Enzymatic synthesis method of N (2)-L-alanyl-L-glutamine

Abstract

The invention relates to an enzymatic synthesis method of glutamine dipeptide, and belongs to the technical field of biosynthesis. In order to solve the problem of poor purity in the prior art, the invention provides an enzymatic synthesis method of N-(2)-L-alanyl-L-glutamine dipeptide, which is characterized by comprising the following steps: reacting L-glutamine with L-alanine methyl ester to obtain N-(2)-L-glutamine dipeptide by using alpha-ester acyltransferase which does not have signal peptide and has an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.3. The invention also provides an enzymatic synthesis method of N-(2)-L-glutamine dipeptide. The L-glutamine and L-alanine methyl ester can be effectively promoted to react so as to be converted into the L-glutamine dipeptide, the enzyme activity is high, the selectivity is high, the purity reaches 99% or above, and the yield reaches 80% or above.

Description

Technical Field

[0001] The invention relates to an enzymatic synthesis method of glutathione, and belongs to the technical field of biosynthesis. Background Art

[0002] Alanyl-glutamine, also known as L-alanyl-L-glutamine, is a biologically active dipeptide composed of alanine and glutamine residues. As a dipeptide, it is a dipeptide that is extremely important in the field of modern medical health. Alanyl-glutamine is very important in maintaining the body's physiological functions. It has the effects of improving intestinal cell activity, immune function, promoting protein and ester synthesis, and regulating ammonia and sugar concentrations in the blood. It can be used clinically for the prevention and treatment of intestinal diseases and lung tissue damage.

[0003] The main biosynthetic pathways currently used for the synthesis of glutathione are biocatalysis and microbial fermentation, which mainly use L-amino acid ligase and α-ester acyltransferase for reaction and synthesis. Among them, the L-amino acid ligase method directly catalyzes the production of glutathione by the bacteria themselves, which has the advantage of the lowest cost in the product formation stage, but the process of extracting high-purity glutathione using this route is complex and difficult, and the yield is also lower than other routes. Another biological method is to use α-ester acyltransferase to catalyze the production of glutathione using L-alanine methyl ester as a substrate. Compared with L-amino acid ligase, α-ester acyltransferase has a higher product concentration and a shorter reaction time, which is more suitable for industrial implementation by fermentation and synthesis enterprises, and the α-ester acyltransferase method is also one of the main research directions at present.

[0004] However, when using the α-ester acyl transferase method, the residual impurities in the enzyme solution have a greater impact on the raw materials and products of the reaction. In order to reduce the host cell residues of Escherichia coli, especially the residual endotoxins, most researchers currently turn to yeast as an expression host to avoid the degradation of raw materials and products. Genetically engineered bacteria also need to knock out genes such as pepA, pepB, pepD, pepN, dpp and dtp, which is not conducive to practical application. In addition, the expression of α-ester acyl transferase reported in existing literature mostly uses genes with long signal peptide sequences. After the gene is expressed in Escherichia coli, the target protein has a long useless sequence, which reduces the expression efficiency, and the composition of the enzyme solution is complex, which is not conducive to the purification of the enzyme; at the same time, the residual host cell components (residues of substances such as endotoxins) in the obtained enzyme solution, as well as the hydrolytic enzymes such as peptidases of the host cells, are not easy to remove, which hydrolyzes the raw materials and products, resulting in poor conversion rate and purity quality of the reaction. Summary of the invention

[0005] In view of the defects in the above prior art, the present invention provides an enzymatic synthesis method of alanine dipeptide, which solves the problem of how to realize the synthesis using a transferase without a signal peptide to achieve easy purification and high catalytic conversion rate.

[0006] The objective of the present invention is achieved through the following technical scheme: an enzymatic synthesis method of alanine dipeptide, characterized in that the method comprises using an α-ester acyltransferase without a signal peptide whose amino acid sequence is shown in SEQ ID NO.1 or SEQ ID NO.3 to react L-glutamine and L-alanine methyl ester to obtain alanine dipeptide.

[0007] The present invention uses for the first time an α-ester acyltransferase without a useful part of a signal peptide as an enzyme catalyzing the reaction of L-glutamine and L-alanine methyl ester, and it is found that it has high activity, the product is easy to purify, and the obtained product has the effect of high purity and high yield. Specifically, the present invention improves the amino acid sequence of α-ester acyltransferase, removes part of the signal peptide amino acid sequence (KNTISCLTLALLSASQLHA), and uses an enzyme without a signal peptide as the required enzyme to promote the reaction of L-glutamine and L-alanine methyl ester. It is found that it also has enzyme activity and can achieve a high product yield; compared with the existing enzymes that all need to use a signal peptide, it provides a new direction to achieve the effect of using an enzyme without a signal peptide and having high catalytic enzyme activity. At the same time, the amino acid sequence of α-ester acyltransferase is further innovated, and the signal peptide (i.e., amino acid without signal peptide) is removed while the amino acid sequence is provided with His-tag or QQQQ sequence, and finally the characteristics of the amino acid sequence shown in SEQ ID NO.1 (sequence with His-tag and without signal peptide) or SEQ ID NO.3 (sequence with QQQQ and without signal peptide) are more conducive to its stable expression and preservation, and can better avoid the hydrolysis of raw materials and products by the residues of hydrolases such as peptidases of host cells in the crude enzyme solution, and reduce the residues of substances such as host endotoxins, so as to achieve the excellent conversion rate when the enzyme is applied to the synthesis of propylglutathione, so that the obtained product has high purity quality, the purity reaches more than 99%, and the yield reaches more than 80%. The structural formula of the above-mentioned propylglutathione is as follows:

[0008]

[0009] In the above-mentioned enzymatic synthesis method of alanine dipeptide, preferably, when the amino acid sequence of the α-ester acyltransferase is SEQ ID NO.1, the nucleic acid sequence of the α-ester acyltransferase is as shown in SEQ ID NO.2; when the amino acid sequence of the α-ester acyltransferase is SEQ ID NO.3, the nucleic acid sequence of the α-ester acyltransferase is as shown in SEQ ID NO.4.

[0010] In the above-mentioned enzymatic synthesis method of glutathione, preferably, the α-ester acyltransferase is selected from a free enzyme or an immobilized enzyme. By adopting the above-mentioned α-ester acyltransferase, the expressed enzyme has the advantages of high purification quality and less influence of foreign enzymes, so that the free enzyme or immobilized enzyme can be used to promote the reaction, and both have good reaction conversion rate, high product yield, and high purity quality. As a further preference, the α-ester acyltransferase is an immobilized enzyme, and the carrier used is selected from a metal affinity carrier, an amino carrier or an epoxy carrier.

[0011] The α-ester acyltransferase uses an immobilized enzyme that has been immobilized, which can better reduce the impact of the impurities in the enzyme solution on the hydrolysis of the raw material and the product. The above-mentioned amino carrier is an activated amino carrier, which can more effectively load the above-mentioned α-ester acyltransferase. When the amino acid sequence carries a His-tag, it can be better adsorbed on an affinity carrier such as Ni-IDA or Ni-NTA for immobilization, making it easier to separate from the enzyme solution obtained after expression in Escherichia coli, better avoiding the adsorption of hydrolases such as peptidases or endotoxins in the host cell, effectively achieving the effect of purification and immobilization of α-ester acyltransferase, and after immobilization, it also has the effect of good stability, can be directly used for the enzymatic synthesis of propylglutathione, and the purity and yield of propylglutathione obtained reach a higher level. It is further preferred that the immobilized enzyme is obtained by the following method:

[0012] The α-ester acyltransferase with SEQ ID NO.1 or SEQ ID NO.3 sequence is fermented and expressed in Escherichia coli (Escherichia coli BL21 (DE3)) using the PET expression system, and then the bacterial cells are collected, and then the crude enzyme liquid is obtained by centrifugation after crushing, and the crude enzyme liquid is immobilized with an immobilization carrier to obtain an immobilized α-ester acyltransferase enzyme. The α-ester acyltransferase containing a His-tag or a QQQQ sequence is mixed with a carrier, so that the enzyme can be effectively adsorbed on the carrier, and other components in the host cell, such as peptidase and other hydrolases, are prevented from being adsorbed together, so that the α-ester acyltransferase with a His-tag or a QQQQ sequence can be separated from other host components such as Escherichia coli, thereby improving the quality of purification.

[0013] In the above enzymatic synthesis method of glutathione, preferably, the reaction temperature is 10°C to 20°C. By using the above α-ester acyltransferase to promote the reaction, it has the advantages of mild reaction and easy operation, and can better ensure the enzyme activity, so that it has the advantage of high catalytic conversion rate. As a further preference, the reaction temperature is 12°C to 15°C.

[0014] In the above enzymatic synthesis method of alanylglutamine, the pH value of the reaction is preferably controlled at 8.0 to 8.5, which can make the α-ester acyltransferase used have better enzyme activity, be more conducive to the enzymatic catalysis to convert the substrate into the product alanylglutamine, and have a high product yield.

[0015] In the above-mentioned enzymatic synthesis method of alanine dipeptide, preferably, the molar ratio of L-glutamine to L-alanine methyl ester is 1:1.0-2.0, which can more effectively utilize the substrate raw materials and improve the utilization rate of the substrate raw materials. As a further preferred method, the molar ratio of L-glutamine to L-alanine methyl ester is 1:1.2-1.5.

[0016] In the above enzymatic synthesis method of alanine, preferably, the mass ratio of L-glutamine to immobilized enzyme is 1:0.5-1.0. It has excellent enzyme catalytic activity and high product conversion rate. As a further preference, the mass ratio of L-glutamine to immobilized enzyme is 1:0.6-0.8.

[0017] In the above enzymatic synthesis method of alanine dipeptide, preferably, the reaction time is 0.5 to 5 hours.

[0018] The chemical reaction equation of the enzymatic synthesis method of propylglutathione is as follows:

[0019]

[0020] In summary, compared with the prior art, the present invention has the following advantages:

[0021] 1. By using an enzyme with no signal peptide amino acids and carrying a His-tag or QQQQ sequence, the α-ester acyltransferase can be better purified and immobilized, which is more conducive to its stable expression and preservation. When used to synthesize glutathione, it has an excellent conversion rate and the obtained product has high purity quality.

[0022] 2. The enzymatic method of the present invention can effectively promote the reaction of substrates L-glutamine and L-alanine methyl ester to convert them into alanine dipeptide, and the enzyme activity is high, with high selectivity, a purity of more than 99%, and a yield of more than 80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a comparative liquid chromatography analysis chart of propylglutathione obtained in Example 9 of the present invention and a standard product.

[0024] Figure 2 It is a mass spectrometry analysis diagram of propylglutathione obtained in Example 9 of the present invention and a standard substance.

[0025] Figure 3 This is the mass spectrometry analysis of the propylglutathione standard.

[0026] Figure 4 This is the hydrogen spectrum analysis diagram of propylglutathione obtained in Example 9 of the present invention.

[0027] Figure 5 This is the hydrogen spectrum analysis diagram of the propylglutathione standard.

[0028] Figure 6 This is the carbon spectrum analysis diagram of propylglutathione obtained in Example 9 of the present invention.

[0029] Figure 7 This is the carbon spectrum analysis diagram of the propylglutathione standard.

[0030] Figure 1 In the figure, the upper part is the spectrum of the propylglutathione standard; the lower part is the spectrum of the propylglutathione sample of Example 13. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further specifically described below through specific embodiments and drawings, but the present invention is not limited to these embodiments.

[0032] Example 1

[0033] The gene sequence shown in SEQ ID NO.2 was synthesized by conventional gene synthesis technology, cloned into plasmid pET30a, inoculated into Escherichia coli BL21 (DE3), single colonies were selected and cultured in LB medium containing 50 μg / ml kanamycin at 37°C, 0.5 mM IPTG was added to induce at 15°C for 16 hours after the OD value reached 0.8, and the bacterial cells were collected by centrifugation, and the obtained bacterial cells were further broken to obtain a crude enzyme solution, i.e., a crude enzyme solution containing α-ester acyltransferase, and the amino acid sequence of α-ester acyltransferase was shown in SEQ ID NO.1 (without signal peptide and with His-tag). The obtained crude enzyme solution was further purified, specifically, the crude enzyme solution was centrifuged to obtain a supernatant.

[0034] Furthermore, the metal affinity carrier can be placed in a chromatography column, and after washing, the supernatant obtained above is added, and the outflow rate is controlled to be 15-20s / drop. After the enzyme solution is completely adsorbed, it is first washed with 15ml of washing solution, and then 10ml of elution solution is added for elution to obtain a purified enzyme solution. The amino acid sequence of α-ester acyltransferase in the purified enzyme solution is shown in SEQ ID NO.1.

[0035] Example 2

[0036] The gene sequence shown in SEQ ID NO.4 was synthesized by conventional gene synthesis technology, cloned into plasmid pET30a, and inoculated into Escherichia coli BL21 (DE3), and single colonies were selected and cultured in LB medium containing 50 μg / ml kanamycin at 37°C with shaking. After the OD value reached 0.8, 0.5 mM IPTG was added and induced at 15°C for 16 hours, and the bacterial cells were collected by centrifugation. The obtained bacterial cells were further broken to obtain a crude enzyme solution, i.e., a crude enzyme solution containing α-ester acyltransferase, and the amino acid sequence of α-ester acyltransferase was shown in SEQ ID NO.3 (hereinafter referred to as T195) (without signal peptide and with QQQQ sequence). The obtained crude enzyme solution was further purified, and specifically, the crude enzyme solution was centrifuged to obtain a supernatant (called T195 supernatant).

[0037] Example 3

[0038] Analysis of the hydrolysis activity of the crude enzyme solution and the purified enzyme solution obtained in Example 1 on L-Ala-OMe

[0039] A 10 g / L L-Ala-OMe solution was prepared with a 0.2 mol / L boric acid solution and added to three 50 ml centrifuge tubes, respectively. The three centrifuge tubes were numbered as follows: no enzyme, crude enzyme solution, and purified enzyme solution obtained in Example 1 (the amount of enzyme protein added to the three centrifuge tubes was the same). The total system was 20 ml. The reaction conditions were controlled at 15° C. and pH 8.0. The reaction was carried out on a shaking table at 200 rpm. After 3 hours of reaction, samples were taken to determine the concentration change.

[0040] The results show that the residual L-Ala-OMe in the three centrifuge tubes without enzyme, with crude enzyme solution and with purified enzyme solution are 8.2, 2.9 and 8.3 g / L respectively. The above analysis data show that the crude enzyme solution contains a high enzyme activity for decomposing L-Ala-OMe, and the enzyme after immobilization and purification by using the above-mentioned α-ester acyltransferase of the present invention (gene sequence such as SEQ ID NO.2, amino acid sequence such as SEQ ID NO.1) has high purity performance and basically does not decompose the raw material L-Ala-OMe.

[0041] Analysis of crude enzyme solution and purified enzyme solution on AG hydrolysis activity

[0042] A 10 g / L glutathione solution was prepared with a 0.2 mol / L boric acid solution and added to three 50 ml centrifuge tubes, respectively. The three centrifuge tubes were numbered as follows: no enzyme, crude enzyme, and purified enzyme such as SEQ ID NO.1 obtained in Example 1 (the amount of protein added was the same), with a total system of 20 ml. The reaction conditions were controlled at 15° C. and pH 8.0, and the shaking reaction was carried out at 200 rpm. After 3 hours of reaction, samples were taken to determine the concentration change.

[0043] The results showed that the residual AG in the three centrifuge tubes without enzyme, with crude enzyme solution and with purified enzyme were 7.9, 4.4 and 8.2 g / L respectively, which also showed that the crude enzyme contained a higher level of impurity enzyme activity that could decompose AG.

[0044] Example 4

[0045] To a 50 mL reaction bottle, add 29 mL of pH = 8.0 PBS buffer and 4 g of activated amino resin (model: 703, manufacturer: Xi'an Lanxiao Technology New Materials Co., Ltd.), control the reaction temperature at 15°C, add 1 ml of the T195 supernatant obtained in Example 2, stir and adsorb for 4 hours, filter, and obtain 3.9 g of immobilized enzyme.

[0046] Example 5

[0047] The reaction of the purified α-ester acyltransferase using the amino acid sequence shown in SEQ ID NO.1

[0048] Weigh 10g L-glutamine and 1.3g boric acid into a beaker, add 20mL purified water, cool to 15°C, start stirring, use 2M sodium hydroxide to adjust the pH of the reaction system to about 8.0, add 10ml of the pure enzyme solution obtained by the method of Example 1, weigh 12g L-alanine methyl ester hydrochloride and dissolve it in 20ml purified water, adjust the pH to 6.60, use a peristaltic pump to slowly pump it into the column, control the time for 1h, and at the same time, add 2M sodium hydroxide to keep the pH at 8.0. The reaction ends after 2h, the total volume is 92ml, and the concentration of alanine dipeptide generated by HPLC liquid phase detection is 93.25g / L.

[0049] The conditions for HPLC liquid phase detection are: detection wavelength 215nm, mobile phase acetonitrile:50mM potassium dihydrogen phosphate = 65:35, column temperature 30°C, flow rate: 1ml / min, injection volume 10μl, column model: ShimNex HE NH2, 5μm, column length: 4.6×250mm.

[0050] Example 6

[0051] The reaction using the crude enzyme solution T195 (α-ester acyltransferase) shown in SEQ ID NO.3

[0052] In a clean reaction bottle, 14 g of L-glutamine, 2 g of boric acid, and 18.2 g of L-alanine methyl ester hydrochloride were weighed, 100 ml of purified water were added, and the pH of the reaction system was adjusted to 8.30. 4 mL of the crude enzyme solution (containing α-ester acyltransferase) obtained by the method of Example 2 was added, and the reaction temperature was controlled to be 15 ° C. The reaction was carried out for 1 h. The total volume at the end of the reaction was 120 ml. The concentration of alanine dipeptide generated by HPLC liquid phase detection was 125.98 g / L.

[0053] Example 7

[0054] 8.2 g of L-glutamine and 2 g of boric acid were added to the reaction column, 100 ml of purified water was added, the pH was adjusted to 8.3 with sodium hydroxide, 5.6 g of the immobilized enzyme prepared according to the method of Example 4 was added, 12.2 g of L-alanine methyl ester hydrochloride was weighed and dissolved in 50 ml of purified water and the pH was adjusted to 6.6, and a peristaltic pump was used to add dropwise to adjust the pH of the reaction system to 8.15. The reaction temperature was controlled at 15° C. for 200 min. The total volume at the end of the reaction was 220 ml. The concentration of alanine dipeptide detected by liquid phase was 50.53 g / L, and the yield of the substrate L-glutamine was 91.26%.

[0055] Example 8

[0056] 10.5 g of the immobilized enzyme prepared by the method of Example 4 was loaded into a column (column diameter ratio of 1:3.5), and then 12.5 g of L-glutamine and 2.5 g of boric acid were added, and purified water was added and stirred. The temperature was lowered to 15° C., and the pH was adjusted to 8.15 using 2 M sodium hydroxide. Then 15.7 g of L-alanine methyl ester hydrochloride was weighed and dissolved in 50 ml of purified water, and the pH was adjusted to 6.60. The mixture was slowly pumped into the column using a peristaltic pump, and 2 M sodium hydroxide was added dropwise to keep the pH at 8.15 for reaction. The reaction was completed after 1.5 h, and the total volume was 130 ml. The concentration of alanine dipeptide produced in the reaction solution was 82.95 g / L using HPLC liquid phase detection.

[0057] HPLC liquid phase conditions are: detection wavelength 215nm, mobile phase acetonitrile: 50mM potassium dihydrogen phosphate = 65:35, column temperature 30℃, flow rate: 1ml / min, injection volume 10μl, column model: ShimNex HE NH2, 5μm, column length: 4.6×250mm. The column loaded with immobilized enzyme can be reused for 20 batches, and the yield can reach 80-90%.

[0058] Example 9

[0059] Collect 200 mL of the reaction solution synthesized by the method of Example 7-8, filter and obtain a filtrate, add 5‰ of the mass of the product to the filtrate, then heat and control the temperature at 50°C for stirring and adsorption for 30 minutes, filter to remove the activated carbon and other adsorbed impurities, take the filtrate and adjust the pH to 7.3 with sodium hydroxide, then put it in a rotary evaporator, and concentrate the volume in a rotary evaporator until a small amount of precipitate is precipitated. After completion, slowly cool to 30°C, and slowly add methanol dropwise with a separatory funnel. The mass ratio of methanol to the above-mentioned concentrated solution is 5:1. Stir slowly. After the addition is complete, cool to 10°C and keep stirring for 1 hour to allow full crystallization. Centrifuge and discard the supernatant to obtain the crude product of propylglutathione. The crude product of propylglutathione is subjected to liquid phase detection. The test results are as follows Figure 1 As shown, the peak area accounts for 98.293%. The obtained product was placed in an oven and dried at 40°C. After drying, liquid phase detection was performed, and the chromatographic purity of the product was 99.196%. The mass spectrum of the product is shown Figure 2 The results of mass spectrometry analysis are shown below, and the molecular weight is 217.22. Other hydrogen and carbon spectra of samples and standards are shown below. Figure 2-Figure 7 shown.

[0060] Example 10

[0061] The gene sequence shown in SEQ ID NO.2 was synthesized by conventional gene synthesis technology and cloned into plasmid pET30a, which was then inoculated into Escherichia coli BL21 (DE3) and Rosetta TM 2(DE3), a single colony was selected and cultured in LB medium containing 50 μg / ml kanamycin at 37°C with shaking, and after the OD value reached 0.8, 0.5 mM IPTG was added and induced at 15°C for 16 hours, and the bacterial cells were collected by centrifugation, and the obtained bacterial cells were further broken to obtain a crude enzyme solution, i.e., a crude enzyme solution containing α-ester acyltransferase, and the amino acid sequence of α-ester acyltransferase is shown in SEQ ID NO. 1. The obtained crude enzyme solution was further purified, specifically, the crude enzyme solution was centrifuged to obtain a supernatant.

[0062] Embodiment 11

[0063] The gene sequence shown in SEQ ID NO.4 was synthesized by conventional gene synthesis technology and cloned into plasmid pET30a, which was then inoculated into Escherichia coli BL21 (DE3) and Rosetta TM2(DE3), a single colony was selected and cultured in LB medium containing 50 μg / ml kanamycin at 37°C with shaking, and after the OD value reached 0.8, 0.5 mM IPTG was added and induced at 15°C for 16 hours, and the bacterial cells were collected by centrifugation, and the obtained bacterial cells were further broken to obtain a crude enzyme solution, i.e., a crude enzyme solution containing α-ester acyltransferase, and the amino acid sequence of α-ester acyltransferase is shown in SEQ ID NO. 3. The obtained crude enzyme solution was further purified, specifically, the crude enzyme solution was centrifuged to obtain a supernatant.

[0064] The specific embodiments described in the present invention are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the attached claims.

[0065] Although the present invention has been described in detail and some specific embodiments have been cited, it is obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention.

Claims

1. An enzymatic synthesis method of glutathione, characterized in that: The method comprises using an alpha-ester acyltransferase without a signal peptide whose amino acid sequence is shown in SEQ ID NO.1 or SEQ ID NO.3 to react L-glutamine and L-alanine methyl ester to obtain alanine dipeptide.

2. The enzymatic synthesis method of glutathione according to claim 1, characterized in that: When the amino acid sequence of the α-ester acyltransferase is SEQ ID NO.1, the nucleic acid sequence of the α-ester acyltransferase is shown as SEQ ID NO.2; when the amino acid sequence of the α-ester acyltransferase is SEQ ID NO.3, the nucleic acid sequence of the α-ester acyltransferase is shown as SEQ ID NO.

4.

3. The enzymatic synthesis method of glutathione according to claim 1, characterized in that: The α-ester acyltransferase is selected from a free enzyme or an immobilized enzyme.

4. The enzymatic synthesis method of glutathione according to claim 3, characterized in that: The α-ester acyl transferase is an immobilized enzyme, and the carrier used is selected from a metal affinity carrier, an amino carrier or an epoxy carrier.

5. The enzymatic synthesis method of alanine dipeptide according to any one of claims 1 to 3, characterized in that: The reaction temperature is 10°C to 20°C.

6. The enzymatic synthesis method of alanine dipeptide according to any one of claims 1 to 3, characterized in that: The pH value of the reaction is controlled at 8.0-8.

5.

7. The enzymatic synthesis method of glutathione according to any one of claims 1 to 3, characterized in that: The molar ratio of L-glutamine to L-alanine methyl ester is 1:1.0-2.0.

Images