Recombinant Escherichia coli Cells Expressing a Modified γ-Glutamyltranspeptidase Gene from Pseudomonas nitroreducens ()
1. Introduction
L-Theanine (γ-glutamylethylamide) is an amino acid derivative approved by the Food and Drug Administration, USA, as a food additive to stimulate the Umami taste. L-Theanine has various additional reported benefits on human health, including improvement of concentration and learning ability [1], reduction of blood pressure [2], improvement of immune responses [3], and promotion of relaxation [4]. L-Theanine is naturally present in specific organisms such as Camellia sinensis (green tea) and the mushroom, Xerocomus badius [5]. It can be obtained by chemical synthesis or extraction from green tea, but both procedures include time-consuming, cost-ineffective, and complicated operational processes [6]. Hence, a biotechnological alternative for the production of L-theanine, using microbial enzymes such as γ-glutamyltranspeptidase (GGT) and glutamine synthetase [7] [8], has attracted significant attention. GGT is a ubiquitous enzyme that catalyzes the hydrolysis of the γ-glutamyl linkages of γ-glutamyl compounds and the transfer of their γ-glutamyl moieties to acceptor substrates [9]. It exists in a wide range of organisms from mammals to bacteria [10]. Bacterial GGTs have been studied in Escherichia coli (EcGGT) [11] [12], Bacillus licheniformis (BlGGT) [13], and Pseudomonas nitroreducens (PnGGT) [14]. The activity of GGTs produced by such cultured bacteria can be exploited to produce L-theanine when provided with L-glutamine and ethylamine as substrates.
We have reported previously that GGT from P. nitroreducens IFO12694 (PnGGT) has a high hydrolysis: transfer activity ratio (H/T ratio; with hydrolysis being the greater activity than transfer) and that this ratio is higher than that of other characterized GGTs [14]. Three-dimensional structural analysis of recombinant PnGGT explored the role of amino acid residues, W385, F417, and W525 in substrate recognition [15]. Mutations made at these positions based on the 3D structural analysis brought about dramatically decreased hydrolysis and increased transfer activities. In this study, we selected the W525D mutant, showing high transfer activity with a lowest H/T ratio among mutant enzymes which were examined and we evaluated high usability of immobilized recombinant E. coli-W525D for L-theanine production.
2. Material and Methods
2.1. Chemicals and Reagents
All analytical grade chemicals for culture media and GGT activity assay were purchased from FUJIFILM Wako Pure Chemical Corporation Ltd. (Osaka, Japan), unless otherwise stated.
2.2. Site-Directed Mutagenesis
The plasmid for the expression of wild-type (pET22b-PnGGT) was constructed previously [14]. PCR was performed using KOD-Plus-Neo DNA polymerase (Toyobo Co., Osaka, Japan), pET22b-PnGGT (template) with W525D primers, the forward primer 5'-GAAGATCAGGGACCGAACATGGGTGACATC-3' and the reverse primer 5'-GATGTCACCCATGTTGTCGCCCTGATCTTC-3', to generate the site-directed mutation. The PCR product was treated with DpnI at 37˚C for 1 h and transformed into E. coli XL-10 gold. Upon confirmation of their DNA sequence, the resulting plasmid was designated as pET22b-W525D.
2.3. Bacterial Strains and Growth Conditions
P. nitroreducens IFO12694 was cultured in the medium described previously [16]. E. coli Rosetta-gami B (RGB) was cultured in a medium containing 0.5% w/v yeast extract, 1% w/v polypeptone, 0.33% w/v (NH4)2SO4, 0.68% w/v KH2PO4, 0.71% w/v Na2HPO4, 0.05% w/v glucose, 0.2% w/v lactose, and 0.015% w/v MgSO4. These strains grew overnight at 37˚C with an agitation of 100 rpm. Antibiotics were used, when required, at the following concentrations: 0.1% v/v ampicillin, 0.1% v/v chloramphenicol, and 0.1% v/v kanamycin.
2.4. Determination of the Best Strain for L-Theanine Production
P. nitroreducens IFO12694 (the original strain), E. coli-PnGGT, E. coli-W525D, and E. coli-pET22b (harboring empty vector) were cultured in optimal conditions to evaluate their GGT activities and L-theanine production. Bacterial cells were harvested by centrifugation at 16,890 g for 10 min at 4˚C. Cell pellet was then suspended in 10-fold (w/v) 10 mM Tris-HCl buffer (pH 8). To precisely analyze their GGT activities and L-theanine production abilities, cell suspension of each strain was adjusted to equal cell density at 600 nm with suspension buffer. To evaluate hydrolysis and transfer activities of each cell-free extract, cell suspension was disrupted with sonication and separated from debris by centrifugation. Prepared cell-free extract from each strain was used for its hydrolysis and transfer activities according to the method described below. For evaluation of L-theanine production, reaction mixture containing 25 mM L-Gln, 250 mM ethylamine, and 100 mM borate buffer (pH 10) was mixed with 100 µL of cell-free extract or 100 µL of cell suspension (free cells) or 200 mg of immobilized cells. L-Theanine formed for 1 h reaction was determined using a high-performance liquid chromatography (HPLC; Shimadzu, Kyoto, Japan) system equipped with a COSMOSIL 5C18-MS-II (Nacalai, Kyoto, Japan) according to the procedure described previously [14].
2.5. Enzyme Assay
The GGT activity was determined according to the procedure of Imaoka et al. [14]. For the hydrolytic activity, the reaction mixture containing 2.5 mM γ-L-glutamyl-p-nitroanilide (GpNA), 100 mM imidazole buffer (pH 9.0), and the enzyme was incubated at 30˚C for 5 - 15 min. The reaction was stopped by adding 10% acetic acid, and the released p-nitroaniline was estimated by measuring absorbance at 410 nm. One unit of the GGT hydrolysis activity was defined as the amount of the enzyme required to catalyze the formation of 1 µmol of p-nitroaniline per min. For the transfer activity, a reaction mixture containing 40 mM L-glutamine (L-Gln), 20 mM hydroxylamine, 100 mM imidazole buffer (pH 7.5), and the enzyme was incubated at 30˚C for 5 - 15 min. The reaction was stopped by adding a solution containing 200 mM FeCl3∙6H2O, 120 mM trichloroacetic acid, 250 mM HCl, and water at a ratio of 8:2:1:13. The formation of γ-L-glutamylhydroxamate was evaluated by measuring the absorbance at 540 nm. One unit of the GGT transfer activity was defined as the amount of the enzyme required to catalyze the formation of 1 µmol of γ-L-glutamylhydroxamate per min.
2.6. Immobilization of the Bacterial Cells
Cell immobilization was performed by the method reported by Lishen et al. [17] with some modifications. The bacterial cells were grown and harvested as described above, and the cell pellet was resuspended in 10 mM Tris-HCl buffer (pH 7.5). The cell suspension was homogeneously mixed with supporting materials such as 4% (w/v) sodium alginate solution at the ratio of 1:1. The mixture was applied to a syringe and dropped into 2% (w/v) CaCl2 solution. The resulting immobilized cell beads were left to harden at 4˚C overnight. Two hundred mg of immobilized beads were used for the optimization of the reaction conditions as described below. Immobilized cells prepared by all methods [18] - [23] were subjected to evaluation of L-theanine production at 40˚C in 100 mM borate buffer (pH 10) for 1 h. L-theanine production (% yield) was analyzed using HPLC according to the method described above.
2.7. Optimization of pH, Temperature, and Molar Ratio of Substrates for L-Theanine Production
Optimization of pH for L-theanine production by immobilized cells expressing W525D PnGGT was examined using 100 mM borate buffer at pH values of 8 to 11. The optimal temperature for L-theanine production was determined using the range 25˚C - 50˚C in conditions of optimal pH. To determine the optimal molar ratio of substrates for L-theanine production, the ratio of L-Gln to ethylamine was varied from 1:0.5 to 1:10. For all optimization experiments, L-theanine formation in the various substrate molar ratios was analyzed by HPLC as described earlier. All assays were performed in triplicate, and average values as well as the standard deviations are presented.
2.8. Analysis of the Residual Activity of Immobilized Cells after Treatment with Disinfectants
According to the method previously described by Bindal and Gupta [24], 200 mg of immobilized cells were treated with sodium hypochlorite (0.05% w/v) and sodium azide (0.05% v/v) for 10 min at room temperature. Immobilized beads were separated from the solution and washed twice with sterilized water prior to analysis of residual activity using the hydrolysis activity assay described earlier. The water washes were spread onto LB-agar plates, and resulting colonies were counted to determine the number of colony-forming units.
2.9. Analysis of the Residual Activity of the Immobilized Cells after Treatment with CaCl2
The degree of stability to which cells are immobilized after treatment with buffer containing CaCl2 was examined according to a method by [25], with modification. Immobilized beads, 200 mg, were incubated in 20 mL of 100 mM imidazole buffer (pH 7.5) containing CaCl2 at concentrations of 2, 10, 50, and 200 mM at room temperature for 1 h. Leakage of cells from the beads was determined by measuring PnGGT hydrolysis activity in the wash buffer.
2.10. Analysis of the Long-Term Stability and Reusability of the Immobilized Cells
The long-term stability and reusability of immobilized cells was investigated according to the method described by [24]. To test reusability, immobilized beads subjected to the enzyme hydrolysis activity assay were separated from the reaction buffer and reused in a repeated assay, performing this cycle 13 times. To assess stability upon storage, the residual hydrolysis activity of the immobilized beads was determined at intervals between 0 and 4 weeks after storage at 4˚C or room temperature.
3. Results and Discussion
3.1. Selection of the Best Strain to Be Used for Immobilization
To evaluate the GGT activities and L-theanine production of P. nitroreducens, E. coli-PnGGT, and E. coli-W525D, we measured the hydrolysis and transfer activities of their cell-free extracts and L-theanine formed using their cell-free extracts, free cells, and immobilized cells as well as E. coli-pET22b as a control. As shown in Table 1, E. coli-W525D showed 9.05 U/mg of transfer activity (UT) in the cell-free extract which was higher than those of P. nitroreducens and E. coli-PnGGT.
Moreover, it also showed 21.42 of UT/UH ratio 100-times higher than those of P. nitroreducens (0.21) and E. coli-PnGGT (0.22). Hydrolysis and transfer activities of E. coli-pET22b were negligible, and it did not show significant effects on GGT activities and L-theanine production of other recombinant strains, E. coli-PnGGT and E. coli-W525D. Consequently, E. coli-W525D exhibited the highest L-theanine production in all cases using cell-free extract, free cells, and immobilized cells among strains examined. Based on the results, E. coli-W525D was selected as a best strain for immobilization study for L-theanine production.
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Table 1. GGT activities and L-theanine production of the strains used in this study.
Protein concentration of the cell-free extract of each strain was adjusted in the range of 3 - 13 mg/mL. Amounts of cells contained in cell suspension and immobilized cells were adjusted to be approximately 100 mg/mL and 50 mg/g immobilized gel, respectively.
3.2. Optimal Reaction Conditions for L-Theanine Synthesis by Immobilized E. coli-W525D
Efficient synthesis of L-theanine by immobilized P. nitroreducens cells has previously been demonstrated [26]. It is clearly more economical to use immobilized cells, obtained from a suspension of harvested enzyme-expressing E. coli cultures, than to use purified enzyme in any biotechnological application. We thus selected E. coli-W525D showing the highest L-theanine-producing ability for such cell immobilization and for the subsequent optimization of reaction conditions for L-theanine production. Upon varying the pH (pH 8 - 11) and temperature (25˚C - 50˚C), immobilized E. coli-W525D demonstrated a pH optimum of 10, as shown in Figure 1(a). The result was consistent with most previous studies on L-theanine production (Table 2), which indicated optimal activity at high alkaline pH [13]. The optimal temperature for L-theanine production was 40˚C, as shown in Figure 1(b), which is slightly higher than most previous data (Table 2). However, there are some method-dependent differences between reports of temperature effects. For example, some reports showed a decrease in the transfer activity at temperatures higher than 37˚C, that might be attributed to a low boiling point of ethylamine [27], whereas work with immobilized cells [28] has indicated an optimal temperature of 50˚C.
3.3. Effect of Substrate Molar Ratio on L-Theanine Production
To further increase the yield of L-theanine produced by E. coli-W525D, we investigated the optimal molar ratio of substrates in the reaction. When the concentration of L-Gln was fixed, the highest amount of L-theanine was obtained by the reaction of L-Gln and ethylamine at a 1:10 molar ratio (Figure 1(c)). In previous studies, the ratio of substrates for optimal L-theanine production showed various values ranging from 1:1.6 to 1:11 [28] [29]. The highest L-theanine production level in these studies was obtained at a ratio of 1:2.5, giving a 94% conversion rate [30], but the ratio is highly dependent on the individual characteristics of each GGT and how it was used in the assays. In our experiments, using immobilized cells, a 1:10 molar ratio of substrates gave the optimum yield of product, with a 90% conversion rate, which is the highest ever reported rate using immobilized cell.
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Figure 1. L-Theanine production in various conditions with immobilized cells of E. coli-W525D mutant. (a) pH-dependence of L-theanine production, pH 8 - 11, (b) Temperature-dependence of L-theanine production, 25˚C - 50˚C, and (c) Dependence of L-theanine production on the substrate molar ratio using L-Gln and ethylamine and ratios from 0.5 to 10. The values given in the figures represent the mean ± SD (n = 3).
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Table 2. Summary of host strains, GGT source, production condition and yields reported in other researches about the process of L-theanine production with GGT.
3.4. Effect of the Immobilizing Material on L-Theanine Production
We investigated the efficiency of various immobilizing materials for L-theanine production, utilizing materials referred to in previous studies: 4% κ-carrageenan [18]; 2% κ-carrageenan [19]; 4% Gelatin, 2% alginate and 16% glyceroli [23]; 2% Alginate in KPB and 1% gelatin [22]; 2.5% alginate and starch [20]; and 2% carrageenan with 1% locus bean gum [21]. High L-theanine conversion rate was obtained when using 2% κ-carrageenan with 1% locus bean gum, 4% κ-carrageenan, and 2% alginate as the immobilizer, as shown in Figure 2. Interestingly, additional supporting materials such as gelatin, glycerol and starch added in combination with alginate led to lower L-theanine production. The observations were consistent with a more open matrix favoring substrate diffusion and accessibility to cellular enzymes. The yield of L-theanine production using 2% alginate was similar to that achieved in a previous study [28], which applied 3% alginate to immobilized cells and resulted in a conversion rate of 87%.
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Figure 2. Effect of immobilization methods on L-theanine production. E. coli-W525D mutant cells were used for immobilization. The numbers indicate the immobilization material used: 1, 4% κ-carrageenan; 2, 2% κ-carrageenan; 3, 4% Gelatin, 2% alginate and 16% glycerol; 4, 2% Alginate in KPB and 1% gelatin; 5, 2.5% Alginate and starch; 6, 2% Carrageenan and 1% locus bean gum; 7, 2% alginate. The values for L-theanine yield reported are representative of the mean ± SD (n = 3).
Stability of immobilized cells and its dependence on the immobilizer materials were evaluated by measuring residual activity for L-theanine production after three-time usages of immobilized cells at different temperatures, 30˚C and 40˚C (Figure 3). Immobilized cells with both 4% and 2% κ-carrageenan demonstrated high stability at 30˚C, but at 40˚C the activity massively decreased after three-time usages. Conversely, cells immobilized by alginate exhibited high stability at both 30˚C and 40˚C. Taken together, the results demonstrated that 2% alginate, with no additional matrix materials, provided the best support for active and stable immobilized cells in the production of L-theanine.
3.5. Effect of CaCl2 on Stability of the Immobilized Cells
Calcium chloride solution is proposed to assist the stability of immobilized cells made using alginate and to prevent loss, or leakage, of active enzyme [25]. Hence, immobilized cells made by alginate with E. coli-W525D were washed with different concentrations of CaCl2 solution and enzyme activity in the wash solution was assayed. Figure 4 shows the relative enzyme activity detected in the CaCl2 washes. For wash solutions of 50 and 200 mM CaCl2, negligible enzyme activity was detected. The result showed that treatment of the immobilized cells with buffer containing CaCl2 at 10 mM or higher was effective for stabilization of the immobilized cells.
3.6. Effect of Disinfectants on the Storage of the Immobilized Cells
After use, immobilized cells were treated with disinfectant to prevent them from microbial contamination and degradation and thus to increase their shelf life. It was, however, also important to assess whether the disinfectants chosen, NaN3 and NaClO, had a negative effect on cellular enzyme activity. Residual GGT activity of immobilized cells decreased to 91% and 95%, respectively, after treatment with wash solutions containing NaN3 and NaClO, thus demonstrating very
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Figure 3. Effect of immobilization methods on the stability of immobilized cells. The residual activity of each sample of immobilized cells was measured after having repeated their use three times for 1 h at each temperature (1. 4% κ-carrageenan, 2. 2% κ-carrageenan, 3. 4% gelatin 2% alginate and 16% glycerol, 4. 2% Alginate in KPB and 1% gelatin, 5. 2.5% Alginate and starch, 6. 2% Carrageenan and 1% locus bean gum and 7. 2% alginate). (a) stability recorded as residual activity at 30˚C and (b) stability recorded as residual activity at 40˚C, Values for L-theanine production represent the mean ± SD (n = 3).
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Figure 4. Effect of CaCl2 concentration on the stability of immobilized cells. Immobilized cells (E. coli-W525D) were washed for 1 h with 100 mM imidazole buffer (pH 7.5) containing CaCl2 at concentrations ranging from 0 to 200 mM. After the treatment, enzyme activity present in the wash solution was measured. The values reported for enzyme activity represent the mean ± SD (n = 3).
good retention of activity. The wash solutions generated no bacterial colonies on agar plates in comparison to a wash solution with no added disinfectant (>200 colonies formed). This result was similar to a previous study [24] which showed 100% residual activity, and supports use of such disinfectants to prolong the shelf life of immobilized cells.
3.7. Reusability and Storage Stability of the Immobilized Cells
After 13 rounds of use, the activity of immobilized cells decreased to 57% with respect to hydrolysis activity (Figure 5(a)). There are some previous reports, where immobilized cells on 3% alginate retained 12.8% activity after ten-time usage [28] and immobilized enzyme on Ca-alginate-κ-carrageenan showed 55% activity after six repeated uses [29]. The shelf life of immobilized cells was investigated at both 4˚C and 25˚C. At 4˚C, enzyme activity of immobilized cells decreased slightly to 98% and 85% after the storage for one week and one month, respectively (Figure 5(b)). At 25˚C, the activity also decreased slightly to 96% and 78% after storage for one week and one month, respectively. Similarly, Bindal and
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Figure 5. Assessment of the reusability and stability of immobilized cells. A) Reusability of immobilized cells; B) Stability of immobilized cells (-●-) after storage at 4˚C and room temperature (-▲-), The values of activity are hydrolysis activity yields and represent the mean ± SD (n = 3).
Gupta [24] observed activity decreases to 82% and 75% after one month at 4˚C and room temperature, respectively. Small decrease in activity observed during the storage might be partially due to microbial contamination, which would be prevented by regular treatment with disinfectant to maintain an aseptic environment.
3.8. L-Theanine Production by W525D
Immobilized E. coli-W525D cells with 2% alginate exhibited 23 mM of L-theanine production from 25 mM L-Gln and 250 M ethylamine, giving a 90% yield. This yield was much higher than that of immobilized E. coli RGB cells expressing PnGGT (wild-type), which showed a 1.43% conversion rate. L-Theanine production by wild-type P. nitroreducens immobilized cells was significantly low under the same condition. The reaction conditions such as L-Gln and ethylamine concentrations were different to those for the enzyme reaction reported previously by Tachiki et al. [31]. As shown in Table 1, however, the expression level of GGT, indicated by the difference of specific activities between the original strain and the recombinant E. coli might be considered to mainly affect the yield of L-theanine production using immobilized cells in this study. Currently, the highest L-theanine conversion rate reported thus far is 94%, for the synthesis using free enzyme from B. subtilis SK11.004 [30]. However, the conversion rate reported for E. coli-W525D in our study are the highest achieved thus far for the synthesis using immobilized cells. The proven benefits of immobilization of E. coli-W525D on alginate, together with the reusability of the immobilized cells and their tolerance to various conditions, demonstrates a high potential for its application to L-theanine production for the food industries, critically avoiding the complex and expensive enzyme purification process.
4. Conclusion
The objective of this research was to increase L-theanine production utilizing the E. coli RGB expression system for production of a W525D mutant of PnGGT, named E. coli-W525D, which displayed increased transfer activity. The immobilized cells expressing E. coli-W525D demonstrated an increased L-theanine production rate compared with that of the other immobilized cells, including wild-type (E. coli-PnGGT). Optimal production of L-theanine was observed in borate buffer, pH 10, at 40˚C and in the presence of a 1:10 molar ratio of substrates L-Gln and ethylamine. The most suitable cell immobilization material, 2% alginate, retained 57% residual activity after reuse 13 times. Furthermore, 85% and 78% residual activity was observed after storage of immobilized cells for a month at 4˚C and room temperature, respectively. These results show that immobilized E. coli-W525D cells achieve one of the best reported conversion rates for biosynthesis of L-theanine and the best yield thus far from immobilized cells rather than enzyme, avoiding any purification steps. The immobilized E. coli-W525D cells thus have potential application in biosynthesis of L-theanine for the food and supplement industries.
Acknowledgements
This work was supported by JICA Innovative Asia Scholarship.