Method for highly selectively extracting phenylalanine by using ionic liquid surfactant / sulfonated calix[4]arene aqueous two-phase system

Abstract

The present invention discloses a method for highly selectively extracting phenylalanine by using an ionic liquid surfactant / sulfonated calix[4]arene aqueous two-phase system. The method comprises adding a solution to be extracted containing phenylalanine into the [C n mim]Br / SC[4] aqueous two-phase system, mixing evenly, and then centrifuging to completely separate the two phases, and collecting the upper phase, which is the extracted phenylalanine. The present invention utilizes the ionic liquid surfactant / sulfonated calix[4]arene aqueous two-phase system to achieve the highly selective extraction of phenylalanine.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic matter separation, and relates to a method for highly selectively extracting phenylalanine by using an ionic liquid surfactant / sulfonated calix[4]arene aqueous two-phase system. Background Art

[0002] Phenylalanine (Phe) is one of the essential amino acids for humans and animals, and has broad application values in the fields of medicine, food, etc. Therefore, it has important practical significance to achieve the highly selective extraction of such biomolecules.

[0003] The aqueous two-phase system (ATPS) is a new type of liquid-liquid extraction technology, which has characteristics such as mild conditions and good stability, and has received wide attention. However, traditional ATPSs, such as polymer-polymer, polymer-salt, polymer-surfactant, etc. ATPSs, have high viscosity and a small polarity range, which is not conducive to the mass transfer of biomolecules and limits the selective extraction efficiency of ATPSs. Ionic liquids are a kind of green solvents with diverse structures and easy modification. It has been found that when ionic liquids are introduced as additives into polymer / salt ATPSs or aqueous two-phase systems based on ionic liquids are constructed, the performance of ATPSs in various aspects will be improved, such as the phase separation time becoming faster and the polarity range of the two phases increasing. Neves et al. (C.M.M.S.Neves et al., Biochemical Engineering Journal, 2019, 141, 239-246) introduced ionic liquids ([Ch]Cl, [C4mim]Cl, [C4mpyr]Cl, [C4mpip]Cl, [N 4444 Cl or [P 4444 Cl) as additives into PEG 400 / (NH4)2SO4 ATPS to extract phenolic compounds, alkaloids and amino acids, and found that the above substances were all enriched in the PEG phase, and the distribution coefficient of phenylalanine was about 2.5. Sousa et al. (R.C.S.Souza et al., Journal of Chemical Technology and Biotechnology, 2018, 93, 1940-1947) used ionic liquid ([C4mpyr]Cl) / K3C6H5O7 / C6H8O7 ATPS to extract dopamine, levodopa, phenylalanine and tyrosine, and found that the distribution coefficients of the above substances were all greater than 1, the distribution coefficient of phenylalanine was about 4, and the selectivity of phenylalanine for the other three substances (dopamine, levodopa and tyrosine) was 1.1, 1, 0.8 respectively. To sum up, it is impossible to achieve the highly selective extraction of phenylalanine by using the existing ATPSs. Summary of the Invention

[0004] The object of the present invention is to provide a method for highly selectively extracting phenylalanine by using an ionic liquid surfactant / sulfonated calix[4]arene ([C n mim]Br / SC[4]) aqueous two-phase system.

[0005] The technical solution for achieving the object of the present invention is as follows:

[0006] A method for highly selectively extracting phenylalanine by using an ionic liquid surfactant / sulfonated calix[4]arene aqueous two-phase system, specifically: adding a solution to be extracted containing phenylalanine into the ionic liquid surfactant / sulfonated calix[4]arene aqueous two-phase system, mixing evenly, and then centrifuging to completely separate the two phases, and collecting the upper phase, which is the extracted phenylalanine; the ionic liquid surfactant is 1-decyl-3-methylimidazolium bromide ([C 10 mim]Br) or 1-dodecyl-3-methylimidazolium bromide ([C 12 mim]Br).

[0007] The structural formula of SC[4] described in the present invention is: [C 10 The structural formula of mim]Br is: [C 12 The structural formula of mim]Br is:

[0008] In the present invention, in the [C n mim]Br / SC[4] aqueous two-phase system, there is no special limitation on the concentrations of [C n mim]Br and SC[4], as long as it is in the two-phase region of the aqueous two-phase system. Specifically, the concentration of [C n mim]Br can be 0.3 wt.% to 40 wt.%, and the concentration of SC[4] can be 1 wt.% to 14 wt.%.

[0009] In a specific embodiment of the present invention, in the [C n mim]Br / SC[4] aqueous two-phase system, the concentration of [C n mim]Br is 15 wt.% to 25 wt.%, and the concentration of SC[4] is 5 wt.% to 10 wt.%. Preferably, the concentration of [C n mim]Br is 20 wt.%, and the concentration of SC[4] is 5 wt.%.

[0010] Compared with the prior art, the present invention has the following advantages:

[0011] The present invention constructs a [C n mim]Br / SC[4] aqueous two-phase system for the first time. At 20 wt.% [C 12Under the [C Description of the Drawings

[0012] Figure 1 For [C n Phase diagram of [C

[0013] Figure 2 For [C 10 Influence result diagram of [C

[0014] Figure 3 For [C 12 Influence result diagram of [C Detailed Description of the Invention

[0015] The present invention will be further described in detail below with reference to the embodiments and the drawings.

[0016] To detect the selectivity of [C n aqueous two-phase system of [C The structural formula of MA is: The structural formula of Trp is: The structural formula of Theo is: The structural formula of Tyr is: The structural formula of Phe is:

[0017] In the following embodiments, deionized water is used as the solvent for preparing the ionic liquid surfactant solution and the sulfonated calix[4]arene solution.

[0018] Van shows a characteristic absorption peak at 280 nm, MA and Phe show characteristic absorption peaks at 257 nm, Trp shows a characteristic absorption peak at 279 nm, Theo shows a characteristic absorption peak at 272 nm, and Tyr shows a characteristic absorption peak at 275 nm. With 10 the upper phase of [C n mim]Br / SC[4]ATPS as the solvent, a series of solutions of Van, MA, Trp, Theo, Tyr, and Phe were prepared. By measuring the absorbance of target molecules at different concentrations at their characteristic absorption peaks, standard curves were obtained. Then, [C

[0019] mim]Br / SC[4]ATPSs containing different target molecules (Van or MA or Trp or Theo or Tyr or Phe) were configured. The ATPSs were vigorously stirred for 15 min, maintained at 25 °C for at least 30 min, and then centrifuged for 5 min to ensure complete separation of the two phases. Finally, the volume of the upper phase of the ATPSs was determined. Using the standard curves, the concentration of Van or MA or Trp or Theo or Tyr or Phe in the upper phase could be obtained, and the distribution coefficient of Van or MA or Trp or Theo or Tyr or Phe was calculated.

[0020] The calculation formula for the distribution coefficient of Van, MA, Trp, Theo, Tyr, and Phe is as follows: 上 V 上 / (C 总 V 总 -C 上 V 上 ),

[0021] where K: the distribution coefficient of the substance; C 总 : the total concentration of the substance in ATPS; V 总 : the total volume of ATPS; C 上 : the concentration of the substance in the upper phase of ATPS; V 上 : the volume of the upper phase of ATPS.

[0022] The calculation formula for selectivity is as follows:

[0023] S1 = K Phe / K van , S2 = K Phe / K MA , S3 = K Phe / K Trp , S4 = K Phe / K Theo , S5 = K Phe / K Tyr ,

[0024] where S: selectivity, KMA : Distribution coefficient of MA, K Phe : Distribution coefficient of Phe, K Trp : Distribution coefficient of Trp, K Van : Distribution coefficient of Van, K Theo : Distribution coefficient of Theo, K Tyr : Distribution coefficient of Tyr.

[0025] Example 1

[0026] [C n mim]Br / SC[4]ATPS phase diagram:

[0027] (1) Prepare a series of [C 10 mim]Br solutions (10wt.% - 50wt.%), add 30wt.% SC[4] mother liquor dropwise to the above solutions, mix well. After turbidity appears, record the weight of the added solution, and the composition of a series of cloud point components can be calculated.

[0028] (2) Prepare a series of SC[4] solutions (1wt.% - 15wt.%), add 50wt.% [C 10 mim]Br mother liquor dropwise to the above solutions, mix well. After turbidity appears, record the weight of the added solution, and the composition of a series of cloud point components can be calculated.

[0029] (3) Change [C 10 mim]Br to [C 12 mim]Br, and repeat steps (1) - (2).

[0030] Example 2

[0031] [C 10 mim]Br / SC[4]ATPS effect on the distribution of target molecules:

[0032] (1) Prepare 15wt.% [C 10 mim]Br / 5wt.% SC[4]ATPS containing 65mM Van (or 150mM MA or 50mM Trp or 40mM Theo or 2mM Tyr or 150mM Phe), mix well, then mix in a constant temperature mixer at 25°C for 15 min and equilibrate for 30 min, centrifuge for 5 min, and detect the absorbance value of the upper phase of ATPS at the characteristic absorption peak of the target molecule. Calculate the concentration of Van (or MA or Trp or Theo or Tyr or Phe) in the upper phase according to the standard curve.

[0033] (2) Change [C 10When the concentration of [[C]]mim]Br is 20 wt.% or 25 wt.%, repeat step (1).

[0034] According to the calculation formula of the distribution coefficient, calculate the distribution coefficients of Van (or MA or Trp or Theo or Tyr or Phe) in each system. The results are as Figure 2 shown. According to the calculation formula of selectivity, calculate the selectivity of Phe relative to other substances. The results are shown in Table 1.

[0035] Example 3

[0036] [C 12 Effect of [[C]]mim]Br / SC[4] ATPS on the distribution of target molecules:

[0037] (1) Prepare 15 wt.% [[C]]mim]Br / 5 wt.% SC[4] ATPS containing 65 mM Van (or 150 mM MA or 50 mM Trp or 40 mM Theo or 2 mM Tyr or 150 mM Phe). After the solid substances are completely dissolved, mix them in a constant temperature mixer at 25 °C for 15 min and then equilibrate for 30 min. Centrifuge for 5 min, and measure the absorbance value of the upper phase of ATPS at the characteristic absorption peak of the target molecule. Calculate the concentration of Van (or MA or Trp or Theo or Tyr or Phe) in the upper phase according to the standard curve. 12

[0038] (2) Change the concentration of [[C]]mim]Br in the above ATPS to 20 wt.% or 25 wt.%, and repeat step (1).(2) Change the concentration of [[C]]mim]Br in the above ATPS to 20 wt.% or 25 wt.%, and repeat step (1). 12 When the concentration of [[C]]mim]Br is 20 wt.% or 25 wt.%, repeat step (1).

[0039] According to the calculation formula of the distribution coefficient, calculate the distribution coefficients of Van (or MA or Trp or Theo or Tyr or Phe) in each system. The results are as Figure 3 shown. According to the calculation formula of selectivity, calculate the selectivity of Phe relative to other substances. The results are shown in Table 1.

[0040] Table 1 Composition of ATPS in each system and its selectivity for Phe

[0041]

[0042] Figure 1 is the aqueous two-phase system constructed by [[C]]mim]Br (n = 10, 12) and SC[4]. It is found that as the alkyl chain length of the ionic liquid surfactant increases, the area of the two-phase region increases. n When the concentration of SC[4] is 5 wt.%, [[C]]mim]Br (n = 10, 12) and SC[4] construct an aqueous two-phase system, and it is found that as the alkyl chain length of the ionic liquid surfactant increases, the area of the two-phase region increases.

[0043] Figure 2 It shows that when the concentration of SC[4] is 5 wt.%, [[C]]mim]Br (n = 10, 12) and SC[4] construct an aqueous two-phase system, and it is found that as the alkyl chain length of the ionic liquid surfactant increases, the area of the two-phase region increases. 10When the concentration of [mim]Br increases from 15 wt% to 25 wt%, the distribution coefficients of Van, MA, and Trp change slightly. The distribution coefficient of Van changes from 0.13 to 0.18, the distribution coefficient of MA changes from 0.17 to 0.34, and the distribution coefficient of Trp changes from 0.16 to 0.46. The distribution coefficients of Theo and Tyr increase slightly. The distribution coefficient of Theo increases from 0.60 to 1.01, and the distribution coefficient of Tyr increases from 1.03 to 1.49. The distribution coefficient of Phe increases significantly, from 1.34 to 5.55. [C 10 When the concentration of [mim]Br increases from 15 wt% to 25 wt%, the selectivity of Phe for other substances first increases and then decreases. S1 increases from 10.62 to 31.57 and then changes to 30.48, S2 increases from 7.79 to 19.78 and then changes to 16.48, S3 increases from 8.33 to 17.94 and then changes to 12.11, S4 increases from 2.25 to 5.62 and then changes to 5.51, S5 increases from 1.30 to 3.91 and then changes to 3.72 (Table 1). The ATPS composition with the best selectivity is 20 wt.% [C 10 [mim]Br / 5 wt.% SC[4].

[0044] Figure 3 It shows that when the concentration of SC[4] is 5 wt.%, [C 12 When the concentration of [mim]Br increases from 15 wt% to 25 wt%, the distribution coefficients of Van, MA, and Trp change slightly. The distribution coefficient of Van changes from 0.06 to 0.05, the distribution coefficient of MA increases from 0.15 to 0.29, and the distribution coefficient of Trp increases from 0.14 to 0.27. The distribution coefficients of Theo and Tyr increase slightly. The distribution coefficient of Theo increases from 0.56 to 0.98, and the distribution coefficient of Tyr increases from 1.28 to 1.78. The distribution coefficient of Phe increases significantly, from 1.84 to 7.02. [C 12 When the concentration of [mim]Br increases from 15 wt% to 25 wt%, the selectivity of Phe for other substances first increases and then decreases. S1 increases from 29.23 to 150.87 and then changes to 133.88, S2 increases from 11.90 to 55.93 and then changes to 23.83, S3 increases from 13.53 to 54.38 and then changes to 26.03, S4 increases from 3.32 to 9.16 and then changes to 7.12, S5 increases from 1.44 to 4.49 and then changes to 3.95 (Table 1). The ATPS composition with the best selectivity is 20 wt.% [C 12 [mim]Br / 5 wt.% SC[4].

Claims

1. A method for selectively extracting phenylalanine using an ionic liquid surfactant / sulfonated calix[4]arene aqueous two-phase system, characterized in that, Specifically: Add the solution to be extracted containing phenylalanine into the ionic liquid surfactant / sulfonated calix[4]arene aqueous two-phase system. After mixing evenly, centrifuge to completely separate the two phases, and collect the upper phase, which is the extracted phenylalanine; the ionic liquid surfactant / sulfonated calix[4]arene aqueous two-phase system uses deionized water as the solvent, the concentration of the ionic liquid surfactant is 15 wt.% - 20 wt.%, and the concentration of the sulfonated calix[4]arene is 5 wt.%; the ionic liquid surfactant is [C 10 mim]Br or [C 12 mim]Br, and the structural formula of the sulfonated calix[4]arene is , [C 10 mim]Br has the structural formula of , [C 12 mim]Br has the structural formula of .

2. The method according to claim 1, characterized in that, In the ionic liquid surfactant / sulfonated calix[4]arene aqueous two-phase system described above, the concentration of the ionic liquid surfactant is 20 wt.%, and the concentration of the sulfonated calix[4]arene is 5 wt.%.

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