A method for chiral separation of amino acids by using Aspergillus sp. CM96 spore-modified capillary column

Abstract

The invention discloses a method for chiral separation of amino acids by Aspergillus sp. CM96 spore-modified capillary column, belonging to the field of bioanalysis. First, a capillary column modified with polyethyleneimine is prepared, and a novel capillary column is prepared by using Aspergillus sp. CM96 spores as chiral ligands, and the enantiomers of amino acids are separated under electrophoresis conditions. Using the enantiomers of proline as template molecules, the baseline separation of the enantiomers of proline can be achieved, and the chiral separation of arginine, histidine, phenylalanine and tryptophan is preliminarily realized. The separation method is simple to operate, and a novel high-selectivity separation and detection technology for amino acid enantiomers is established.

Description

Technical Field

[0001] The present invention belongs to the field of bioanalysis, and particularly relates to a method for chiral separation of amino acids by using Aspergillus sp. CM96 spore modified capillary column. Background Art

[0002] Since a pair of enantiomers have different physiological effects and pharmacological toxicities, chiral resolution has important practical significance. Relying on cell-based chiral stationary phases is conducive to exploring the recognition mechanism between chiral molecules and cells at the cellular level and can achieve a broader spectrum of chiral separation. However, the immobilization efficiency of cells and cell activity are easily affected by various factors in the microenvironment, and it is difficult to prepare cell-modified chiral stationary phases with excellent and stable performance.

[0003] Capillary electrochromatography (CEC) technology combines the high selectivity of high-performance liquid chromatography and the high efficiency of capillary electrophoresis. It can use microliter-level reagents to achieve rapid separation of various samples, so CEC is more environmentally friendly and economical. As a rapid, efficient, and low-sample-consumption technology, CEC has broad application prospects in the field of chiral separation.

[0004] The present invention selects Aspergillus sp. CM96 spores as chiral selectors. There is no literature report on the application of this chiral selector. The technical advantages of using Aspergillus sp. CM96 spores as chiral selectors to separate amino acids such as proline are as follows: the chiral selectivity of Aspergillus sp. CM96 spores for amino acids can be explored at the cellular level, and it can provide certain reference for the study of the chiral interaction mechanism between cells and amino acids. Fungi are an important type of eukaryotic microorganism in nature. The study of cell chirality not only helps to reveal the significance of chirality in vitro, but also has important significance for understanding the value of chirality existing in nature. Compared with vegetative cells, spores have special stability to resist the influence of external factors, which lays a foundation for preparing chiral stationary phases with stable performance. Developing a method for chiral separation of amino acid enantiomers is crucial for exploring chiral recognition in nature. Since the chiral preference of amino acid enantiomers may be an important indicator of life signs, it is of great significance for the present invention to further evaluate the interaction between amino acid enantiomers and the basic unit of life, cells. At present, there is little research on the chiral-specific interaction between amino acids and fungal spores. The present invention explores the chiral selectivity of Aspergillus sp. CM96 spores for amino acids at the cellular level. Summary of the Invention

[0005] The object of the present invention is to broaden the current range of chiral selectors available. Aspergillus sp. CM96 spores are used as chiral selectors. The Aspergillus sp. CM96 spores are adsorbed onto a capillary column, and the resulting Aspergillus sp. CM96 spore-modified capillary column is used for chiral separation of amino acids. Baseline separation of proline, arginine, histidine, phenylalanine, and tryptophan has also been preliminarily achieved.

[0006] To solve its technical problems, the technical solution adopted by the present invention is as follows:

[0007] First, the capillary is pretreated with alkali and acid. A γ-glycidoxypropyltrimethoxysilane solution is introduced into the capillary column, and after capping, it is reacted at 110 °C for 10 hours. It is rinsed with toluene and anhydrous methanol to remove impurities, and an epoxy-functionalized capillary column is obtained. Subsequently, a polyethyleneimine solution is introduced, and after capping, it is reacted at 25 °C for 10 hours and rinsed with water until neutral to obtain a polyethyleneimine-modified capillary column.

[0008] The Aspergillus sp. CM96 spore suspension is poured into the column, and further, a paraformaldehyde solution is poured into the column. It is reacted at room temperature for 1 hour, and the unreacted residues are removed by rinsing with a buffer solution, that is, rinsed with an electrophoresis buffer solution (20 mM citric acid-sodium citrate electrophoresis buffer solution, pH 5.0) to obtain an Aspergillus sp. CM96 spore-modified capillary column.

[0009] The mass fraction of the paraformaldehyde solution is 4%, and the solvent of the paraformaldehyde solution is 20 mM phosphate buffer. The role of paraformaldehyde is to increase the stability of the immobilization of Aspergillus sp. CM96 spores and improve the separation repeatability. If paraformaldehyde is not added, the separation effect deteriorates and the stability deteriorates.

[0010] The present invention further explores the influence of different environmental systems on the separation of amino acid enantiomers by the Aspergillus sp. CM96 spore-modified capillary column and discusses the interaction force between Aspergillus sp. CM96 spores and amino acid enantiomers.

[0011] The method for chiral separation of amino acids by the Aspergillus sp. CM96 spore-modified capillary column is as follows: The amino acid is dissolved in an electrophoresis buffer solution with a pH value of 4.5 - 7.0 and a concentration of 20 - 30 mM to prepare an amino acid solution. The capillary column is equilibrated with the electrophoresis buffer solution for 30 min and then injected. Injection is carried out at 10 kV for 3 seconds, and the amino acid enantiomers are separated under the condition of a 10 kV capillary electrophoresis separation voltage.

[0012] Further optimized to 20 mM citric acid - sodium citrate electrophoresis buffer with a pH value of 6.0. The pH value of the citric acid - sodium citrate electrophoresis buffer regulates the electroosmotic mobility and electrophoretic mobility, improving the separation efficiency.

[0013] The separation conditions are further preferably as follows: the capillary electrophoresis separation voltage is 10 kV; the buffer concentration is 20 mM; the injection volume is 10 kV, 3 s; the buffer pH value is 6.0; under these conditions, the interaction force between Aspergillus sp. CM96 spores and amino acid enantiomers is strong, and a better chiral recognition effect can be obtained.

[0014] Furthermore, the amino acid is one of proline, arginine, histidine, phenylalanine, and tryptophan.

[0015] After adopting the above technical solution, the beneficial effects obtained by the present invention are as follows: A method for chiral separation of multiple amino acids by Aspergillus sp. CM96 spore - modified capillary column provided by the present invention establishes a more broad - spectrum chiral separation system, which is simple to operate and further expands the application of capillary electrochromatography technology in the field of chiral separation. Description of the Drawings

[0016] Figure 1 : Influence of the pH value of the electrophoresis buffer on the separation of proline enantiomers by Aspergillus sp. CM96 spore - modified capillary column. Among them, the buffer pH values are a, 4.5; b, 5.0; c, 5.5; d, 6.0; e, 6.5; f, 7.0.

[0017] Figure 2 : Influence of the buffer concentration on the separation of proline enantiomers by Aspergillus sp. CM96 spore - modified capillary column. Among them, the buffer concentrations are a, 10 mM; b, 15 mM; c, 20 mM; d, 25 mM; e, 30 mM.

[0018] Figure 3 : Influence of the separation voltage on the separation of proline enantiomers by Aspergillus sp. CM96 spore - modified capillary column. Among them, the separation voltages are a, 5 kV; b, 10 kV; c, 15 kV; d, 20 kV.

[0019] Figure 4 : Influence of the injection volume on the separation of proline enantiomers by Aspergillus sp. CM96 spore - modified capillary column. Among them, the injection volumes are a, 10 kV, 1 s; b, 10 kV, 3 s; c, 10 kV, 6 s.

[0020] Figure 5:Repeatability of the separation of proline enantiomers by Aspergillus sp. CM96 spore-modified capillary column. Among them, a-c, within-day repeatability; c-e, between-day repeatability.

[0021] Figure 6 :Electrochromatograms of the separation of various amino acid enantiomers by Aspergillus sp. CM96 spore-modified capillary column. a, arginine; b, histidine; c, phenylalanine; d, tryptophan. Figure 7 :Electrochromatogram of the separation of proline enantiomers by an unmodified Aspergillus sp. CM96 spore capillary column.

[0022] Figure 8 :Electrochromatogram of the separation of tyrosine enantiomers by Aspergillus sp. CM96 spore-modified capillary column. Detailed implementation mode

[0023] The present invention will be further described with reference to the following examples, but it should be understood that these examples are only for illustrative purposes and should not be construed as limiting the implementation of the present invention.

[0024] The Aspergillus sp. CM96 spore suspension can be obtained by preparing it from the laboratory using known methods.

[0025] Example 1

[0026] 1. Preparation of Aspergillus sp. CM96 spore-modified capillary column

[0027] The quartz capillary was rinsed with pure water for 15 minutes, rinsed with 1 mol / L sodium hydroxide for 45 minutes, rinsed with pure water for 1 hour, then rinsed with 0.1 mol / L hydrochloric acid for 45 minutes, rinsed with pure water and anhydrous methanol for 30 minutes respectively, and blown with nitrogen for 1 hour for standby. A toluene solution of 60% mass fraction γ-glycidoxypropyltrimethoxysilane was introduced into the capillary column, the column was filled for 1 hour, and after capping, it was reacted at 110 °C for 10 hours, rinsed with toluene and anhydrous methanol for 30 minutes respectively, and dried with nitrogen to obtain an epoxy-functionalized capillary column. Subsequently, a solution of 20% mass fraction of polyethyleneimine was introduced, the column was filled for 1 hour, the solvent of the polyethyleneimine solution was 100 mM phosphate buffer (pH 8), and after capping, it was reacted at 25 °C for 10 hours and rinsed with water until neutral. The concentration was 2×10 6The column was filled with an Aspergillus sp. CM96 spore suspension at cells / mL. Further, a 4% paraformaldehyde solution (solvent: 20 mM phosphate buffer) was filled into the column, and the reaction was carried out for 1 hour. The unreacted residues were removed by rinsing with 20 mM phosphate buffer (pH 5), and then rinsed with a citric acid-sodium citrate electrophoresis buffer (20 mM citric acid-sodium citrate electrophoresis buffer, pH 5.0) for 1 hour to obtain a capillary column modified with Aspergillus sp. CM96 spores.

[0028] 2. Capillary electrochromatography analysis

[0029] The prepared capillary column modified with Aspergillus sp. CM96 spores was used to separate proline enantiomers. 200 mL of 20 mM citric acid-sodium citrate electrophoresis buffer was prepared. The purchased proline (Macklin (Shanghai, China)) was dissolved in the electrophoresis buffer (20 mM citric acid-sodium citrate electrophoresis buffer) to prepare a sample solution at 1 mg / mL at room temperature. By optimizing the pH value of the citric acid-sodium citrate electrophoresis buffer (pH 4.5 - 7.0), the electroosmotic mobility and electrophoretic mobility were adjusted to improve the separation efficiency. At the same time, the electrophoresis separation conditions including voltage (5 - 20 kV), citric acid-sodium citrate electrophoresis buffer concentration (10 - 30 mM), and injection volume (10 kV, 1 - 6 s) were optimized to obtain the best electrophoresis conditions for separating proline enantiomers.

[0030] Example 2

[0031] According to the method of Example 1, the preferred value of the pH of the citric acid-sodium citrate electrophoresis buffer was confirmed. In this example, the effect of the buffer pH on the separation of proline enantiomers by the Aspergillus sp. CM96 spore-modified capillary column was investigated. Figure 1 The pH values of the buffer were a, 4.5; b, 5.0; c, 5.5; d, 6.0; e, 6.5; f, 7.0.

[0032] From Figure 1 the preferred results, it can be seen that as the pH of the electrophoresis buffer decreased from 7.0 to 4.5, the retention time of proline enantiomers increased. When the pH value was 6.5 or 7.0, proline carried a negative charge, and there was an electrostatic repulsion between it and the spores in the Aspergillus sp. CM96 spore-modified capillary column, resulting in a shorter retention time. When the pH value decreased from 6.0 to 4.5, as the pH decreased, the positive charge of proline increased, so the electrostatic interaction between proline and the spores in the Aspergillus sp. CM96 spore-modified capillary column was enhanced, and the retention of proline was enhanced. From Figure 1It can be seen that when the pH value of the buffer solution is 4.5, the resolution of the proline electrochromatogram is the largest, but the peak shape is not very good and there is a tailing phenomenon. When the pH value is 6.0, the peak shape of the proline electrochromatogram is better and the resolution is also relatively large, achieving baseline separation. Therefore, the optimal pH value of the buffer solution is selected as 6.0.

[0033] Example 3

[0034] According to the method of Example 1, the optimal value of the buffer solution concentration was confirmed. In this example, the effect of the buffer solution concentration on the separation of proline enantiomers by Aspergillus sp. CM96 spore-modified capillary column was investigated. Under the condition of the optimal buffer solution pH value of 6.0, the concentration of citric acid-sodium citrate electrophoresis buffer solution was optimized ( Figure 2 ). Among them, the buffer solution concentrations were a, 10 mM; b, 15 mM; c, 20 mM; d, 25 mM; e, 30 mM.

[0035] As the buffer solution concentration increases, the resolution of the proline enantiomers increases. The increase in the buffer solution concentration will decrease the magnitude of the electroosmotic flow, resulting in an increase in the retention of proline. When the buffer solution concentration is 30 mmol / L -1 , it has a greater resolution, but the stronger retention of proline will cause a decrease in column efficiency. When the selected buffer solution concentration is lower than 15 mmol / L -1 , it cannot play a separation role. Therefore, the buffer solution concentration of 20 mmol / L -1 is selected as the optimal value, at which time baseline separation can be achieved and the peak shape is better.

[0036] Example 4

[0037] According to the method of Example 1, the optimal value of the operating voltage was confirmed. In this example, the effect of the operating voltage on the separation of proline enantiomers by Aspergillus sp. CM96 spore-modified capillary column was investigated. Among them, the separation voltages were a, 5 kV; b, 10 kV; c, 15 kV; d, 20 kV. From Figure 3 the preferred test results, it can be seen that as the voltage increases, the retention time of proline shows a decreasing trend. It is speculated that the reason for this phenomenon is that high voltage will cause a large electroosmotic flow and electrophoresis rate. As Figure 3 shown, although there will be a higher separation column efficiency when the voltage is 20 kV, the weak interaction between Aspergillus sp. CM96 spores and proline will lead to a decrease in resolution. Considering the better chiral separation ability and high column efficiency, the resolution is the largest when the voltage is 10 kV.

[0038] Example 5

[0039] According to the method of Example 1, the optimal value of the injection volume was confirmed. In this example, the effect of the injection volume on the separation of proline enantiomers by Aspergillus sp. CM96 spore-modified capillary column was investigated( Figure 4 ).

[0040] As the injection volume increased, it would cause chromatographic peak tailing and a decrease in column efficiency. The theoretical plate number of the electrophoretogram of proline enantiomers showed a decreasing trend with the increase of the injection volume. Since a higher sample volume would cause peak tailing and a decrease in column efficiency, the resolution of proline enantiomers would decrease at a higher sample volume. Finally, the optimal value of the injection volume was determined to be 10 kV, 3 s.

[0041] In summary, the optimal separation conditions for separating proline enantiomers are as follows: the separation voltage of capillary electrophoresis is 10 kV; the concentration of the electrophoresis buffer is 20 mM; the injection volume is 10 kV, 3 s; the pH value of the electrophoresis buffer is 6.0.

[0042] Figure 5 To study the repeatability of the separation of proline enantiomers by Aspergillus sp. CM96 spore-modified capillary column under the optimal separation conditions, as Figure 5 shown, under the optimal separation conditions, this separation system has high stability.

[0043] Example 6

[0044] According to the method of Example 1, the prepared Aspergillus sp. CM96 spore-modified capillary column was used to separate arginine, histidine, phenylalanine and tryptophan enantiomers. 200 mL of 20 mM citric acid-sodium citrate buffer electrophoresis buffer (pH value 6.0) was prepared. The purchased tryptophan (Macklin (Shanghai, China)), phenylalanine (Macklin (Shanghai, China)), histidine (Macklin (Shanghai, China)), and arginine (Macklin (Shanghai, China)) were respectively dissolved in the electrophoresis buffer and prepared into a 1 mg / mL sample solution at room temperature. The separation conditions were the same as the optimal conditions for separating proline. As Figure 6 shown, this separation system has good chiral separation performance for arginine, histidine, phenylalanine and tryptophan.

[0045] Example 7

[0046] According to the method of Example 1, the unmodified Aspergillus sp. CM96 spore-modified capillary column obtained by preparation was used to separate proline enantiomers. 200 mL of 20 mM citric acid-sodium citrate buffer electrophoresis buffer was prepared. The purchased proline (Macklin (Shanghai, China)) was dissolved in the electrophoresis buffer and prepared into a 1 mg / mL sample solution at room temperature. The optimal electrophoresis separation conditions for proline were selected for analysis. As Figure 7 shown, the polyethyleneimine-modified capillary column (unmodified Aspergillus sp. CM96 spores) has no chiral separation efficiency for proline enantiomers ( Figure 7 ).

[0047] Example 8

[0048] According to the method of Example 1, the Aspergillus sp. CM96 spore-modified capillary column obtained by preparation was used to separate tyrosine enantiomers. 200 mL of 20 mM citric acid-sodium citrate buffer electrophoresis buffer (pH 6.0) was prepared. The purchased tyrosine (Macklin (Shanghai, China)) was dissolved in the electrophoresis buffer and prepared into a 1 mg / mL sample solution at room temperature. The separation conditions were the same as the optimal conditions for separating proline. As Figure 8 shown, there were signs of separation of tyrosine in this separation system, but baseline separation was not achieved.

[0049] It can be seen from the above examples that the capillary electrochromatography technology based on Aspergillus sp. CM96 spores provides a rapid and efficient analysis platform for the separation of proline enantiomers.

[0050] Inspired by the above ideal embodiments based on the present invention, through the above description, relevant staff can completely make various changes and modifications without departing from the technical idea of the present invention. The technical scope of the present invention is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A Aspergillus method for chiral separation of amino acids by using a CM96 spore-modified capillary column, comprising the following steps: Step 1: Modification of capillary column: The capillary column is pretreated with alkali and acid, an organosilane solution is introduced into the capillary column for reaction, then a polyethyleneimine solution is introduced for reaction, and it is rinsed with water until neutral to obtain a polyethyleneimine-modified capillary column; Step 2: Aspergillus Modification of sp. CM96 spores: Pour Aspergillus the sp. CM96 spore suspension into the capillary column modified with polyethyleneimine in Step 1, then pour the paraformaldehyde solution into the column for reaction, and after the reaction, rinse with the electrophoresis buffer to obtain Aspergillus the capillary column modified with sp. CM96 spores; Step 3: Capillary electrophoresis detection: The Aspergillus sp. CM96 spore-modified capillary column was applied to the separation of amino acid enantiomers: A citric acid-sodium citrate electrophoresis buffer was prepared, and the amino acid was dissolved in the electrophoresis buffer to prepare an amino acid solution at room temperature. Through Aspergillus the sp. CM96 spore-modified capillary column, the separation of amino acid enantiomers was achieved under electrophoresis conditions; wherein the amino acid was one of arginine, histidine, phenylalanine and tryptophan; Among them, the pH value of the citric acid-sodium citrate electrophoresis buffer is 4.5 - 7.0, and the concentration of the electrophoresis buffer is 20 - 30 mM; the electrophoresis separation conditions are a voltage of 5 - 20 kV, an injection time of 1 - 6 s, and an injection voltage of 10 kV.

2. According to claim 1, a Aspergillus method for chiral separation of amino acids by CM96 spore-modified capillary column, characterized in that The organosilane solution described in Step 1 is a 60% by mass concentration of γ-glycidoxypropyltrimethoxysilane solution, and the polyethyleneimine solution has a mass concentration of 20%.

3. A method according to claim 2 for chiral separation of amino acids using a sp. CM96 spore-modified capillary column, characterized in that, Aspergillus The modification of the capillary column described in Step 1 is specifically: The capillary column is pretreated with alkali and acid, the γ-glycidoxypropyltrimethoxysilane solution is introduced into the capillary column, and after capping, it reacts at 110 °C for 10 hours, and is rinsed with an organic solvent to remove impurities to obtain an epoxy-functionalized capillary column; Then a polyethyleneimine solution is introduced, and after capping, it reacts at 25 °C for 10 hours, and is rinsed with water until neutral to obtain a polyethyleneimine-modified capillary column.

4. A method for chiral separation of amino acids using a sp. CM96 spore-modified capillary column according to claim 1, characterized in that, Aspergillus The mass concentration of the paraformaldehyde solution described in Step 2 is 4%; the reaction of filling the paraformaldehyde solution into the column is carried out at room temperature for 1 hour.

5. A method for chiral separation of amino acids using a capillary column modified with sp. CM96 spores according to claim 1, characterized in that, Aspergillus The capillary electrophoresis separation voltage described in Step 3 is 10 kV; the concentration of the electrophoresis buffer is 20 mM; the injection voltage is 10 kV, the injection time is 3 s; the pH value of the electrophoresis buffer is 6.0.

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