Method for efficiently and directionally enriching activity promoting peptides of ethanol dehydrogenase and acetaldehyde dehydrogenase

Abstract

The invention discloses a method for directionally enriching and screening peptide fragments capable of effectively promoting the activity of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) from complex biological enzymatic hydrolysate, and belongs to the field of biotechnology and active peptide screening. The method comprises the following steps: co-immobilizing ADH1B and ALDH2 on the same solid-phase carrier in a high-activity manner by adopting a segmented pH sequential coupling technology to prepare a dual-enzyme affinity carrier; the carrier is used for adsorbing peptide fragments capable of being specifically combined with double enzymes in a complex peptide library; step-by-step elution is carried out by utilizing a specific competitor of ADH1B and ALDH2, so that two types of targeted peptide enriched components are respectively obtained; the specificity is verified through reverse screening and alcohol metabolism enzyme activity promotion function determination. According to the invention, high-efficiency parallel screening and synergistic combination discovery of alcohol metabolism key pathway double-target active peptides are realized for the first time, and an innovative technical platform is provided for developing a multi-target and high-efficiency alcohol metabolism regulator.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and functional peptide screening, specifically to a parallel screening method and a dedicated solid-phase affinity carrier capable of simultaneously enriching peptides that promote the activity of alcohol dehydrogenase 1B (ADH1B) and aldehyde dehydrogenase 2 (ALDH2). Background Technology

[0002] Alcohol metabolism is a crucial detoxification pathway in the human body, and its efficiency primarily depends on the sequential action of two key enzymes: first, alcohol dehydrogenase (ADH) oxidizes ethanol to acetaldehyde, and then aldehyde dehydrogenase (ALDH) further oxidizes acetaldehyde to harmless acetic acid. These two enzymes play vital roles in the body's alcohol metabolism process. Their activities collectively determine an individual's alcohol metabolism capacity and are closely related to liver protection, cardiovascular health, and cancer risk.

[0003] In the human alcohol dehydrogenase (ADH) family, type I ADH (including ADH1A, ADH1B, and ADH1C) are the main isoenzymes catalyzing ethanol oxidation in the liver. Among them, ADH1B is considered a key factor influencing individual alcohol metabolism rate and alcohol-related disease risk due to its high-activity genetic polymorphism in East Asian populations. For example, the enzyme encoded by the ADH1B*2 (Arg48His) allele has extremely high activity and is one of the main determinants of differences in "alcohol flushing response" and alcohol metabolism rate among East Asian populations. Therefore, selecting ADH1B as an immobilization target ensures that the screened bioactive peptides directly target the most physiologically and population-specific key enzyme in the first step of alcohol metabolism, thereby enhancing the translational value of the screening results.

[0004] Within the human ALDH enzyme family, mitochondrial ALDH2 is the core enzyme responsible for acetaldehyde clearance in alcohol metabolism. Compared to other isoenzymes, ALDH2 exhibits the highest catalytic efficiency for acetaldehyde, making it a well-defined target for drug and functional factor development. Furthermore, recombinant human ALDH2 protein is readily available in high-purity, high-activity forms. Similar to ADH1B, selecting recombinant human ALDH2 protein, rather than other isoenzymes, to prepare immobilized enzymes enhances the specificity and transformative potential of the screening results.

[0005] Bioactive peptides are potential ADH and ALDH regulators due to their high activity, low toxicity, and good biocompatibility. These bioactive peptides are typically derived from the enzymatic hydrolysis products of proteins; however, the composition of these hydrolysates is extremely complex, containing thousands of peptides with different sequences, lengths, and properties, of which only a few possess ADH and ALDH activation functions. Furthermore, when faced with highly complex peptide mixtures (such as animal and plant protease hydrolysates), direct activity assays often suffer from poor sensitivity and low screening efficiency due to numerous interfering substances and low abundance of the target peptide. Traditional separation and purification methods (such as single chromatographic techniques) can purify peptides, but they lack specific targeting of the target function, are cumbersome, and lack strong specificity, often failing to achieve both high resolution and activity targeting, resulting in low yields of the target peptide, indiscriminate screening, and low efficiency.

[0006] Therefore, developing a method to stably, efficiently, and actively co-immobilize ADH1B and ALDH2 on the same carrier, and thereby establishing a parallel screening platform that can enrich dual-target active peptides and their synergistic combinations from complex peptide libraries in a single operation, has significant scientific value and application prospects for discovering a new generation of highly efficient and synergistic alcohol metabolism regulators.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a simple, specific, and efficient method for enriching peptides that promote the activity of alcohol dehydrogenase and aldehyde dehydrogenase from complex polypeptide mixtures. This method utilizes a dual-enzyme co-immobilization carrier as an affinity adsorbent, and through specific binding, washing, and competitive elution, directly obtains peptides capable of regulating the activity of alcohol dehydrogenase and aldehyde dehydrogenase.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A method for enriching peptides that promote the activity of alcohol dehydrogenase and acetaldehyde dehydrogenase, characterized by comprising the following steps: 1. Activation of the solid-phase support: The solid-phase support was washed with 0.1M 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES buffer). The support was resuspended in HEPES buffer, and EDC-HCl and NHS were added sequentially. The reaction was carried out at room temperature in the dark with stirring. After the reaction was completed, the support was immediately washed rapidly with pre-cooled 0.1M NaHCO3 buffer (pH 8.3), and then used for the coupling of ADH1B and ALDH2 proteins. 2. Preparation of the dual-enzyme co-immobilized affinity vector: A segmented pH sequential coupling strategy was employed to covalently couple alcohol dehydrogenase 1B (ADH1B) and aldehyde dehydrogenase 2 (ALDH2) onto the same solid-phase carrier to maximize the preservation of dual-enzyme activity and minimize steric hindrance. The specific steps were as follows: The activated solid-phase carrier was washed with coupling buffer (0.1M NaHCO3 solution at pH 8.3). Coupling was first performed under slightly acidic conditions close to the isoelectric point of ADH1B, followed by coupling under weakly alkaline conditions conducive to the conformational stability of ALDH2. During coupling, the carrier was shaken and incubated to allow the free amino groups of the enzyme protein to form covalent bonds with the active groups on the carrier. After coupling, the carrier was washed with coupling buffer to remove unbound protein. Subsequently, the carrier was incubated with blocking buffer for 2 hours to block the remaining active sites on the carrier. Finally, the enzymes were thoroughly washed with phosphate-buffered saline (PBS) at pH 7.4–8.0, and the resulting co-immobilized affinity vector was resuspended in PBS buffer and stored at 4°C for later use. The coupling efficiency and enzyme activity retention were assessed by measuring the protein concentration in the supernatant before and after immobilization and the residual activity of the immobilized enzyme. 3. Affinity adsorption enrichment of target peptides: The enzymatic hydrolysate containing complex peptides to be screened is mixed with the dual-enzyme co-immobilized affinity carrier prepared in step 2 and incubated in PBS buffer. Certain specific sequences of peptides in the complex peptide hydrolysate are precisely complementary to the functional sites on the enzyme surface through their spatial conformation. They are stably bound mainly by intermolecular forces such as hydrogen bonds, ionic bonds, and hydrophobic interactions, thereby adsorbing peptides in the mixed peptide solution that can specifically bind to ADH1B and ALDH2 onto the immobilized enzyme. 4. Washing to remove impurities: After incubation, collect the dual-enzyme co-immobilized affinity vectors with adsorbed peptides. Wash repeatedly with PBS buffer containing 0.1-1.0M NaCl to thoroughly remove unbound and non-specifically bound peptides. 5. Competitive Elution and Collection: Utilizing the high specificity and affinity of different competing agents for their respective enzyme active sites, peptide separation based on the target site is achieved. First, elution is performed using a first elution buffer containing an ADH1B-specific competitive agent, collecting the first elution fraction rich in ADH1B-binding peptides. Subsequently, elution is performed using a second elution buffer containing an ALDH2-specific competitive agent, collecting the second elution fraction rich in ALDH2-binding peptides. During elution, incubation is carried out with gentle shaking for 10-30 minutes to allow the specifically bound peptides to be competitively dissociated. The peptides from the first and second elution fractions are mixed to obtain a solution enriched with potential ADH1B and ALDH2 activity-enhancing peptides. 6. Reverse screening verification: Set up a control experiment and use the same method to prepare vectors that only immobilize ADH1B, vectors that only immobilize ALDH2, and vectors that immobilize irrelevant proteins. Perform steps 2 to 5 in parallel and verify the specificity of peptide binding by comparing the differences in the eluted components. 7. Peptide Sequence Identification and Verification: The enriched product obtained in step 5 and the eluted product obtained in step 6 were sequenced by liquid chromatography-tandem mass spectrometry. The amino acid sequences of the peptides in the samples were compared and analyzed to exclude non-specifically binding peptides. The identified core peptide sequences were chemically synthesized, and their individual activation activities for alcohol dehydrogenase and aldehyde dehydrogenase were measured to verify their enzyme activity promoting function. Finally, highly efficient alcohol dehydrogenase and aldehyde dehydrogenase activity promoting peptides were confirmed.

[0011] Preferably, in step 1, the solid support is magnetic agarose microspheres, and more preferably carboxylated magnetic agarose microspheres.

[0012] Preferably, in step 1, the group used to activate the solid support is selected from N-hydroxysuccinimide (NHS) ester group.

[0013] Preferably, in step 2, the covalent coupling is achieved through an amide bond formation reaction mediated by N-hydroxysuccinimide (NHS) / carbodiimide (EDC).

[0014] Preferably, in step 2, the segmented pH sequential coupling method specifically involves: first coupling ADH1B under pH 5.5-7.0 conditions, then washing, and finally coupling ALDH2 under pH 7.5-9.0 conditions.

[0015] Preferably, in step 2, the coupling molar ratio of ADH1B to ALDH2 is 1:2 to 3:1.

[0016] Preferably, in step 2, the coupling reaction temperature of the purified recombinant human ADH1B enzyme and ALDH2 enzyme with the solid-phase carrier is 4℃-25℃, and the reaction time is 2h-6h.

[0017] Preferably, in step 3, the incubation conditions for the enzymatic hydrolysate containing complex multiple peptide segments and the dual-enzyme co-immobilized affinity vector prepared in step 2 are oscillation incubation at 4℃-37℃ for 30-90 minutes.

[0018] Preferably, in step 4, when using magnetic microspheres, the dual-enzyme co-immobilized affinity carriers that have adsorbed peptides are collected by magnetic separation.

[0019] Preferably, in step 5, the ADH1B specific competitor is selected from ethanol, NAD+, etc. + Or a combination thereof; the ALDH2 specific competitor is selected from acetaldehyde, NAD+, etc. +Known ALDH2 allosteric agonists (Alda-1) or combinations thereof are used to specifically enrich active peptides acting on this key regulatory site.

[0020] Preferably, in step 6, the irrelevant protein used in the control group is bovine serum albumin (BSA).

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) Screening efficiency doubled: For the first time, a dual-target affinity interface for ADH1B and ALDH2 was successfully constructed on the same solid-phase support, enabling simultaneous screening of active peptides targeting two key rate-limiting enzymes in the alcohol metabolism pathway in one experiment, significantly improving screening efficiency. (2) Discovering synergistic peptides: The unique parallel screening mode provides a unique platform for discovering synergistic peptides that can act on multiple nodes of the metabolic pathway at the same time. These peptides may enhance the efficiency of the entire metabolic pathway through coordination. (3) Innovative vector design: Through an innovative segmented pH-controlled sequential coupling method, the coupling microenvironment of ADH1B and ALDH2 is optimized according to their different biochemical characteristics, which effectively solves the problems of activity loss and spatial interference in the co-immobilization of the two enzymes and ensures the high performance of the affinity vector. (4) More functionally targeted: Stepwise specific competitive elution combined with reverse screening verification ensures the target specificity of enriched peptides, reduces false positives, and provides a high-purity candidate library for subsequent identification and functional studies. Attached Figure Description

[0022] Figure 1 This is a diagram illustrating the principle of collecting enrichment products.

[0023] Figure 2 This is a schematic diagram of the enzymatic hydrolysate under the action of different proteases.

[0024] Figure 3 This is a comparison chart of the degree of hydrolysis of enzymatic hydrolysates under the action of different proteases.

[0025] Figure 4 This graph shows the activation rates of ethanol dehydrogenase and acetaldehyde dehydrogenase in three samples: the original enzyme hydrolysate, the enriched product, and the reverse elution product.

[0026] Figure 5 This is a graph showing the aspartate aminotransferase (AST) activity results of three samples: the original enzyme hydrolysate, the enriched product, and the reverse elution product. Detailed Implementation

[0027] It should be understood that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. To enable those skilled in the art to more clearly understand the technical solutions of the present invention, the technical solutions of the present invention will be described in detail below with reference to specific embodiments. The experimental materials used in the embodiments of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels.

[0028] The first embodiment of this invention provides a method for preparing a dual-enzyme co-immobilized affinity vector. First, carboxylated magnetic agarose microspheres are activated with NHS using EDC-HCl to generate stable NHS esters with amino-reactive properties. A segmented pH sequential coupling strategy is employed. The activated magnetic agarose microspheres are first incubated with recombinant human ADH1B enzyme dissolved in coupling buffer at pH 5.5-7.0 with shaking. The free primary amino groups (-NH2) on the enzyme surface react with the NHS ester under mild alkaline conditions to form stable amide bonds. After coupling, the microspheres are thoroughly washed. Then, they are incubated with recombinant human ALDH2 enzyme dissolved in coupling buffer at pH 7.5-9.0 with shaking. After coupling, the microspheres are washed and blocked to consume any remaining active groups on the microsphere surface that have not reacted with the enzyme protein. This prevents covalent or non-specific binding with non-target peptides or other proteins in subsequent screening steps, thereby reducing background interference and false positives, and preparing stable dual-enzyme co-immobilized magnetic agarose microspheres. The magnetic microspheres were suspended in PBS buffer and stored at 4°C for later use.

[0029] The second embodiment of the present invention provides a method for preparing and collecting enriched products. An enzymatic hydrolysate containing complex peptides to be screened is mixed with prepared dual-enzyme co-immobilized magnetic agarose microspheres and incubated in PBS buffer. Certain specific sequences of peptides in the complex polypeptide hydrolysate are precisely complementary to functional sites on the enzyme surface through their spatial conformation, and are stably bound mainly by intermolecular forces such as hydrogen bonds, ionic bonds, and hydrophobic interactions. This allows peptides in the mixed peptide solution that specifically bind to ADH1B and ALDH2 to be adsorbed onto the immobilized enzyme. After incubation, the magnetic microspheres adsorbed with peptides are collected by magnetic separation. After multiple washes with PBS buffer containing 0.1-1.0M NaCl, elution is first performed using a first elution buffer containing an ADH1B-specific competitive agent, collecting the first elution fraction rich in ADH1B-binding peptides; subsequently, elution is performed using a second elution buffer containing an ALDH2-specific competitive agent, collecting the second elution fraction rich in ALDH2-binding peptides. During elution, incubate with gentle shaking for 10-30 minutes to allow the specifically bound peptides to be competitively dissociated. Mix the peptides from the first and second elution fractions to obtain a solution enriched with potential ADH1B and ALDH2 activity-enhancing peptides.

[0030] The third embodiment of the present invention provides a reverse screening and functional verification process. To exclude peptides that may bind nonspecifically to the solid-phase support, a reverse control experiment is set up. Vectors immobilized only ADH1B, vectors immobilized only ALDH2, and vectors immobilized irrelevant proteins are prepared using the same method, and parallel experiments are conducted under the same conditions. By comparing the differences in the elution components, the specificity of peptide binding is verified.

[0031] To make the technical solution of the present invention clearer, the following detailed description is provided through several specific embodiments.

[0032] Example 1: Activation of magnetic agarose microspheres Take 1 mL of carboxylated magnetic agarose microspheres and wash them three times with 0.1 M HEPES buffer. Resuspend the microspheres in 2 mL of HEPES buffer, and add EDC-HCl and NHS sequentially to final concentrations of 20 mM and 10 mM, respectively. Stir the mixture at room temperature in the dark for 20 minutes. After the reaction is complete, immediately wash the microspheres four times with pre-cooled 0.1 M NaHCO3 buffer (pH 8.3), and then use the mixture for conjugation of recombinant human ADH1B and ALDH2 proteins.

[0033] Example 2: Preparation of magnetic microspheres co-immobilized with each ADH1B / ALDH2 enzyme using a segmented pH sequential coupling method. (1) Coupling of ADH1B Take 1 mL of activated magnetic microspheres, wash with 1 mM HCl, and then wash with 0.1 M NaHCO3 buffer. Dissolve 1.5 mg of recombinant human ADH1B protein in 2 mL of 0.1 M PBS buffer, and incubate with all 1 mL of pretreated microspheres at 4 °C with gentle rotation for 2 hours. After incubation, perform magnetic separation by placing the reaction tube on a magnetic rack and collecting the supernatant to determine the amount of unbound protein. The ADH1B coupling efficiency was calculated to be approximately 75%. Wash the magnetic microspheres thoroughly three times with PBS buffer to completely remove uncoupled ADH1B and reaction byproducts, obtaining ADH1B-pre-immobilized microspheres. (2) Coupling of ALDH2 1 mg of recombinant human ALDH2 protein was dissolved in 2 mL of 0.1 M Tris-HCl buffer (pH 7.8) and incubated with the "ADH1B-pre-immobilized microspheres" obtained in the previous step at 4 °C for 2 hours. After incubation, the reaction tube was magnetically separated by placing it on a magnetic rack and collecting the supernatant to determine the amount of unbound protein. The ALDH2 coupling efficiency was calculated to be approximately 60%. The microspheres were washed three times with Tris-HCl buffer (pH 7.8) to completely remove uncoupled ALDH2 and reaction byproducts, yielding the dual-enzyme-coupled microspheres. (3) Closed Add 1M ethanolamine-HCl solution (pH 8.5) to the microspheres and incubate gently by rotation at room temperature for 2 hours to covalently block all unreacted active ester groups on the microspheres. Magnetic separation is then performed, and the blocking solution is discarded. The microspheres are then thoroughly washed with PBS buffer. The resulting microspheres are the prepared ADH1B / ALDH2 dual-enzyme co-immobilized magnetic microspheres, which are suspended in PBS buffer and stored at 4°C for later use. Enzyme activity assays showed that the activity retention rates of ADH1B and ALDH2 immobilized on the same microspheres were approximately 68% and 62% of their initial free enzyme activities, respectively.

[0034] Example 3: Parallel enrichment of dual-enzyme activity-enhancing peptides from corn protein hydrolysate Different proteases were used to perform enzymatic hydrolysis of corn gluten meal, and the degree of hydrolysis was used as an indicator to screen the proteases. Figure 3 It can be seen that the enzymatic hydrolysis effect of flavor protease on corn protein is significantly higher than that of the other five enzymes, with a degree of hydrolysis reaching 42.00±1.01%. Therefore, the enzymatic hydrolysate under the action of flavor protease was selected for subsequent experiments. Mix 2 mL of corn protein hydrolysate (peptide concentration approximately 5 mg / mL) with 200 μL of the ADH1B / ALDH2 dual-enzyme co-immobilized magnetic microsphere suspension prepared in Example 2, and bring the total volume to 3 mL with PBS buffer. Incubate at room temperature for 60 minutes. Magnetic separation is performed, the supernatant is discarded, and the magnetic beads are thoroughly washed. Add 0.5 mL of a solution containing 50 mM ethanol and 10 mM NAD. + Incubate the microspheres with PBS buffer and gently shake for 15 minutes at room temperature. Perform magnetic separation and collect the supernatant as the first elution fraction (E1). Wash the microspheres once quickly with PBS. Add 0.5 mL of buffer containing Alda-1 and incubate for 15 minutes at room temperature. Perform magnetic separation and collect the supernatant as the second elution fraction (E2). Desalt and concentrate fractions E1 and E2 separately using ultrafiltration centrifuge tubes with a molecular weight cutoff of 10 kDa. Mix fractions E1 and E2 at a 1:1 volume ratio to obtain a mixed solution (E1+E2). Freeze-dry each of E1, E2, and (E1+E2) separately for subsequent analysis.

[0035] Example 4: Reverse Screening Control Following the method in Example 1, supports immobilized only with ADH1B, supports immobilized only with ALDH2, and BSA-immobilized magnetic microspheres were prepared. Parallel experiments were conducted under the same conditions to obtain elution products, which were designated as control products A, B, and C, respectively.

[0036] Example 5 Functional Verification The activities of alcohol dehydrogenase, aldehyde dehydrogenase, and aspartate aminotransferase (AST) in three samples—the original enzyme hydrolysate, the enriched product (E1+E2 fraction), and the reverse elution product with BSA immobilization only—were determined using the Waller-Hoch method and kits for aldehyde dehydrogenase and AST, respectively. The activation rates of alcohol dehydrogenase and aldehyde dehydrogenase, and the activities of AST were calculated using the following formulas. Figure 4 As shown, the reverse elution products, due to the lack of binding to functional peptides, exhibited only 5.47±1.55% activation of alcohol dehydrogenase and 5.27±1.29% activation of acetaldehyde dehydrogenase. Compared to the original hydrolysate (55.06±1.24%) and ALDH activation (59.52±0.75%), the enriched products, due to their high purity of ADH1B and ALDH2 activity-promoting peptides, showed significantly higher ADH and ALDH activation rates, reaching 90.29±0.96% and 89.83±1.32%, respectively. Similarly, as... Figure 5As shown, the reverse elution product, not having bound to functional peptides, showed little difference in AST activity compared to the model group, at approximately 39.50±0.72 U / gprot. However, the enriched product, possessing high-purity ADH1B and ALDH2 activity-promoting peptides, exhibited superior hepatoprotective and alcohol-detoxifying effects, resulting in the greatest reduction in AST activity. Compared to the model group (41.53±0.97 U / gprot) and the original enzyme hydrolysate (31.34±1.69 U / gprot), it decreased to 23.12±1.54 U / gprot. Aspartate aminotransferase activity (U / gprot) = Enzyme activity (Kamen's units) * 0.482 ÷ Protein concentration

[0037] In summary, the technical solution of this invention, through the innovative design of a method capable of enriching alcohol dehydrogenase and aldehyde dehydrogenase activating peptides, can efficiently enrich potential alcohol dehydrogenase and aldehyde dehydrogenase activity-promoting peptides from complex peptide solutions, providing an effective technical means for the discovery and development of related functional peptides.

[0038] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for enriching peptides that promote the activity of alcohol dehydrogenase and aldehyde dehydrogenase, characterized in that, Includes the following steps: a) Activation of the solid support: First, the solid support was washed, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl) and N-hydroxysuccinimide (NHS) were added sequentially for activation. The reaction was carried out at room temperature in the dark with stirring. After the reaction was completed, the solid support was immediately washed with pre-cooled 0.1M NaHCO3 buffer (pH 8.3) to obtain the activated solid support. b) Preparation of dual-enzyme co-immobilized affinity vector: A segmented pH sequential coupling strategy was adopted to covalently couple alcohol dehydrogenase 1B (ADH1B) and acetaldehyde dehydrogenase 2 (ALDH2) to the same solid-phase support to maximize the preservation of dual enzyme activity and reduce steric hindrance. After the coupling reaction was completed, the remaining active sites on the support were blocked with 1M ethanolamine-HCl solution (pH 8.5) to prepare the dual-enzyme co-immobilized affinity vector. c) Parallel affinity adsorption: The complex polypeptide digest to be screened is incubated with the dual-enzyme carrier in a buffer solution to adsorb peptides that can specifically bind to ADH1B and ALDH2. d) Wash with buffer to remove unbound impurities; e) Stepwise competitive elution and collection: First, elute and collect the first fraction with a first eluent containing an ADH1B competitive agent, and then elute and collect the second fraction with a second eluent containing an ALDH2 competitive agent; f) Reverse screening verification: Using an immobilized vector coupled only with ADH1B protein, ALDH2 protein and irrelevant protein as a control, the same enzyme digest was treated under the same conditions, and the obtained elution product and the enriched product of step e were sequenced. The peptide amino acid sequences of the two samples were compared and analyzed to exclude non-specific binding peptides in order to identify high-confidence ADH1B and ALDH2 specific binding peptides. g) Functional verification: Mix the first component with the second component and determine its effect on the activity of alcohol dehydrogenase and acetaldehyde dehydrogenase.

2. The method according to claim 1, characterized in that, The solid support mentioned in step a) is preferably magnetic agarose microspheres.

3. The method according to claim 1, characterized in that, The covalent coupling described in step b) adopts a segmented pH sequential coupling method: first, ADH1B is coupled under pH conditions of 5.5-7.0, and then ALDH2 is coupled under pH conditions of 7.5-9.

0.

4. The method according to claim 1 or 2, characterized in that, The coupling molar ratio of ADH1B to ALDH2 in step b) is 1:2 to 3:

1.

5. The method according to claim 1, characterized in that, In step e), the ADH1B competitor is selected from ethanol, NAD+, etc. + Or a combination thereof; the ALDH2 competitor is selected from acetaldehyde, NAD+, etc. + Known ALDH2 allosteric agonists (Alda-1) or combinations thereof.

6. A dual-enzyme co-immobilization affinity vector for use in the method according to any one of claims 1-5, characterized in that, It consists of magnetic agarose microspheres with ADH1B and ALDH2 covalently coupled to the surface, wherein the molar ratio of ADH1B to ALDH2 is 1:2 to 3:

1.

7. A peptide composition enriched by the method according to any one of claims 1-5, characterized in that, It contains ADH1B activity-promoting peptide and ALDH2 activity-promoting peptide.

8. The use of the dual-enzyme co-immobilized affinity vector of claim 6 in the preparation of products for screening or discovering alcohol metabolism regulators.

9. The use of the peptide composition of claim 7 in the preparation of functional foods, health products or medicines for promoting alcohol metabolism, relieving hangovers or preventing alcohol-related diseases.

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