Efficient asymmetric biosynthesis of (R)-(−)-epinephrine in hydrophilic ionic liquid-containing systems

Abstract

The strain Acinetobacter sp. UN-16 with adrenaline dehydrogenase could catalyze adrenalone to (R)-(−)-epinephrine. For the asymmetric biosynthesis of (R)-(−)-epinephrine with whole cells of Acinetobacter sp. UN-16, ten different hydrophilic ionic liquids were successfully tested. The diverse water–ionic liquid systems showed significant but different influences on the initial reaction rate, yield, and reduction activity. Of all the tested ionic liquids, [HOOCEMIM]NO₃ showed good biocompatibility, moderately increased cell membrane permeability, markedly improved reaction efficiency and best biotransformation results.

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Biological catalysts consist of free/immobilized enzyme as well as whole-cells.¹ Compared to the free enzyme, the immobilization of the enzyme on/in a solid support can improve the stability and recyclability of the biocatalyst, along with having the advantages of a lower risk of product contamination with enzyme residues and low or no allergenicity.² However, numerous biological reactions are complex redox reaction and coenzyme regeneration is necessary in the process. Moreover, they may also have limitations such as not being stable enough under industrially relevant conditions (presence of organic solvents and high temperatures) or not having enough activity, selectivity or specificity towards the target industrial products.³ An optimized co-immobilized multi-enzymatic system may be very competitive, with a much higher volumetric activity and no problems of permeability or membrane breakage, but it is more expensive.⁴

Whole-cell biocatalysts are considered to be an alternative option and an elegant way of producing fine biological products.⁵ In contrast with isolated enzyme and immobilized enzymes, whole-cell biocatalysts show greater stability and convenience with no need for enzyme purification and coenzyme addition, or an additional system for coenzyme regeneration and with less enzyme inactivation, as the enzymes are kept within the natural environment of a living cell.⁶ However, due to permeability barriers, whole-cell biocatalyzed reactions are generally much slower than reactions catalyzed by free enzymes.⁷

Multiphase processes have been established to address these limitations, and green solvents such as supercritical fluids and ionic liquids are promising alternatives. Among the modern solvents applicable in “green biotechnologies”,⁸ ionic liquids have attracted increasing attention due to the improvement in process economics, reaction activity, selectivity and yield.⁹ As a new type of green materials, ionic liquids have been widely used as co-solvents in biological catalysis for the past few years because of their unique advantages, such as having almost no vapor pressure, and consequently being less volatile.¹⁰ However, some studies have shown that ionic liquids could damage some microbial cells,¹¹ which proved that ionic liquids have a definite toxicity to cells. Therefore, in order to find suitable ionic liquids for biological catalytic reactions in which the ionic liquids are used as the medium, the toxic effects of ionic liquids on microbial cells have to be taken into account.

In this study, when adrenalone was used as the substrate, we focused on the evaluation of 10 different hydrophilic ionic liquids as the co-solvents for the asymmetric biosynthesis of (R)-(−)-epinephrine by the bacterium Acinetobacter sp. UN-16 (reaction shown in Fig. S1†) and discussed the effects of these ionic liquids on the biological catalytic reactions and its mechanism. Moreover, we found that the use of ionic liquids with the strain shows great promise, and the maximum yield was enhanced. The strain Acinetobacter sp. UN-16 along with another called Kocuria rhizophila had been isolated by our research group previously and conserved in our laboratory.¹² The former one, the bacterial strain G92, which can produce (R)-epinephrine by asymmetric reduction of adrenalone in high stereoselectivity (enantiomeric excess, >99%) and high product yield (31.2%), was isolated from wet land of the Min river. The 16S rDNA sequence of G92 showed that this strain belongs to Acinetobacter and it was named Acinetobacter sp. G92. In order to achieve a higher product yield, UV mutagenesis, nitrosoguanidine (NTG) mutagenesis and UV-NTG-combined mutagenesis were employed for the strain screening. A mutant named Acinetobacter sp. UN-16, which afforded a high product yield of 54.9%, was obtained via UV-NTG-combined mutagenesis and used for the following study. The types of carbon and nitrogen source and inorganic salts in the fermentation medium of Acinetobacter sp. UN-16 cells were determined by a single factor method. The fermentation medium composition ratio was optimized by response surface methodology, and the optimum medium contained: glycerol (9.12 g L⁻¹), beef extract (7.82 g L⁻¹), NH₄H₂PO₄ (5.65 g L⁻¹), K₂HPO₄ (0.1 g L⁻¹), MgSO₄ (0.1 g L⁻¹), MnSO₄ (0.01 g L⁻¹) and ZnSO₄ (0.01 g L⁻¹). With the optimal culture medium, the shaking flask fermentation conditions were sequentially optimized, and the optimized conditions were as follows: inoculating amount of 1% v/v, 7.4 pH, fermentation volume of 20 mL, adrenalone concentration of 8 mmol L⁻¹, adrenalone addition time of 18 h, fermentation termination time of 22 h, culture temperature of 34 °C, and a rotation speed of 200 rpm. The yield for (R)-epinephrine was increased to 68.2% with the optimized medium and fermentation conditions.

In order to take advantage of the benefits provided by ionic liquids and explore the effects of structural differences in ionic liquids, which are known to influence biotransformations in related systems,¹³ we evaluated ten different hydrophilic ionic liquids as a co-solvent for the bioreduction of adrenalone with Acinetobacter sp. UN-16 cells. Under water culture conditions, the Acinetobacter sp. UN-16 cell seed solution was first cultured to logarithmic phase and then 200 μL of the seed solution was added to a 50 mL conical flask containing 20 mL of fermentation medium access (pH 7.4). After 18 h of culture (34 °C, 200 rpm) in an aseptic operation room, 8 mM (R)-epinephrine and 7.5% (v/v) of the ionic liquid were added into the conical flask to culture for further 22 h. The fermented liquid (1 mL) was taken and centrifuged for 5 min at 10 000 rpm. The supernatant was filtered through a water filter membrane (0.22 μm) and then analyzed by high performance liquid chromatography (HPLC) and high performance capillary electrophoresis (HPCE), respectively (Table 1).

Table 1 Full names of ionic liquids and their abbreviations in this paper

Full name of ionic liquid	Abbreviation
1-Ethyl-3-methylimidazolium chloride	[EMIM]Cl
1-Hydroxyethyl-3-methyl imidazolium nitrate	[HOEMIM]NO3
1-Butyl-3-methylimidazolium chloride	[BMIM]Cl
1-Heptyl-3-methylimidazolium chloride	[C7MIM]Cl
1-Ethyl-3-methylimidazolium nitrate	[EMIM]NO3
1-Ethyl-3-methylimidazolium tetrafluoroborate	[EMIM]BF4
1-Hydroxyethyl-3-methylimidazolium chloride	[HOEMIM]Cl
1-Hydroxyethyl-3-methylimidazolium trifluoromethanesulfonate	[HOEMIM]OTF
1-Ethyl-2,3-dimethylimidazolium nitrate	[EMMIM]NO3
1-Carboxymethyl-3-methylimidazolium nitrate	[HOOCEMIM]NO3

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As shown in Table 2, the bioreduction showed a variety of results when using different ionic liquids. The presence of the [HOEMIM]⁺-based ionic liquids [HOEMIM]NO₃ and [HOEMIM]Cl, accelerated significantly the biological catalysis reaction; in contrast, the reaction slowed down clearly and the achieved yield decreased significantly with the addition of [HOEMIM]OTF. Moreover, [EMIM]⁺-based ionic liquids containing different anions (NO₃⁻, Cl⁻, BF₄⁻) also showed remarkable effects on the biocatalysis. Surprisingly, Acinetobacter sp. UN-16 cells displayed a poor reduction activity in the solution containing [HOEMIM]OTF, as indicated by the very low product yield and initial reaction rate. This was probably due to the OTF⁻ anion being easily converted to hydrofluoric acid within the solution and [HOEMIM]OTF also showed comparatively high toxicity to the cells. All of these results indicated that different anions associated with the same cation could greatly affect the biological catalysis reaction and that ionic liquids with Cl⁻ and NO₃⁻ anions (especially NO₃⁻) were superior to the ionic liquids with other anions (BF₄⁻, OTF⁻) for achieving a high initial reaction rate and maximum yield.

Table 2 Asymmetric biosynthesis of (R)-(−)-epinephrine by Acinetobacter sp. UN-16 cells in various ionic liquids

Reaction medium	Initial reaction rate (μmol min^−1)	Yield (%)	Product ee (%)
Water	40.8	68.2	68.2
[EMIM]Cl	44	35	35
[BMIM]Cl	41.2	20.6	20.6
[C7MIM]Cl	21.6	10.8	10.8
[EMIM]NO3	47.8	57.4	57.4
[EMIM]BF4	9.1	8.1	8.1
[HOEMIM]Cl	44.4	45.8	45.8
[HOEMIM]NO3	48.6	70	70
[HOEMIM]OTF	2.8	6.3	6.3
[EMMIM]NO3	43.3	47.6	47.6
[HOOCEMIM]NO3	55.7	80	80

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As for the NO₃⁻-based ionic liquids, the yield and the initial reaction rate varied a lot, and this anion had little effect on the biocatalysis. Moreover, it was found that the obtained product was always (R)-(−)-epinephrine (i.e., ee (R); >99%), indicating that adding whichever of these ionic liquids into the systems did not alter the enantioselectivity of the product. Furthermore, when the cation was [HOOCEMIM]⁺, both the yield and the initial reaction rate were clearly enhanced. This result shows that the carboxymethyl ([HOOCEMIM]⁺) functionalized ionic liquid noticeably promotes the efficiency of biological catalysis.

Subsequently, we evaluated the biocompatibility of these ionic liquids with Acinetobacter sp. UN-16 cells by directly measuring the sugar metabolic activity retention. As can be seen in Fig. 1, the MAR values were lower in all the tested ionic liquid-containing systems than in the aqueous system, suggesting that all the examined ionic liquids were to some extent toxic to Acinetobacter sp. UN-16 cells. MAR values for various ionic liquid-containing systems varied greatly for each ionic liquid. [EMIM]BF₄ and [HOEMIM]OTF exhibited the worst biocompatibility with the cells. Their MAR values (25% and 22%) were far lower than for the rest. On the other hand, [HOOCEMIM]NO₃ exhibited the best biocompatibility with Acinetobacter sp. UN-16 cells, having the highest MAR value of 92%. These results suggest that the addition of [HOOCEMIM]NO₃ into the reaction system could substantially lower the toxic effects towards Acinetobacter sp. UN-16 cells.

Fig. 1 Metabolic activity retention (MAR, %) of Acinetobacter sp. UN-16 cells after 22 h culture in various ionic liquid-containing systems. * each data point was the average value of three experiments.

These ionic liquids might increase the cell membrane permeability and allow the substrate and product to pass more quickly in and out of the cells, resulting in an acceleration of the biocatalysis. Thus, it is meaningful to investigate the influence of the various ionic liquids on the cell membrane permeability of Acinetobacter sp. UN-16. Nucleic acids and proteins were assumed as the indicators of the release of intracellular components after removal of the cells, and their changes were recorded by ultraviolet spectrophotometry. This way, the OD values were taken as a direct measure of the ionic liquids' effect on cell membrane permeability.

As can be seen in Fig. 2, the addition of various hydrophilic ionic liquids to an aqueous system increased the membrane permeability of Acinetobacter sp. UN-16 cells, as the OD₂₆₀ and OD₂₈₀ values were clearly higher in ionic liquid-containing systems than in the aqueous systems. [HOEMIM]OTF gave the highest OD₂₆₀ and OD₂₈₀ values, implying that it greatly increased the cell membrane permeability. However, in the [HOEMIM]OTF-containing systems, there were very high toxic effects towards the cells (Fig. 1) and a very poor catalytic efficiency (Table 2). One possible explanation for this is that [EMIM]OTF-based ionic liquids might damage the cell membrane too seriously for the cells to maintain their catalytic activity. The lowest OD₂₆₀ and OD₂₈₀ values were observed for the [HOOCEMIM]NO₃-based system, which also had the best biotransformation results and the lowest toxicity towards the cells. These results show that a substantial increase in membrane permeability has a net negative effect on the reaction, although a moderate increase in cell membrane permeability is compatible with a high reaction efficiency.

Fig. 2 Effects of various ionic liquids on cell permeability. * OD₂₆₀ and OD₂₈₀ values given here show the increase in OD₂₆₀ and OD₂₈₀ for the medium from 0 h to 22 h; each data point represents the average value of three experiments.

Normally, the increase in cell membrane permeability caused by the ionic liquids may lead to harmful effects on the biological catalysis because of cell death and a lower availability of reducing equivalents for the reaction. Flow cytometry (FCM) was accepted as a particularly simple and accurate technique for measuring the membrane integrity of Acinetobacter sp. UN-16 cells after being incubated in various ionic liquid-containing systems for 22 h, and propidium iodide (PI) was used as a cell fluorescein dye. The results for cell membrane integrity are shown in Fig. S2.†

With the addition of various ionic liquids into the aqueous system, different types of ionic liquids exerted different effects on the cell membrane integrity, and the cell membrane integrity decreased significantly. As for the [EMIM]⁺-based ionic liquids, the cell membrane integrity was clearly dependent on the typical anion NO₃⁻, Cl⁻ and Br⁻, which were suitable for the [HOEMIM]⁺-based ionic liquids. In comparison with [HOEMIM]Cl and [HOEMIM]NO₃, [HOEMIM]OTF gave the worst cell membrane integrity (less than 40.3%, Fig. S2f†) and the OD values (Fig. 2) were relatively high. However, the microbial cells, in fact, displayed a very low activity and a very poor catalytic efficiency (Table 2). For the Cl⁻-based ionic liquids, cell membrane integrity decreased remarkably with the elongation of the alkyl chain. Moreover, the catalytic activity of the cell was also clearly reduced with increasing alkyl chain length. In addition, the cell membrane integrity was relatively higher for [HOOCEMIM]NO₃ than for the rest, which was in good agreement with the better biocompatibility of [HOOCEMIM]NO₃ with Acinetobacter sp. UN-16 cells. Moreover, [HOOCEMIM]NO₃ furnished much lower OD₂₆₀ and OD₂₈₀ values than [HOEMIM]NO₃ (Fig. 2). For all the ionic liquids, the highest cell membrane integrity and the lowest OD₂₆₀ and OD₂₈₀ values were recorded for [HOOCEMIM]NO₃, which could be correlated with the best performance of the cells in the [HOOCEMIM]NO₃-containing system. To sum up, only a moderate increase in cell membrane permeability could enhance the reaction efficiency, and [HOOCEMIM]NO₃ is the most suitable ionic liquid for the reaction, which is consistent with the previous conclusions.

In summary, the asymmetric biosynthesis of valuable (R)-(−)-epinephrine was successfully carried out by Acinetobacter sp. UN-16 cells in ten different ionic liquid-containing systems. The diverse water-miscible ionic liquids showed different influences on the asymmetric biosynthesis of (R)-(−)-epinephrine. Of all the tested ionic liquids, the efficiency of the bioreduction with Acinetobacter sp. UN-16 cells could be substantially enhanced in a [HOOCEMIM]NO₃-containing system as compared to both an aqueous system and the other ionic liquid-containing systems. Due to a markedly improved cell integrity and a moderate increase in cell membrane permeability, [HOOCEMIM]NO₃ proved to be the best ionic liquid for the bioreduction of adrenalone to (R)-(−)-epinephrine. The results clearly indicated that the Acinetobacter sp. UN-16 cell biocatalytic process with [HOOCEMIM]NO₃ is very promising for the efficient preparation of (R)-(−)-epinephrine. In subsequent articles, we will dive deeper into each feature and demonstrate how immobilization or co-immobilization of enzymes may be an alternative to further optimize the results in contrast to the whole-cell biocatalysis.

References

1. (a) V. M. Balcao and M. Vila, Adv. Drug Delivery Rev., 2015, 93, 25; (b) S. Cantone, V. Ferrario, L. Corici, C. Ebert, D. Fattor, P. Spizzo and L. Gardossi, Chem. Soc. Rev., 2013, 42, 6262; (c) R. A. Sheldon and S. van Pelt, Chem. Soc. Rev., 2013, 42, 6223.
2. (a) H. Vaghari, H. Jafarizadeh-Malmiri, M. Mohammadlou, A. Berenjian, N. Anarjan, N. Jafari and S. Nasiri, Biotechnol. Lett., 2016, 38, 223; (b) O. Barbosa, C. Ortiz, A. Berenguer-Murcia, R. Torres, R. C. Rodrigues and R. Fernandez-Lafuente, Biotechnol. Adv., 2015, 33, 435; (c) F. Secundo, Chem. Soc. Rev., 2013, 42, 6250; (d) C. Garcia-Galan, A. Berenguer-Murcia, R. Fernandez-Lafuente and R. C. Rodrigues, Adv. Synth. Catal., 2011, 353, 2885.
3. (a) U. Guzik, K. Hupert-Kocurek and D. Wojcieszynska, Molecules, 2014, 19, 8995; (b) K. Min and Y. J. Yoo, Biotechnol. Bioprocess Eng., 2014, 19, 553; (c) R. C. Rodrigues, C. Ortiz, A. Berenguer-Murcia, R. Torres and R. Fernandez-Lafuente, Chem. Soc. Rev., 2013, 42, 6290; (d) R. DiCosimo, J. McAuliffe, A. J. Poulose and G. Bohlmann, Chem. Soc. Rev., 2013, 42, 6437.
4. (a) F. Kazenwadel, M. Franzreb and B. E. Rapp, Anal. Methods, 2015, 7, 4030; (b) E. T. Hwang and M. B. Gu, Eng. Life Sci., 2013, 13, 49; (c) K. Hernandez and R. Fernandez-Lafuente, Enzyme Microb. Technol., 2011, 48, 107; (d) R. Fernandez-Lafuente, Enzyme Microb. Technol., 2009, 45, 405; (e) P. V. Iyer and L. Ananthanarayan, Process Biochem., 2008, 43, 1019.
5. (a) J. Heyland, N. Antweiler, J. Lutz, T. Heck, B. Geueke, H.-P. E. Kohler, L. M. Blank and A. Schmid, Microb. Biotechnol., 2010, 3, 74; (b) Z. J. Xiao, P. X. Du, W. Y. Lou, H. Wu and M. H. Zong, Chem. Eng. Sci., 2012, 84, 695.
6. (a) H. Gao, I.-W. Kim, J.-H. Choi, E. Khera, F. Wen and J.-K. Lee, Biochem. Eng. J., 2015, 96, 23; (b) J. H. Park, S. H. Lee, G. S. Cha, D. S. Choi, D. H. Nam, J. H. Lee, J.-K. Lee, C.-H. Yun, K. J. Jeong and C. B. Park, Angew. Chem., Int. Ed., 2015, 54, 969.
7. (a) R. Scopel, C. F. da Silva, A. M. Lucas, J. J. Garcez, A. T. do Espirito Santo, R. N. Almeida, E. Cassel and R. M. F. Vargas, Fluid Phase Equilib., 2016, 417, 203; (b) M. Armand, F. Endres, D. R. MacFarlane, H. Ohno and B. Scrosati, Nat. Mater., 2009, 8, 621.
8. T. Torimoto, T. Tsuda and K. Okazaki, Adv. Mater., 2010, 22, 1196.
9. (a) N. Galonde, K. Nott, A. Debuigne and A. Deleu, J. Chem. Technol. Biotechnol., 2012, 87, 451; (b) F. van Rantwijk and R. A. Sheldon, Chem. Rev., 2007, 107, 2757.
10. T. P. Pham, C. W. Cho and Y. S. Yun, Water Res., 2010, 44, 352.
11. (a) Y. Li, H. Cao, Q. Ouyang, C. Li, L. Zheng and X. Zhang, Chem. J. Chin. Univ., 2014, 35, 1057; (b) A. Zhao, C. Zhao, M. Li, J. Ren and X. Qu, Anal. Chim. Acta, 2014, 809, 128; (c) A. Romero, A. Santos and J. Tojo, J. Hazard. Mater., 2008, 151, 268.
12. B. Chen, K. Yang, P. Chen, W. Ruan and X. Xu, Comput. Appl. Chem., 2009, 26, 935.
13. (a) S. A. Gangu, L. R. Weatherley and A. M. Scurto, Curr. Org. Chem., 2009, 13, 1242; (b) Y.-N. Li, X.-A. Shi, M.-H. Zong, C. Meng, Y.-Q. Dong and Y.-H. Guo, Enzyme Microb. Technol., 2007, 40, 1305; (c) S. Bräutigam, D. Dennewald, M. Schürmann, J. Lutje-Spelberg, W. R. Pitner and D. Weuster-Botz, Enzyme Microb. Technol., 2009, 45, 310; (d) W. Wang, M.-H. Zong and W.-Y. Lou, J. Mol. Catal. B: Enzym., 2009, 56, 70.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra22140h

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