Prolyl endopeptidase with multiple cleavage sites and application of prolyl endopeptidase in degradation of gluten immunogenic 33-mer peptide

Abstract

The invention discloses prolyl endopeptidase with multiple cleavage sites and application of the prolyl endopeptidase in degradation of gluten immunogenic 33-mer peptide, and belongs to the technical field of enzyme engineering and food biology. New prolyl endopeptidase (PEP) and application thereof are screened from human intestinal metagenome assembly genomes (MAGs), the coverage degree of hydrolysate sequences of the prolyl endopeptidase C9-2 and C11-1 to 33-mer peptide reaches 100%, cleavage sites are richer, short fragments can be generated through multi-site cleavage, the immunogenicity of the 33-mer peptide can be eliminated to a greater extent, and the prolyl endopeptidase (PEP) can be used for preparing the prolyl endopeptidase. The strain can be applied to preparation of anti-celiac disease foods, intestinal immune regulation preparations and gluten food desensitization processing, and has important clinical and industrial values.

Description

Technical Field

[0001] This invention relates to a prolyl endopeptidase with multiple cleavage sites and its application in the degradation of gluten immunogenic 33-mer peptide, belonging to the fields of enzyme engineering and food biotechnology. Background Technology

[0002] Gluten protein is a unique storage protein complex found in grains such as wheat, barley, and rye. It is mainly composed of prolamins and glutenins, accounting for about 80% of the total grain protein. With its unique viscoelasticity, extensibility, and gas-holding properties, it is indispensable in the processing of food products such as bread, noodles, and beer, and has significant economic value.

[0003] However, gluten intake poses a serious health threat to certain populations, including celiac disease, an autoimmune enteropathy caused by gluten, with a global prevalence of approximately 1%. Human proteases cannot completely hydrolyze gluten, producing various peptides. The 33-mer peptide, derived from α-prolamin, is a core immunogenic epitope. This peptide is rich in proline and glutamine, and its dense pyrrolidine ring structure makes it resistant to intestinal enzymatic degradation. It can also penetrate the intestinal epithelial barrier. After being catalyzed by tissue transglutaminase, it enhances its affinity for human leukocyte antigen molecules, triggering an immune response and leading to symptoms such as intestinal inflammation, diarrhea, and malnutrition. It is the core carrier and therapeutic target of gluten toxicity.

[0004] Prolyl endopeptidase (PEP, EC: 3.4.21.26) specifically cleaves the C-terminal peptide bond of proline residues, making it an ideal enzyme for degrading 33-mer peptides. However, existing microbial PEP sources have significant limitations. PEP derived from *Flavobacterium meningitidis* is pH-sensitive, with its activity dropping sharply in gastric acid; while PEP derived from *Aspergillus niger* is acid-resistant, its ability to degrade immunogenic peptides is somewhat insufficient. Therefore, developing novel prolyl endopeptidases that efficiently degrade gluten immunogenic peptides, have abundant cleavage sites, and are adapted to the application environment is of great significance. Summary of the Invention

[0005] The first object of the present invention is to provide a prolyl endopeptidase capable of degrading 33-mer peptides at multiple cleavage sites, wherein the amino acid sequence of the prolyl endopeptidase is shown in any one of SEQ ID NO.1 to SEQ ID NO.4.

[0006] In one embodiment, the amino acid sequence of the prolyl endopeptidase is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0007] The present invention also provides a gene encoding the prolyl endopeptidase.

[0008] The present invention also provides a recombinant vector carrying the prolyl endopeptidase gene.

[0009] In one embodiment, the vector includes, but is not limited to, pET series plasmids.

[0010] In one embodiment, the gene sequence encoding prolyl endopeptidase C9-2 as shown in SEQ ID NO.1 is as shown in SEQ ID NO.5.

[0011] In one embodiment, the gene sequence encoding prolyl endopeptidase C11-1 as shown in SEQ ID NO.2 is as shown in SEQ ID NO.6.

[0012] The present invention also provides a recombinant Escherichia coli that expresses the prolyl endopeptidase.

[0013] In one implementation, pET-SUMO is used as the expression vector.

[0014] In one embodiment, the recombinant Escherichia coli genetically engineered bacteria uses Escherichia coli BL21(DE3) as the expression host.

[0015] The present invention provides a method for preparing the prolyl endopeptidase, wherein the recombinant Escherichia coli is cultured in a culture medium for a period of time and the expression of the enzyme is induced by IPTG.

[0016] In one embodiment, the method involves culturing the recombinant *E. coli* in a seed culture medium at 37 °C and 220 rpm to obtain a seed solution; the prepared seed solution is then transferred to a fermentation medium at a ratio of 1% (v / v) and cultured until OD (Organic Demand). 600 =0.6, then add isopropyl-β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and cool to 28 °C, then induce expression at 150 rpm for 10-14 h.

[0017] This invention also provides the application of prolyl endopeptidase in the degradation of gluten 33-mer peptides.

[0018] In one embodiment, the application includes, but is not limited to, the preparation of anti-celiac disease foods and intestinal immune conditioning agents.

[0019] In one implementation, the application includes, but is not limited to, gluten desensitization.

[0020] Beneficial effects: 1. This invention provides prolyl endopeptidases C9-2 and C11-1, derived from human intestinal metagenomic genome assembly, with bidirectional cleavage capabilities. The number of cleavage sites far exceeds that of the reference enzyme, achieving 100% hydrolysis coverage, overcoming the limitation of existing enzymes with a single cleavage mode and significantly reducing the immunogenicity of 33-mer peptide. The aforementioned prolyl endopeptidases exhibit good stability at 40 °C; among them, prolyl endopeptidase C9-2 exhibits higher enzyme catalytic efficiency.

[0021] 2. The method of this invention utilizes bioinformatics technology to deeply mine the prolyl endopeptidase gene of human intestinal microorganisms. Based on the method of this invention, novel prolyl endopeptidases with excellent performance can be obtained rapidly, and it also provides feasible ideas for the discovery of other novel enzymes. Attached Figure Description

[0022] Figure 1 Phylogenetic analysis of 16 enzymes. All enzymes expressed heterologously in two rounds, with HC as the known reference sequence (UniProt ID: P27195). Taking C9-1 and C9-2 as examples: C9-1 represents the ninth sequence with the highest cluster degree value, and C9-2 represents the ninth sequence with the second highest cluster degree value.

[0023] Figure 2 For comparison of crude enzyme activity (enzymes with activity in crude enzyme solution among 16 enzymes).

[0024] Figure 3 This is a gel image of protein purification. Lane M is the protein marker; lane 1 is the HC crude enzyme solution (supernatant after cell lysis); lane 2 is the HC purification solution (elution buffer after nickel column purification of crude enzyme solution); lane 3 is the HC ultrafiltrate (purification solution concentrated in an ultrafiltration tube and diluted 30 times before loading); lane 4 is the C2-3 crude enzyme solution (supernatant after cell lysis); lane 5 is the C2-3 purification solution (purification solution concentrated in an ultrafiltration tube and diluted 30 times before loading); and lane 6 is the C2-3 ultrafiltrate (purification solution concentrated in an ultrafiltration tube and diluted 30 times before loading). Lane 7 is C9-2 crude enzyme solution (supernatant after cell lysis), Lane 8 is C9-2 purified solution (elution buffer after nickel column purification of crude enzyme solution), Lane 9 is C2-3 ultrafiltrate (purified solution concentrated in ultrafiltration tube and diluted 30 times before loading), Lane 10 is C11-1 crude enzyme solution (supernatant after cell lysis), Lane 11 is C11-1 purified solution (purified solution purified in nickel column purification and eluent), and Lane 12 is C11-1 ultrafiltrate (purified solution concentrated in ultrafiltration tube and diluted 30 times before loading).

[0025] Figure 4 The enzyme activities of four prolyl endopeptidases were compared between crude enzyme solution, purified solution, and ultrafiltrate.

[0026] Figure 5The results of the enzymatic properties study of prolyl endopeptidases include: (A) the optimal temperature of the four prolyl endopeptidases; (B) the thermal stability of the four prolyl endopeptidases at 40 °C; (C) the thermal stability of the four prolyl endopeptidases at 50 °C; (D) the optimal pH of the four prolyl endopeptidases; and (E) the pH stability of the four prolyl endopeptidases.

[0027] Figure 6 These are secondary mass spectra of four prolyl endopeptidases. Detailed Implementation

[0028] In the following examples, gene synthesis, nickel column purchase, sequencing, peptide synthesis, and LC-MS were all obtained from Shanghai Sangon Biotech Co., Ltd. Suc-Ala-Pro-pNA was purchased from MCE Biotech. Staining solutions and buffers used in protein SDS-PAGE were purchased from Beyotime Biotech Co., Ltd.

[0029] (a) Culture medium LB liquid medium: NaCl 10 g / L, yeast extract 5 g / L, peptone 10 g / L.

[0030] LB solid medium: NaCl 10 g / L, yeast extract 5 g / L, peptone 10 g / L, agar powder 15 g / L.

[0031] (II) Detection Methods Method for determining prolyl endopeptidase activity: Enzyme activity was determined using 410 nm ultraviolet light absorption spectrometry. Suc-Ala-Pro-pNA was used as the substrate to measure prolyl endopeptidase activity. The reaction system (200 μL) consisted of 60 μL 1.0 mM Suc-Ala-Pro-pNA and 110 μL buffer. The mixture was preheated at 37 °C for 5 min, followed by the addition of 30 μL of enzyme solution. After immediate mixing, the reaction was incubated at 37 °C for 30 min, and then terminated by heating at 100 °C for 10 min. After cooling to room temperature, the mixture was centrifuged at 4 °C and 6000 × g for 5 min to remove denatured proteins. The supernatant was then used for OD410 nm determination.

[0032] A 10 mM p-nitroaniline stock solution was prepared and diluted to a series of concentrations of 0, 5, 10, 25, 50, 75, 100, 200, 300, and 400 μM. 200 μL of each solution was plotted in a 96-well plate, and the OD410 was measured. A concentration-absorbance linear regression equation was established to calculate the number of moles of p-nitroaniline produced by prolyl endopeptidase hydrolyzing the substrate Suc-Ala-Pro-pNA.

[0033] One unit of enzyme activity is defined as the amount of enzyme required per minute to catalyze the hydrolysis of the substrate Suc-Ala-Pro-PNA to produce 1 μmol of PNA under standard conditions (37°C, pH 7.4). Specific activity is expressed in U / mg.

[0034] (III) Sequence Description The amino acid sequences of prolyl endopeptidases C9-2, C11-1, HC, and C2-3 are shown in SEQ ID NO. 1-4, respectively, and the nucleotide sequences encoding prolyl endopeptidases C9-2, C11-1, HC, and C2-3 are shown in SEQ ID NO. 5-8, respectively.

[0035] Example 1: Selection of candidate prolyl endopeptidases Twenty prolyl endopeptidases were retrieved from the Swiss-Prot database and a local database was built using diamond (v2.1.10). Using this self-built database as a reference, diamond blastp (e<10) was performed against 478,588 MAGs in the GMR database. -5 Homologous sequence alignment was performed. To narrow down the range of candidate genes, conserved domains were annotated in the self-built library sequences using the Interpro database, and the corresponding HMM files for each domain were downloaded. HMMsearch (v3.4) was used for domain filtering (e<10). -5 Redundant sequences were removed using CD-HIT (v4.8.1) 95% sequence identity clustering. The aforementioned sequences were merged with the 20 sequences used in the database construction and uploaded to EFI - Enzyme Similarity Tool (https: / / efi.igb.illinois.edu / efi-est / ). An Alignment Score of 120 and a sequence similarity threshold >35% were set to generate a PEP sequence similarity network (SSN), which was then visualized using Cytoscape (v3.10.3).

[0036] The first eleven clusters in the SSN network were selected, and the degree values ​​of the nodes in each cluster were calculated using the Analyse network algorithm and sorted from highest to lowest. Genes with higher degree values ​​were considered to have a higher degree of association with other genes in the cluster, and the gene with the highest degree was defined as the hub gene of that cluster. First round of selection: (1) Hub genes with the highest degree values ​​in each cluster, a total of 11; (2) The known active prolyl endopeptidase reference sequence (UniProt ID: P27195), located in Cluster 2; (3) The second highest degree sequence in Cluster 2, excluding the hub. Second round of supplementary selection: In order to reduce the "topology-function" bias, one sequence with a degree value second only to the hub was selected from each of the above 3 active clusters. A total of 16 candidate sequences were selected in the second round of selection.

[0037] Phylogenetic analysis was performed on 16 sequences, and the results are as follows: Figure 1 As shown, sequences within the same cluster are all located in adjacent branches.

[0038] Example 2: Heterologous expression and crude enzyme activity assay Candidate genes were optimized using E. coli preferred codons, synthesized by Shanghai Sangon Biotech, and cloned into the pET-SUMO expression vector, retaining a 6×His tag at the C-terminus of the target gene. The synthesized plasmid powder was dissolved in sterile ddH2O to a final volume of 100 ng / μL. - ¹, Gently mix 2 μL of the mixture with 100 μL of BL21(DE3) chemocompetent cells and incubate on ice for 30 min; after heat shock at 42 ℃ for 90 s, immediately incubate on ice for 2 min; add 900 μL of LB liquid medium and incubate at 37 ℃ and 200 rpm for 1 h. Spread 30 μL of the mixture onto a medium containing 50 μg of LB broth. - ¹Kanamycin was cultured on LB agar plates at 37 °C inverted for 12–18 h. Single colonies were randomly picked and verified by colony PCR. Positive clones were then inoculated into 5 mL of LB-Kan (50 μg / mL). - ¹) Liquid culture medium, cultured overnight at 37 ℃ and 220 rpm with shaking, to be used as seed culture.

[0039] Seed culture was inoculated at 1% (V / V) into 200 mL of LB-Kan (50 μg / mL). - ¹), incubate at 37 ℃ and 220 rpm until OD. 600=0.6, add IPTG to a final concentration of 0.5 mM, and immediately transfer to 28 ℃, 150 rpm for 12 h to induce expression. After induction, collect the bacterial cells by centrifugation at 4 ℃, 6000 ×g for 5 min, discard the supernatant, and resuspend in pre-cooled PBS. Add lysis buffer at 1 / 9 (V / V) of the bacterial volume and vortex to mix thoroughly. Centrifuge the lysis buffer at 6000 ×g, 4 ℃ for 15 min, and take the whole bacterial culture, supernatant, and precipitate resuspending solution, respectively, and mix with 2× SDS loading buffer 1:1. Boil at 95 ℃ for 10 min, and verify the expression by SDS-PAGE, with pET-SUMO empty vector as a negative control. The results showed that the supernatant of the above candidate genes was effectively expressed.

[0040] The activity of the crude enzyme solutions (lysis supernatant) was determined. The results showed that only the crude enzyme solutions of HC, C2-1, C2-2, C2-3, C9-1, C9-2, C11-1, and C11-2 showed a yellow color during co-incubation with Suc-Ala-Pro-pNA, indicating that these enzymes successfully hydrolyzed the substrate to release p-nitroaniline, while the other enzymes showed no activity. The enzyme activities of the crude enzyme solutions with the above-mentioned active sequences were compared, and the results are as follows: Figure 2 As shown.

[0041] Example 3: Purification and Enzyme Activity Comparison of Prolyl Endopeptidase The lysis supernatant prepared in Example 2 was filtered through a 0.45 μm microporous membrane and loaded onto a HyPur T Ni-NTA6FF (His-Tag) pre-packed gravity column. Non-specifically bound contaminating proteins were washed away using a gradient of PBS containing 50 mM and 100 mM imidazole, followed by elution of the target protein with PBS containing 250 mM imidazole. The eluent was transferred to a 30 kD ultrafiltration tube, centrifuged at 3000 ×g at 4 °C to a final concentration of <200 μL, and PBS was added to make up to 3 mL. This process was repeated three times to remove imidazole and contaminating proteins. The protein concentration of the final ultrafiltrate was determined by the BCA method, aliquoted, and stored at -20 °C for later use. SDS-PAGE was used to verify the purification effect.

[0042] Using the purification method described above, four prolyl endopeptidases (HC, C2-3, C9-2, and C11-1) with relatively high crude enzyme activity prepared in Example 2 were selected for purification by ultrafiltration. The SDS-PAGE results are as follows: Figure 3 As shown, a single band is clearly visible, indicating good purification effect.

[0043] The specific enzyme activities of the crude enzyme solution, purified solution, and ultrafiltrate of the four prolyl endopeptidases were compared, and the results are shown in Table 1 and... Figure 4 As shown.

[0044] Table 1. Enzyme activity ratios of crude enzyme solution, purified solution, and ultrafiltrate for different enzymes.

[0045] Example 4: Enzymatic Properties Analysis of Prolyl Endopeptidase 1. Effect of temperature on prolyl endopeptidase activity Temperature can alter the rate of enzyme catalytic reactions and also lead to a decrease or inactivation of enzyme protein activity. The relative enzyme activities of prolyl endopeptidases HC, C2-3, C9-2, and C11-1 at different temperatures were determined according to the following steps.

[0046] The activity of ultrafiltration enzyme at 20 ℃, 30 ℃, 40 ℃, 50 ℃, and 60 ℃ was determined using PBS (pH 7.4). The highest enzyme activity was taken as 100%, and the relative enzyme activities of the remaining groups were calculated. The results are as follows: Figure 5 As shown in Figure A, the optimal temperature for HC, C2-3, and C9-7 is 40℃, while the optimal temperature for C11-1 is 30℃.

[0047] Using the activity of prolyl endopeptidase without heat treatment as 100%, the residual enzyme activity of ultrafiltration enzyme was measured after incubation at 40 °C and 50 °C for 4 h, respectively. The results are as follows: Figure 5 As shown in B~5C, all four enzymes showed good stability at 40 °C, with residual enzyme activity still greater than 60% after 4 h of incubation. Although their thermal stability decreased at 50 °C, C9-2 and C11-1 showed slower inactivation rates than the reference enzyme HC.

[0048] 2. Effect of pH on prolyl endopeptidase activity Enzymatic reactions have their optimal pH range. Too high or too low a pH value will affect the activity of the enzyme in catalytic reactions. In this example, the optimal reaction pH of prolyl endopeptidase HC, C2-3, C9-2, and C11-1 will be determined.

[0049] At 37 °C, substrate working solutions were prepared using either 50 mM Na₂HPO₄-citric acid buffer (pH 3.0–8.0) or 50 mM glycine-sodium hydroxide buffer (pH 9.0–11.0). The highest enzyme activity was considered 100%, and the relative enzyme activities of the remaining groups were calculated. The results are as follows: Figure 5 As shown in D, the optimal reaction pH for HC, C2-3, C9-2, and C11-1 are 9.0, 8.0, 7.0, and 5.0, respectively.

[0050] The enzyme solution was diluted appropriately with the aforementioned buffer solution (pH 3.0-11.0) and incubated at 37 °C for 30 min. It was then immediately cooled. The prolyl endopeptidase activity at the optimal pH was taken as 100%, and the residual enzyme activity in the remaining groups was calculated. The results are as follows: Figure 5E. Except for C11-1, HC, C2-3 and C9-2 maintained more than 80% of their relative activity after incubation in the neutral to weakly alkaline range (pH 7-10), showing good adaptability to weakly alkaline environments; while C11-1 showed a unique pH response pattern, with its activity increasing sharply at pH > 4, reaching the highest enzyme activity at pH 5, and the residual activity being less than 50% when the pH rose to 8. This characteristic suggests that it is more suitable for weakly acidic environments.

[0051] 3. Effect of substrate concentration on enzyme-catalyzed reaction rate Substrate concentration significantly affects the efficiency of enzymatic reactions. Under otherwise constant conditions, the effect of substrate concentration on reaction rate follows a rectangular hyperbolic relationship. At low substrate concentrations, the reaction rate is directly proportional to the substrate concentration, indicating a first-order reaction. As the substrate concentration increases, the reaction rate no longer increases proportionally, indicating a mixed-order reaction. When the substrate concentration reaches a certain level, the reaction rate no longer increases, reaching its maximum, indicating a zero-order reaction.

[0052] Under the optimal pH and temperature conditions for the enzyme, a series of substrates (Suc-Ala-Pro-pNA, MCE, HY-P4490) with concentrations ranging from 0.1 to 10 mM were prepared, specifically at concentrations of 0.1, 0.3, 0.5, 0.8, 1.0, 1.2, 1.4, 1.7, 2.0, 2.5, 3.0, 4.0, 5.0, and 10.0 mM. OD was continuously monitored using a microplate reader immediately after the enzyme solution was added. 410 The initial velocity (v0, ΔOD) in the linear interval within the first 3 minutes of data collection was recorded. 410 min - ¹) The absorbance values ​​were converted to μmol / min using the p-nitroaniline standard curve. - ¹. Nonlinear regression was performed using GraphPad Prism 9.5, and the data was fitted to the Michaelis-Menten equation to determine the Michaelis constants. K m), maximum reaction rate ( V max), catalytic constant ( k cat) and catalytic efficiency ( k cat / K m), the results are shown in Table 2.

[0053] Table 2 Catalytic kinetic parameters of different enzymes

[0054] The results showed that the catalytic efficiency was C9-2 > HC > C2-3 > C11-1, indicating that C9-2 had the most outstanding conversion rate and binding affinity for the substrate.

[0055] Example 5: Determination of 33-mer peptide products and cleavage site analysis by prolyl endopeptidase To further analyze the enzymatic cleavage fragments and cleavage sites of the 33-mer peptide, the cleavage pattern was characterized using LC-MS / MS combined with a database retrieval system.

[0056] 33-mer peptide (LQLQPFPQPQLPYPQPQLPYPQPQPQLPYPQPQPF) was synthesized by Shanghai Sangon Biotech Co., Ltd., with a purity >98%. The reaction system (total volume 600 μL) consisted of 180 μL of 2 mg / mL peptide solution, 90 μL of 0.1 mg / mL ultrafiltration enzyme solution, and was brought to a final volume with 50 mM optimal pH buffer. The reaction was incubated at the optimal temperature for 4 h, and then immediately terminated by adding 0.1% (v / v) formic acid. The mixture was centrifuged at 6000 ×g, 4 °C for 5 min, and the supernatant was analyzed by LC-MS / MS.

[0057] Database searches further confirmed the integrity of the hydrolysis products relative to the original 33-mer peptide, as shown in the secondary mass spectra. Figure 6 As shown in the figure, the detection product peptide of C2-3 only showed 75.76% coverage of the 33-mer peptide. This partial coverage may be due to instrument limitations—small molecular weight peptide fragments may not be effectively ionized, resulting in incomplete splicing. The figure only shows the tandem mass spectra of peptide fragments with higher scores and better quality; although partial coverage exists, it ensures the reliability of cleavage site characterization. The product peptides of HC, C9-2, and C11-1 can be completely spliced ​​back to the original 33-mer peptide, achieving 100% sequence coverage. The cleavage site analysis of the four prolyl endopeptidases is shown in Table 3.

[0058] Table 3. Cleavage sites of 33-mer peptide by different enzymes

[0059] The results showed significant differences in the cleavage sites of the four enzymes, with a marked increase in complexity. HC exhibited the simplest cleavage site spectrum, C2-3 showed a slight expansion, C9-2 further increased to 7 cleavage sites, and C11-1 displayed the most comprehensive cleavage spectrum, containing 9 cleavage sites. With the increase in cleavage site diversity, the expansion path of the cleavage sites showed a clear pattern: starting with the core cleavage between proline (P) and a hydrophobic amino acid, it gradually expanded to the peptide bond interaction between glutamine (Q) and leucine (L), ultimately achieving cleavability of all peptide bonds at the amino and carboxyl terms of proline (P) within the 33-mer peptide in the C11-1 sample. This phenomenon intuitively reflects the shift in the enzyme's cleavage mechanism from a single carboxyl-terminal preference to a bidirectional cleavage mode involving both the carboxyl and amino terms, indicating a significant improvement in the enzymatic cleavage range and a greater reduction in the immunogenicity of the 33-mer peptide.

[0060] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Prolyl endopeptidase, characterized in that, The amino acid sequence is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The gene encoding the prolyl endopeptidase of claim 1.

3. The gene according to claim 2, characterized in that, The gene sequence encoding prolyl endopeptidase C9-2 as shown in SEQ ID NO.1 is shown in SEQ ID NO.5; the gene sequence encoding prolyl endopeptidase C11-1 as shown in SEQ ID NO.2 is shown in SEQ ID NO.

6.

4. A recombinant vector carrying the gene of claim 2 or 3.

5. The recombinant vector according to claim 4, characterized in that, The vectors include, but are not limited to, pET series plasmids.

6. A recombinant Escherichia coli, characterized in that, The recombinant Escherichia coli expressed the prolyl endopeptidase described in claim 1.

7. The recombinant Escherichia coli according to claim 6, characterized in that, pET-SUMO was used as the expression vector, and Escherichia coli BL21(DE3) was used as the host.

8. A method for preparing the prolyl endopeptidase according to claim 1, characterized in that, The recombinant Escherichia coli of claim 6 or 7 is cultured in a culture medium for a period of time, and the expression of the enzyme is induced by IPTG.

9. The use of the prolyl endopeptidase of claim 1 in the degradation of gluten 33-mer peptides.

10. The application according to claim 9, characterized in that, The applications include, but are not limited to, the preparation of anti-celiac disease foods, intestinal immune conditioning agents, or gluten-free food desensitization products.

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