Amino acid detection method based on HPLC method and application thereof

Abstract

The invention discloses an amino acid detection method based on an HPLC (High Performance Liquid Chromatography) method and application thereof, and belongs to the technical field of detection methods. According to the amino acid detection method based on the HPLC method, the accuracy of amino acid detection is greatly improved by designing the elution procedure and the sample disc temperature of the HPLC method, the method has a good separation effect on amino acid, the separation time is short, meanwhile, the stability of amino acid derivative products is obviously improved, and the method is suitable for industrial production. And the detection efficiency is improved. The amino acid detection method based on the HPLC method provided by the invention can be directly used for activity detection of amino acid synthetase and qualitative and quantitative detection of proteolysis amino acid.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of detection methods, and particularly relates to an amino acid detection method based on an HPLC method and application thereof. BACKGROUND

[0002] Common methods for detecting amino acids in plants include an amino acid analyzer method and an HPLC method. The amino acid analyzer adopts a post-column ninhydrin derivatization detection method, which has low throughput and can only complete the detection of 30-40 samples in 24 hours. Moreover, when detecting various free amino acids containing amides, the method cannot be completed at one time and needs to use two mobile phase systems, and has poor flexibility. The HPLC method adopts a pre-column derivatization method for amino acid detection. A commonly used derivatization reagent is o-phthalaldehyde (OPA). OPA and a thiol reagent can react with primary amino acids under alkaline conditions to produce fluorescent products. The derivatization product has strong ultraviolet absorption at a wavelength of 338 nm. The method has high flexibility, can adjust the detection time according to the types of amino acids to be detected, and does not need to replace the mobile phase and the chromatographic column. However, due to the short derivatization time of OPA, the derivatization product is unstable and prone to inaccurate detection and quantification.

[0003] Therefore, there is an urgent need for a method capable of accurately detecting the types and contents of amino acids to solve the above problems. SUMMARY

[0004] To solve the above technical problems, the application provides an amino acid detection method based on an HPLC method and application thereof.

[0005] To achieve the above object, the application provides the following technical solutions.

[0006] The application provides an amino acid detection method based on an HPLC method, which adopts high-performance liquid chromatography to detect amino acids.

[0007] The chromatographic conditions of the high-performance liquid chromatography are as follows.

[0008] The mobile phase A is a sodium acetate solution and methanol with a volume ratio of 90:10, and the mobile phase B is a sodium acetate solution and methanol with a volume ratio of 40:60.

[0009] Elution procedure: gradient elution, 0-2min, 95%A and 5%B; 2-12min, 95%A down to 43%A, 5%B up to 57%B; 12-13min, 43%A down to 0%A, 57%B up to 100%B; 13-17min, maintain 0%A and 100%B; 17-19min, 0%A up to 95%A, 100%B down to 5%B; 19-24min, maintain 95%A and 5%B; or: gradient elution, 0-1min, 100%A and 0%B; 1-6min, 100%A down to 90%A, 0%B up to 10%B; 6-11min, maintain 90%A and 10%B; 11-16min, 90%A down to 55%A, 10%B up to 45%B; 16-20min, maintain 55%A and 45%B; 20-32min, 55%A down to 0%A, 45%B up to 100%B; 32-36min, maintain 0%A and 100%B; 36-37min, 0%A up to 100%A, 100%B down to 0%B; 37-38min, maintain 100%A and 0%B;

[0010] Sample tray temperature: 8℃.

[0011] Technical principle: the present application greatly improves the accuracy of amino acid detection by designing the elution procedure and sample tray temperature of high performance liquid chromatography, which has good separation effect on amino acids and short separation time, and significantly improves the stability of amino acid derivative products, thereby improving the detection efficiency.

[0012] Further, the concentration of sodium acetate solution in the mobile phase A and the mobile phase B is 20mmol / L, and the pH value of the sodium acetate solution is 6.8.

[0013] Further, the chromatographic column of the high performance liquid chromatography is Agilent ZORBAX Eclipse AAA column, the flow rate is 1.0mL / min, the column temperature is 40℃, the detection wavelength is 338nm, and the injection amount is 10μL.

[0014] The present application provides an application of the amino acid detection method based on HPLC in the detection of protein hydrolysis amino acids.

[0015] Further, the application of the amino acid detection method based on HPLC in the detection of protein hydrolysis amino acids comprises the following steps:

[0016] (1) 19 kinds of amino acids are prepared into amino acid standard solution, boric acid buffer and o-phthalaldehyde solution are added to the amino acid standard solution to perform a derivatization reaction, a filter membrane is used, and then high performance liquid chromatography is used for detection; a standard curve is drawn with the peak area of the amino acid derivative in the detection result as the vertical coordinate Y and the mass of the amino acid derivative as the horizontal coordinate X;

[0017] (2) a protein hydrolysis amino acid sample solution to be detected is prepared, a derivatization reaction is performed on the protein hydrolysis amino acid sample solution to be detected and o-phthalaldehyde solution, and then high performance liquid chromatography is used for detection; the peak area in the detection result is substituted into the standard curve drawn in step (1), and the content of each amino acid in the protein hydrolysis amino acid sample to be detected is calculated.

[0018] Further, in step (1), the 19 kinds of amino acids are selected from Asp, Glu, Asn, Ser, Gln, Gly, Thr, Arg, His, Tyr, Ala, Cys, Trp, Met, Val, Phe, Lys, Ile and Leu.

[0019] Further, in step (2), the process of preparing the protein hydrolysis amino acid sample solution to be detected is as follows: a protein raw material is added into concentrated hydrochloric acid to perform hydrolysis, and after evaporation and drying, the protein raw material is dissolved in dilute hydrochloric acid, a filter membrane is used, and the protein hydrolysis amino acid sample solution to be detected is obtained.

[0020] Further, the dosage ratio of the protein raw material to concentrated hydrochloric acid is 100 mg:10 mL; the concentration of the concentrated hydrochloric acid is 6 mol / L; and / or,

[0021] The hydrolysis temperature is 110 DEG C, and the hydrolysis time is 24 h.

[0022] The application further provides an application of the amino acid detection method based on the HPLC method in the detection of amino acid synthetase activity.

[0023] Further, the application of the amino acid detection method based on the HPLC method in the detection of amino acid synthetase activity comprises the following steps: a reaction solution of an amino acid synthetase to be detected is prepared, boric acid buffer and o-phthalaldehyde solution are added to the reaction solution of the amino acid synthetase to be detected to perform a derivatization reaction, a filter membrane is used, and then high performance liquid chromatography is used to detect the activity of the amino acid synthetase.

[0024] Compared with the prior art, the application has the following advantages and technical effects:

[0025] The amino acid detection method based on the HPLC method provided by the application has good separation effect on amino acids, short separation time, significantly improves the stability of amino acid derivative products, and further improves the detection efficiency.

[0026] The HPLC-based amino acid detection method provided by this invention greatly improves the accuracy of amino acid detection and can be directly used for the detection of amino acid synthase activity and the qualitative and quantitative detection of protein hydrolysed amino acids. Attached Figure Description

[0027] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0028] Figure 1 Chromatograms of 19 amino acid derivatives in the amino acid standard solutions of Examples 1-2 and Comparative Examples 1-2 are shown, where 1 is Asp; 2 is Glu; 3 is Asn; 4 is Ser; 5 is Gln; 6 is Gly; 7 is Thr; 8 is Arg; 9 is His; 10 is Tyr; 11 is Ala; 12 is Cys; 13 is Trp; 14 is Met; 15 is Val; 16 is Phe; 17 is Lys; 18 is Ile; and 19 is Leu.

[0029] Figure 2 The chromatograms are obtained by HPLC detection of the amino acid derivatives in Example 3 after 6 hours and 8 hours of storage, where 1 is Asp; 2 is Glu; 3 is Asn; and 4 is Ser.

[0030] Figure 3 The chromatograms are obtained by HPLC detection of the amino acid derivatives in Comparative Example 3 after 6 hours and 8 hours of storage, where 1 represents Asp; 2 represents Glu; 3 represents Asn; and 4 represents Ser.

[0031] Figure 4 Chromatograms of the derivatives of the control reaction solution (a) and the enzyme-catalyzed reaction solution (b) in Application Example 1, wherein 1 is L-Asp; 2 is L-Glu; 3 is L-Asn; and 4 is L-Gln.

[0032] Figure 5 The chromatograms are of the corn protein hydrolysate test sample in Application Example 2 and the amino acid mixed standard in Example 1. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0035] This invention provides an amino acid detection method based on HPLC, which uses high performance liquid chromatography to detect amino acids;

[0036] The chromatographic conditions for the high-performance liquid chromatography method are as follows:

[0037] Mobile phase: Mobile phase A is a sodium acetate solution and methanol with a volume ratio of 90:10, and mobile phase B is a sodium acetate solution and methanol with a volume ratio of 40:60.

[0038] Elution program: Gradient elution, 0-2 min, 95% A and 5% B; 2-12 min, 95% A decreases to 43% A, 5% B increases to 57% B; 12-13 min, 43% A decreases to 0% A, 57% B increases to 100% B; 13-17 min, maintain 0% A and 100% B; 17-19 min, 0% A increases to 95% A, 100% B decreases to 5% B; 19-24 min, maintain 95% A and 5% B; or: Gradient elution, 0-1 min, 100% A and 0% B; 1-6 min, 100% A Reduce to 90% A, increase from 0% B to 10% B; 6-11 min, maintain 90% A and 10% B; 11-16 min, reduce 90% A to 55% A, increase from 10% B to 45% B; 16-20 min, maintain 55% A and 45% B; 20-32 min, reduce 55% A to 0% A, increase from 45% B to 100% B; 32-36 min, maintain 0% A and 100% B; 36-37 min, increase from 0% A to 100% A, reduce from 100% B to 0% B; 37-38 min, maintain 100% A and 0% B.

[0039] Sample tray temperature: 8℃.

[0040] In a preferred embodiment, the concentration of sodium acetate solution in both mobile phase A and mobile phase B is 20 mmol / L, and the pH value of sodium acetate solution in both is 6.8.

[0041] In a preferred embodiment, the sodium acetate solution is prepared by dissolving sodium acetate powder in ultrapure water, adjusting the pH of the solution with hydrochloric acid after complete dissolution, and filtering the solution through a membrane to obtain the sodium acetate solution; the pore size of the filter membrane is 0.22 μm.

[0042] In a preferred embodiment, the chromatographic column used in the high-performance liquid chromatography is an Agilent ZORBAX EclipseAAA column, the flow rate is 1.0 mL / min, the column temperature is 40 °C, the detection wavelength is 338 nm, and the injection volume is 10 μL.

[0043] This invention provides an application of the HPLC-based amino acid detection method described above in the detection of amino acids from protein hydrolysis.

[0044] In a preferred embodiment, the application of the HPLC-based amino acid detection method in the detection of amino acids from protein hydrolysis includes the following steps:

[0045] (1) Prepare amino acid standard solutions of 19 amino acids, add borate buffer and o-phthalaldehyde solution to the amino acid standard solutions to carry out derivatization reaction, filter membrane, and then detect by high performance liquid chromatography; plot a standard curve with the peak area of ​​amino acid derivatives in the detection results as the ordinate Y and the mass of amino acid derivatives in the amino acid standard solution as the abscissa X.

[0046] (2) Prepare a sample solution of hydrolyzed amino acids of the protein to be tested. After derivatization reaction of the sample solution of hydrolyzed amino acids of the protein to be tested and o-phthalaldehyde solution, perform high performance liquid chromatography detection. Substitute the peak area in the detection result into the standard curve drawn in step (1) and calculate the content of each amino acid in the sample of hydrolyzed amino acids of the protein to be tested.

[0047] In a preferred embodiment, in step (1), the 19 amino acids are selected from Aspartic acid, Glu, Asn, Serine, Gln, Gly, Threonine, Arg, His, Tyr, Ala, Cys, Tryptophan, Met, Val, Phe, Lys, Ile, and Leu.

[0048] In a preferred embodiment, in step (1), the concentration of the amino acid standard solution is 2.5 mmol / L; the amino acid standard solution is prepared by using 0.1 mol / L HCl to prepare an amino acid standard solution from a mixture containing 19 amino acid powders; the amino acid standard solution is stored at 4°C in the dark; and the concentration of each amino acid in the amino acid standard solution is 2.5 mmol / L.

[0049] In a preferred embodiment, in step (1), the concentration of the o-phthalaldehyde (OPA) solution is 5 mg / mL; the o-phthalaldehyde solution is prepared by dissolving OPA powder in methanol, then adding mercaptoethanol, and then diluting with borate buffer to obtain the o-phthalaldehyde solution; the o-phthalaldehyde solution is stored at 4°C in the dark.

[0050] In a preferred embodiment, in step (1), the pH value of the borate buffer is 9.5.

[0051] In a preferred embodiment, in step (1), the volume ratio of the amino acid standard solution, borate buffer, and phthalaldehyde solution is 1:3:2.

[0052] In a preferred embodiment, in step (1), the pore size of the filter membrane is 0.22 μm; the material of the filter membrane is polytetrafluoroethylene (PTFE).

[0053] In a preferred embodiment, in step (1), the chromatographic conditions of the high-performance liquid chromatography are the same as those in the aforementioned amino acid detection method based on HPLC.

[0054] In a preferred embodiment, in step (2), the process of preparing the test protein hydrolyzed amino acid sample solution is as follows: the protein raw material is added to concentrated hydrochloric acid for hydrolysis, evaporated and dried, dissolved in dilute hydrochloric acid, filtered through a membrane, and the test protein hydrolyzed amino acid sample solution is obtained.

[0055] In a preferred embodiment, the ratio of the protein raw material to concentrated hydrochloric acid is 100 mg: 10 mL; the concentration of the concentrated hydrochloric acid is 6 mol / L.

[0056] In a preferred embodiment, the hydrolysis temperature is 110°C and the hydrolysis time is 24 hours.

[0057] In a preferred embodiment, the concentration of the dilute hydrochloric acid is 0.02 mol / L.

[0058] In a preferred embodiment, the filter membrane has a pore size of 0.22 μm; the filter membrane is made of polytetrafluoroethylene (PTFE).

[0059] In a preferred embodiment, in step (2), the chromatographic conditions for the high-performance liquid chromatography detection are the same as those in the aforementioned amino acid detection method based on HPLC.

[0060] The present invention also provides an application of the HPLC-based amino acid detection method described above in the detection of amino acid synthase activity.

[0061] In a preferred embodiment, the application of the HPLC-based amino acid detection method in the detection of amino acid synthase activity includes the following steps: preparing a reaction solution of the amino acid synthase to be tested, adding borate buffer and o-phthalaldehyde solution to the reaction solution of the amino acid synthase to be tested for derivatization reaction, and then detecting the amino acid synthase activity by high performance liquid chromatography after filtration.

[0062] In a preferred embodiment, the amino acid synthase is selected from ZmASN4 protein; the reaction solution of the amino acid synthase to be tested is prepared as follows: 100 mmol / L Tris-HCl buffer solution (pH=7.6), 10 mmol / L ATP, 10 mmol / L MgCl2, 20 mmol / L L-Asp, 10 mmol / L L-Gln and 1 μg of purified ZmASN4 protein are thoroughly mixed to a total volume of 50 μL. The mixture is placed in a PCR instrument and reacted at 37℃ for 15 min, then at 99℃ for 5 min. The reaction is then terminated, and the mixture is centrifuged at 12000 r / min for 10 min. The supernatant is collected to obtain the reaction solution of the amino acid synthase to be tested.

[0063] In a preferred embodiment, the volume ratio of the reaction solution of the amino acid synthase to be tested, the borate buffer, and the phthalaldehyde solution is 1:3:2.

[0064] In this embodiment of the invention, room temperature refers to "25±2℃".

[0065] Unless otherwise specified, all raw materials used in the embodiments of this invention were purchased through commercial channels.

[0066] In the following examples and comparative examples, sodium acetate was purchased from Sigma-Aldrich; o-phthalaldehyde (OPA, catalog number B24663) and borate buffer (pH = 9.5, catalog number B885515) were purchased from Maclean's; hydrochloric acid was of analytical grade; and ZmASN4 protein was obtained by expression and purification of Saccharomyces cerevisiae strain sc288c.

[0067] Example 1

[0068] An HPLC-based method for amino acid detection includes the following steps:

[0069] Dissolve 2.13g of sodium acetate powder in 1.3L of ultrapure water. After complete dissolution, adjust the pH of the solution to 6.8 with hydrochloric acid. Use a vacuum filtration system with a membrane pore size of 0.22μm to obtain a sodium acetate solution with a concentration of 20mmol / L.

[0070] Nineteen amino acid powders were mixed to obtain an amino acid powder mixture; then, the amino acid powder mixture was prepared into an amino acid standard solution (i.e., amino acid mixed standard) using 0.1 mol / L HCl and stored at 4°C protected from light. The 19 amino acids were Asp, Glu, Asn, Ser, Gln, Gly, Thr, Arg, His, Tyr, Ala, Cys, Trp, Met, Val, Phe, Lys, Ile, and Leu, and the concentration of each amino acid in the amino acid standard solution was 2.5 mmol / L.

[0071] Dissolve 10 mg of OPA powder in 200 μL of methanol (chromatographic grade), then add 100 μL of mercaptoethanol, and dilute to 2 mL with borate buffer (pH = 9.5) to obtain a stock solution of OPA with a concentration of 5 mg / mL. Store at 4°C protected from light.

[0072] Take 20 μL of amino acid standard solution with a concentration of 2.5 mmol / L, add 60 μL of borate buffer (pH = 9.5), mix well, and then add 40 μL of OPA stock solution with a concentration of 5 mg / mL to carry out derivatization reaction. Filter through a 0.22 μm PTFE membrane to obtain amino acid derivatives, and then place them in a sample vial containing an inner tube for HPLC detection.

[0073] The chromatographic conditions for HPLC detection were as follows: Agilent ZORBAX Eclipse AAA column, 4.6 × 150 mm, 5 μm; mobile phase A was 20 mM sodium acetate solution (pH = 6.8) and methanol at a volume ratio of 90:10, and mobile phase B was 20 mM sodium acetate solution (pH = 6.8) and methanol at a volume ratio of 40:60; flow rate was 1.0 mL / min; column temperature was 40 °C; sample tray temperature was 8 °C; detection wavelength was 338 nm; injection volume was 10 μL; elution program was gradient elution, 0 -2 min, 95% A and 5% B; 2-12 min, 95% A decreases to 43% A, 5% B increases to 57% B; 12-13 min, 43% A decreases to 0% A, 57% B increases to 100% B; 13-17 min, maintain 0% A and 100% B; 17-19 min, 0% A increases to 95% A, 100% B decreases to 5% B; 19-24 min, maintain 95% A and 5% B.

[0074] Example 2

[0075] An HPLC-based amino acid detection method differs from Example 1 in that the elution program is as follows: gradient elution, 0-1 min, 100% A and 0% B; 1-6 min, 100% A decreases to 90% A, 0% B increases to 10% B; 6-11 min, maintain 90% A and 10% B; 11-16 min, 90% A decreases to 55% A, 10% B increases to 45% B; 16-20 min, maintain 55% A and 45% B; 20-32 min, 55% A decreases to 0% A, 45% B increases to 100% B; 32-36 min, maintain 0% A and 100% B; 36-37 min, 0% A increases to 100% A, 100% B decreases to 0% B; 37-38 min, maintain 100% A and 0% B; other steps are the same as in Example 1.

[0076] Comparative Example 1

[0077] An HPLC-based method for amino acid detection differs from Example 1 in that the elution program is as follows: gradient elution: 0-14 min, 86% A decreases to 80% A, 14% B increases to 20% B; 14-18 min, 80% A decreases to 70% A, 20% B increases to 30% B; 18-18.1 min, 70% A decreases to 55% A, 30% B increases to 45% B; 18.1-24 min, 55% A decreases to 50% A. 45%B increases to 50%B; 24-26 min, 50%A decreases to 30%A, 50%B increases to 70%B; 26-30 min, 30%A decreases to 0%A, 70%B increases to 100%B; 30-32 min, maintain 0%A and 100%B; 32-32.1 min, 0%A increases to 86%A, 100%B decreases to 14%B; 32.1-39 min, maintain 86%A and 14%B; other steps are the same as in Example 1.

[0078] Comparative Example 2

[0079] An HPLC-based amino acid detection method differs from Example 1 in that the elution program is as follows: gradient elution, 0-1.2 min, 92% A decreases to 82% A, 8% B increases to 18% B; 1.2-14 min, maintain 82% A and 18% B; 14-22 min, 82% A decreases to 75% A, 18% B increases to 25% B; 22-42.5 min, 75% A decreases to 65% A, 25% B increases to 35% B; 42.5-49 min, 65% A decreases to 30% A, 35% B increases to 70% B; 49-50.5 min, maintain 30% A and 70% B; 50.5-51.5 min, 30% A increases to 92% A, 70% B decreases to 8% B; other steps are the same as in Example 1.

[0080] Figure 1 The chromatograms are of derivatives of 19 amino acids in the amino acid standard solutions of Examples 1-2 and Comparative Examples 1-2, where 1 is Asp; 2 is Glu; 3 is Asn; 4 is Ser; 5 is Gln; 6 is Gly; 7 is Thr; 8 is Arg; 9 is His; 10 is Tyr; 11 is Ala; 12 is Cys; 13 is Trp; 14 is Met; 15 is Val; 16 is Phe; 17 is Lys; 18 is Ile; and 19 is Leu. Figure 1 It can be seen that Examples 1 and 2 have good separation effects on the standard solution containing 19 amino acids, but the separation effect of Example 1 is better than that of Example 2, while the separation effects of Comparative Examples 1 and 2 are poor. The separation time of Example 1 is 24 min, which is shorter than that of Example 2.

[0081] Example 3

[0082] An HPLC-based method for amino acid detection differs from Example 1 in that the preparation method of the amino acid standard solution (i.e., amino acid mixed standard) is different. Specifically, the preparation method of the amino acid standard solution is as follows: four amino acid powders are mixed to obtain an amino acid powder mixture; then, the amino acid powder mixture is prepared into an amino acid standard solution (i.e., amino acid mixed standard) using 0.1 mol / L HCl and stored at 4°C in the dark; wherein, the four amino acids are Asp, Glu, Asn, and Ser, and the concentration of each amino acid in the amino acid standard solution is 2.5 mmol / L; other aspects are the same as in Example 1.

[0083] Comparative Example 3

[0084] An amino acid detection method based on HPLC differs from Example 3 in that the sample pan temperature is set to room temperature; otherwise, it is the same as Example 3.

[0085] Figure 2 The chromatograms are obtained by HPLC detection of the amino acid derivatives in Example 3 after 6 hours and 8 hours of storage, where 1 represents Asp; 2 represents Glu; 3 represents Asn; and 4 represents Ser. From... Figure 2 As can be seen from the example, by setting the sample pan temperature to 8°C, Example 3 significantly improved the stability of amino acid derivatives, thereby improving the detection efficiency.

[0086] Figure 3 The chromatograms are obtained by HPLC detection of the amino acid derivatives in Comparative Example 3 after 6 hours and 8 hours of storage, where 1 represents Asp; 2 represents Glu; 3 represents Asn; and 4 represents Ser. From... Figure 3 As can be seen, after the amino acid derivatives were left for 8 hours and then subjected to HPLC detection using the method in Comparative Example 3, the stability of the amino acid derivatives decreased sharply, making it impossible to quantitatively detect the amino acids.

[0087] Application Example 1

[0088] An HPLC-based amino acid detection method is applied to the detection of maize asparagine synthase ZmASN4 activity. The specific steps are as follows:

[0089] (1) Thoroughly mix 100 mmol / L Tris-HCl buffer (pH = 7.6), 10 mmol / L ATP, 10 mmol / L MgCl2, 20 mmol / L L-Asp, 10 mmol / L L-Gln, and 1 μg of purified ZmASN4 protein to a total volume of 50 μL. Place the mixture in a PCR instrument and react at 37℃ for 15 min, then at 99℃ for 5 min. Terminate the reaction, centrifuge at 12000 r / min for 10 min, and collect the supernatant to obtain the enzymatic reaction solution. A reaction system without ZmASN4 protein was used as a control reaction solution.

[0090] (2) Take 20 μL of the supernatant obtained in step (1), add 60 μL of borate buffer and mix well, then add 40 μL of OPA stock solution for derivatization reaction. After passing through a 0.22 μm PTFE filter membrane, place it in a sample vial containing an inner tube for HPLC detection. Inject 10 μL of each sample and measure 3 times. The preparation method of OPA stock solution and the chromatographic conditions for HPLC detection are the same as in Example 1.

[0091] Figure 4 The chromatograms are of the derivatives from the control reaction solution (a) and the enzyme-catalyzed reaction solution (b) in Example 1, where 1 represents L-Asp; 2 represents L-Glu; 3 represents L-Asn; and 4 represents L-Gln. Figure 4 It can be seen that the detection method provided by the present invention can achieve complete separation of reaction products L-Asn and L-Glu from reaction substrates L-Asp and L-Gln.

[0092] Application Example 2

[0093] An application of an HPLC-based amino acid detection method in the detection of amino acids from protein hydrolysis, the specific steps of which are as follows:

[0094] (1) Nineteen amino acids were prepared into amino acid standard solutions with a concentration of 2.5 mmol / L. 60 μL of borate buffer (pH = 9.5) was added to 20 μL of the 2.5 mmol / L amino acid standard solutions, mixed well, and then 40 μL of OPA stock solution with a concentration of 5 mg / mL was added to carry out derivatization reaction. The derivatization reaction was carried out by filtering through a 0.22 μm PTFE membrane to obtain amino acid derivatives. Then, the derivatives were placed in sample vials containing inner tubes at injection volumes of 1 μL, 3 μL, 6 μL, 8 μL, and 10 μL, respectively, for HPLC detection. Each sample was measured 3 times and the average value was taken. A standard curve was plotted with the peak area of ​​the amino acid derivatives in the HPLC detection results as the ordinate Y and the mass of the amino acid derivatives as the abscissa X. The results are shown in Table 1. The preparation methods of the amino acid standard solutions and OPA stock solutions were the same as in Example 1. Other chromatographic conditions for HPLC detection were the same as in Example 1.

[0095] (2) 10 mL of 6 mol / L HCl was added to 100 mg of corn kernel powder, and the mixture was hydrolyzed in an oven at 110 °C for 24 h. After evaporation and drying with nitrogen, the product was dissolved in 0.02 mol / L HCl, shaken and mixed, and then filtered through a 0.22 μm PTFE membrane to obtain the corn protein hydrolysate test sample. 20 μL of the above corn protein hydrolysate test sample was taken, and 60 μL of borate buffer (pH = 9.5) was added. After mixing, 40 μL of 5 mg / mL OPA stock solution was added to carry out the derivatization reaction, and then HPLC detection was performed. The preparation method of OPA stock solution and the chromatographic conditions for HPLC detection were the same as in Example 1. The detection results are shown in Example 1. Figure 5 Substitute the peak area corresponding to each amino acid in the detection results into the standard curve drawn in step (1) to obtain the mass m of each amino acid in the 20 μL sample to be tested, and then substitute the mass m of each amino acid into the formula. The content of each amino acid in corn kernel powder was calculated, where the dilution factor was 6 times, the total volume was 10 mL, and the total mass was 100 mg; the results are shown in Table 2.

[0096] Table 1 Standard Curve

[0097] Amino acid Retention time (min) Standard curve [R 2 ]] Asp 3.726 y = 4E+08x - 44989 0.9927 Glu 6.104 y = 6E+08x - 44681 0.9835 Asn 8.964 y = 1E+09x - 68121 0.9867 Ser 10.074 y = 4E+08x - 58785 0.9402 Gln 10.705 y = 6E+08x - 43411 0.9864 Gly 12.711 y = 4E+08x - 73700 0.9183 Thr 12.969 y = 5E+08x - 45785 0.9794 Arg 13.246 y = 1E+09x - 128780 0.9824 His 13.760 y = 6E+08x - 63550 0.9890 Tyr 14.396 y = 9E+08x - 46619 0.9947 Ala 14.522 y = 3E+08x - 45444 0.9305 Cys 14.715 y = 7E+07x - 5611.3 0.9929 Trp 15.473 y = 7E+08x - 38612 0.9916 Met 15.879 y = 1E+09x - 82067 0.9862 Val 16.181 y = 8E+08x - 37299 0.9967 Phe 16.417 y = 1E+09x - 31201 0.9982 Lys 16.681 y = 8E+08x - 17340 0.9988 Ile 17.793 y = 9E+08x - 42170 0.9949 Leu 18.435 y = 8E+08x - 24733 0.9987

[0098] In Table 1, E+07x=10 7 x; E+08x=10 8 x; E+09x=10 9 x.

[0099] Figure 5 This is a chromatogram of the corn protein hydrolysate sample from Example 2 and the amino acid mixed standard from Example 1. From... Figure 5 As can be seen from the results, the corn protein hydrolysate test sample contains Asp, Glu, Ser, Gly, Thr, Arg, His, Tyr, Ala, Cys, Met, Val, Phe, Lys, Ile, and Leu.

[0100] Table 2. Content of various amino acids in corn kernel powder

[0101] Amino acid Peak area Content (mg / 100 mg dry weight) Asp 85782 0.98 Glu 241935 1.43 Ser 14543 0.55 Gly 3644 0.58 Thr 30664 0.46 Arg 59367 0.56 His 8875 0.36 Tvr 75938 0.41 Ala 10217 0.56 Cys 1003 0.28 Met 7546 0.27 Val 179839 0.81 Phe 89927 0.36 Lys 30826 0.18 Ile 108731 0.50 Leu 187422 0.80

[0102] As can be seen from Table 2, except for Asn and Gln, two amide amino acids, which cannot be detected after hydrochloric acid hydrolysis, and Trp, which requires alkaline hydrolysis for detection, the remaining 16 amino acids in corn hydrolysate can be detected using the method of this invention after hydrochloric acid hydrolysis.

[0103] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for amino acid detection based on HPLC, characterized in that, High performance liquid chromatography (HPLC) was used for the detection of amino acids. The chromatographic conditions for the high-performance liquid chromatography method are as follows: Mobile phase: Mobile phase A is a sodium acetate solution and methanol with a volume ratio of 90:10, and mobile phase B is a sodium acetate solution and methanol with a volume ratio of 40:

60. Elution program: Gradient elution, 0-2 min, 95% A and 5% B; 2-12 min, 95% A decreases to 43% A, 5% B increases to 57% B; 12-13 min, 43% A decreases to 0% A, 57% B increases to 100% B; 13-17 min, maintain 0% A and 100% B; 17-19 min, 0% A increases to 95% A, 100% B decreases to 5% B; 19-24 min, maintain 95% A and 5% B. Alternatively: Gradient elution: 0-1 min, 100% A and 0% B; 1-6 min, 100% A decreases to 90% A, 0% B increases to 10% B; 6-11 min, maintain 90% A and 10% B; 11-16 min, 90% A decreases to 55% A, 10% B increases to 45% B; 16-20 min, maintain 55% A and 45% B; 20-32 min, 55% A decreases to 0% A, 45% B increases to 100% B; 32-36 min, maintain 0% A and 100% B; 36-37 min, 0% A increases to 100% A, 100% B decreases to 0% B; 37-38 min, maintain 100% A and 0% B. Sample tray temperature: 8℃.

2. The amino acid detection method based on HPLC according to claim 1, characterized in that, The concentration of sodium acetate solution in both mobile phase A and mobile phase B is 20 mmol / L, and the pH value of sodium acetate solution in both is 6.

8.

3. The amino acid detection method based on HPLC according to claim 1, characterized in that, The high-performance liquid chromatography (HPLC) method used an Agilent ZORBAX Eclipse AAA column, with a flow rate of 1.0 mL / min, a column temperature of 40 °C, a detection wavelength of 338 nm, and an injection volume of 10 μL.

4. The application of the HPLC-based amino acid detection method as described in any one of claims 1-3 in the detection of amino acids in protein hydrolysis.

5. The application according to claim 4, characterized in that, Includes the following steps: (1) Prepare amino acid standard solutions of 19 amino acids, add borate buffer and o-phthalaldehyde solution to the amino acid standard solutions to carry out derivatization reaction, filter membrane, and then detect by high performance liquid chromatography; plot standard curve with peak area of ​​amino acid derivatives in the detection results as Y on the ordinate and mass of amino acid derivatives as X on the abscissa. (2) Prepare a sample solution of hydrolyzed amino acids of the protein to be tested. After derivatization reaction of the sample solution of hydrolyzed amino acids of the protein to be tested and o-phthalaldehyde solution, perform high performance liquid chromatography detection. Substitute the peak area in the detection result into the standard curve drawn in step (1) and calculate the content of each amino acid in the sample of hydrolyzed amino acids of the protein to be tested.

6. The application according to claim 5, characterized in that, In step (1), the 19 amino acids are selected from Asp, Glu, Asn, Ser, Gln, Gly, Thr, Arg, His, Tyr, Ala, Cys, Trp, Met, Val, Phe, Lys, Ile and Leu.

7. The application according to claim 5, characterized in that, In step (2), the process of preparing the test protein hydrolyzed amino acid sample solution is as follows: the protein raw material is added to concentrated hydrochloric acid for hydrolysis, evaporated and dried, dissolved in dilute hydrochloric acid, filtered through a membrane, and the test protein hydrolyzed amino acid sample solution is obtained.

8. The application according to claim 7, characterized in that, The ratio of the protein raw material to concentrated hydrochloric acid is 100 mg: 10 mL; the concentration of the concentrated hydrochloric acid is 6 mol / L; and / or, The hydrolysis temperature is 110℃, and the hydrolysis time is 24h.

9. The application of the HPLC-based amino acid detection method as described in any one of claims 1-3 in the detection of amino acid synthase activity.

10. The application according to claim 9, characterized in that, Includes the following steps: A reaction solution for the amino acid synthase to be tested was prepared. Borate buffer and o-phthalaldehyde solution were added to the reaction solution to carry out a derivatization reaction. After filtration through a membrane, the activity of the amino acid synthase was detected by high performance liquid chromatography.

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