Preparation method of acid-resistant high-activity dipeptidyl peptidase Ⅲ mutant capable of simultaneously degrading four aflatoxins and application of dipeptidyl peptidase Ⅲ mutant in corn syrup

Abstract

This invention discloses a method for preparing an acid-resistant, highly active dipeptidyl peptidase III mutant capable of simultaneously degrading four aflatoxins and its application in corn steep liquor, belonging to the fields of enzyme engineering technology and food safety. This invention yields a double mutant containing F375L. This mutant exhibits both high catalytic activity and strong acid resistance, effectively degrading the four major aflatoxin subtypes (B1, B2, G1, and G2) under weakly acidic conditions. K m The degradation value was reduced to 1 / 2 to 1 / 3 of that of the wild type, and the catalytic efficiency was increased by 2.2 to 4.8 times compared with the wild type. In a quaternary mixed pollution system, the degradation rate of the four toxins reached up to 88.12% in 24 hours, with no significant substrate competition inhibition. In the real matrix of corn steep liquor, the degradation rate of aflatoxin B1 in 24 hours reached 84.32%, and the final residue met the limit requirements of my country's national food safety standards. It can completely degrade low concentrations of aflatoxin B2, G1, and G2.

Description

Technical Field

[0001] This invention relates to a method for preparing an acid-resistant, highly active dipeptidyl peptidase III mutant capable of simultaneously degrading four aflatoxins and its application in corn steep liquor, belonging to the fields of enzyme engineering technology and food safety. Background Technology

[0002] Aflatoxins (AFs) are highly toxic secondary metabolites produced by fungi such as Aspergillus flavus and Aspergillus parasiticus. The four main subtypes, AFB1, AFB2, AFG1, and AFG2, often exist in food substrates and feed as part of a complex contamination process, seriously threatening food safety and human health. The International Agency for Research on Cancer (IARC) has classified aflatoxins as Group 1 carcinogens. Their contamination not only causes enormous agricultural and economic losses but also accumulates continuously in animals and humans through the food chain, leading to a series of health hazards, including liver toxicity, immunotoxicity, and genotoxicity.

[0003] Currently, methods for degrading aflatoxin are mainly classified into three categories: physical, chemical, and biological methods. Among them, enzymatic degradation has become an ideal approach for aflatoxin detoxification due to its high efficiency, specificity, environmental friendliness, and lack of food safety risks. Existing research has confirmed that dipeptidyl peptidase III (ADPP III) from Aspergillus terreus has specific degradation activity against aflatoxin, targeting and destroying the toxic structure of aflatoxin to convert it into low-toxicity / non-toxic products. Compared with degrading enzymes such as laccase and peroxidase, it has the advantages of strong catalytic specificity, no dependence on cofactors, and no non-specific oxidation of nutrients in food, thus possessing good application potential.

[0004] However, wild-type ADPP III suffers from two major application bottlenecks: First, its catalytic efficiency is insufficient. Its 24-hour degradation rates for AFB1 and AFB2 are only 21.57±1.92% and 20.32±2.98%, respectively, and for AFG1 and AFG2, only 42.62±4.53% and 40.8±2.14%, respectively, which is insufficient to meet practical detoxification requirements. Second, it has poor adaptability to acidic environments. The optimal pH for wild-type ADPP III is 7.0, and its activity significantly decreases under weakly acidic conditions of pH 5.0–6.0. ​​Since aflatoxin-contaminated food and feed substrates such as corn steep liquor and silage are mostly in weakly acidic environments, this enzyme cannot be adapted to industrial applications. Therefore, molecularly modifying ADPP III through enzyme engineering to simultaneously improve its catalytic efficiency and adaptability to acidic environments has significant theoretical and practical value. Summary of the Invention

[0005] Addressing the core shortcomings of wild-type ADPP III, such as low catalytic efficiency and significant activity reduction under weakly acidic conditions, this invention utilizes substrate channel engineering and surface charge remodeling strategies to obtain a dipeptidyl peptidase III mutant, F375L / K570Q, which possesses both high catalytic activity and strong acid resistance. The invention also provides its encoding gene, recombinant expression vector, recombinant genetically engineered bacteria, preparation method, and application of this mutant in aflatoxin degradation. The aim is to provide a novel, efficient, and stable enzyme preparation for the biodegradation of aflatoxin in food and feed.

[0006] To achieve the above-mentioned objectives, the core technical solution adopted by this invention is as follows: This invention provides a dipeptidyl peptidase III mutant, wherein the dipeptidyl peptidase III mutant is, The enzyme obtained by mutating phenylalanine at position 375 of dipeptidyl peptidase III, as shown in SEQ ID NO.1, to leucine, is named F375L. Alternatively, it can be obtained by mutating glycine at position 391 of dipeptidyl peptidase III, as shown in SEQ ID NO.1, to phenylalanine, and named: G391F; Alternatively, the amino acid sequence of dipeptidyl peptidase III, as shown in SEQ ID NO.1, is obtained by mutating phenylalanine at position 375 to leucine and lysine at position 491 to asparagine, and named as: F375L / K491N. Alternatively, the amino acid sequence of dipeptidyl peptidase III, as shown in SEQ ID NO.1, is obtained by mutating phenylalanine at position 375 to leucine and lysine at position 570 to glutamine, and named as: F375L / K570Q. Alternatively, the amino acid sequence of dipeptidyl peptidase III, as shown in SEQ ID NO.1, is obtained by mutating phenylalanine at position 375 to leucine and lysine at position 600 to asparagine, and named as: F375L / K600N. Alternatively, the amino acid sequence of dipeptidyl peptidase III, as shown in SEQ ID NO.1, is obtained by mutating phenylalanine at position 375 to leucine and lysine at position 643 to asparagine, and named as: F375L / K643N. Alternatively, the amino acid sequence of dipeptidyl peptidase III, as shown in SEQ ID NO.1, is obtained by mutating phenylalanine at position 375 to leucine and lysine at position 644 to glutamine, and named as: F375L / K644Q. Alternatively, the amino acid sequence of dipeptidyl peptidase III, as shown in SEQ ID NO.1, is obtained by mutating phenylalanine at position 375 to leucine and arginine at position 612 to glutamine, and named as: F375L / R612Q. Alternatively, the amino acid sequence of dipeptidyl peptidase III, as shown in SEQ ID NO.1, can be obtained by mutating phenylalanine at position 375 to leucine and arginine at position 620 to glutamine, and named F375L / R620Q.

[0007] The amino acid sequence (SEQ ID NO.1) of the dipeptidyl peptidase III parent enzyme is as follows: MDDNIKQHYLADSPPTVVRLEIKSHFDNLSDPKLRKYAHFMSRAAFEGTRITLRQVSPESEPIYDLIVSLYRACNGNWNELAQNTNVSDEHLRFFLEYAAQFLGNCGNYKGFGDSKFIPRLPVESLEALASVTPETKAAFQKANSTGGGIYETKEQSLMHLGYSEAGHMT TYYPDSPSITKDEITAIGDLLEKKGLPLENTRLKKTPSGDFELLIASGISSPPSRDRRDLGDVHTLELEGNLKGKKLRLVFGDHIEEMAKIAHSIKLAEINAANDTQKRMLDAYAKSFGTGSIEAFKESQRVWVKDQKPVLETNIGFVETYRDPHGVRGEWEGFVALVNLER TRAFGKLVDSAESMIPKLPWGQDFEKDKFLSPDFTSLEVLSFQSSGIPAGINLPNYDDIRQNLGFKNVSLGNVLGAKAPDEPVPFIAEKDLEVYRRCRDPAFEVQVGIHELLGHGTGKLLQETSSGQYNFDISNPPVSPVTKKPVNSWYKPGQTWSSVFGAIASSYEECRA ECVAMVLGCDFGILKIFGFGDGTVDLNNEAGDVLFAAYLQMARAGLVALEFWDPKTQKWGQAHMQARYSILRTFLDAGDNFVKLAYTKEDLSDLEIHLDRSKILTHGRPAVEKYLQKLHVYKSTADVEAGKKLYDDITSVDEWWGTNVRDIVLKNKIPRKAAALEHHHHHH The gene sequence (SEQ ID NO.2) of the dipeptidyl peptidase III parent enzyme is as follows: This invention also provides a highly catalytically active, acid-resistant dipeptidyl peptidase III mutant, F375L / K570Q. The mutant is based on dipeptidyl peptidase III (ADPP Ⅲ) derived from Aspergillus terreus with GenBank accession number PP861154, with double-point mutations F375L and K570Q occurring in its amino acid sequence. Specifically, F375L represents a mutation of phenylalanine at position 375 of the parental sequence to leucine, and K570Q represents a mutation of lysine at position 570 of the parental sequence to glutamine.

[0008] In one embodiment, the amino acid sequence of the mutant F375L / K570Q is shown in SEQ ID NO.3.

[0009] The amino acid sequence of F375L / K570Q is as follows (SEQ ID NO.3): MDDNIKQHYLADSPPTVVRLEIKSHFDNLSDPKLRKYAHFMSRAAFEGTRITLRQVSPESEPIYDLIVSLYRACNGNWNELAQNTNVSDEHLRFFLEYAAQFLGNCGNYKGFGDSKFIPRLPVESLEALASVTPETKAAFQKANSTGGGIYETKEQSLMHLGYSEAGHMT TYYPDSPSITKDEITAIGDLLEKKGLPLENTRLKKTPSGDFELLIASGISSPPSRDRRDLGDVHTLELEGNLKGKKLRLVFGDHIEEMAKIAHSIKLAEINAANDTQKRMLDAYAKSFGTGSIEAFKESQRVWVKDQKPVLETNIGFVETYRDPHGVRGEWEGFVALVNLER TRAFGKLVDSAESMIPKLPWGQDFEKDKFLSPDLTSLEVLSFQSSGIPAGINLPNYDDIRQNLGFKNVSLGNVLGAKAPDEPVPFIAEKDLEVYRRCRDPAFEVQVGIHELLGHGTGKLLQETSSGQYNFDISNPPVSPVTKKPVNSWYKPGQTWSSVFGAIASSYEECRA ECVAMVLGCDFGILKIFGFGDGTVDLNNEAGDVLFAAYLQMARAGLVALEFWDPKTQQWGQAHMQARYSILRTFLDAGDNFVKLAYTKEDLSDLEIHLDRSKILTHGRPAVEKYLQKLHVYKSTADVEAGKKLYDDITSVDEWWGTNVRDIVLKNKIPRKAAALEHHHHHH The present invention also provides a gene encoding the mutant F375L / K570Q.

[0010] In one embodiment, the nucleotide sequence of the gene is shown in SEQ ID NO.4.

[0011] The gene sequence of F375L / K570Q is as follows (SEQ ID NO.4): The present invention also provides a gene encoding the above-mentioned dipeptidyl peptidase III mutant or a recombinant vector carrying the gene.

[0012] The present invention also provides recombinant cells expressing the dipeptidyl peptidase III mutant or carrying the gene or the recombinant vector.

[0013] In one embodiment, the recombinant cells are bacteria or fungi as host cells.

[0014] The present invention also provides a recombinant enzyme catalyst containing the above-mentioned dipeptidyl peptidase III mutant, which is any one of the following forms: (1) Culture recombinant expression transformants containing dipeptidyl peptidase III mutant and isolate transformant cells containing recombinant dipeptidyl peptidase III mutant enzyme; (2) Culture recombinant expression transformants containing dipeptidyl peptidase III mutant, isolate transformant cells containing recombinant dipeptidyl peptidase III mutant enzyme, and break the transformant cells containing the recombinant dipeptidyl peptidase III mutant enzyme to obtain cell lysate. (3) Cultivate recombinant expression transformants containing dipeptidyl peptidase III mutants, isolate transformant cells containing recombinant dipeptidyl peptidase III mutant enzymes, break the transformant cells containing the recombinant dipeptidyl peptidase III mutant enzymes, obtain cell lysate, and freeze-dry the cell lysate of the recombinant dipeptidyl peptidase III mutant enzymes to obtain lyophilized enzyme powder.

[0015] The present invention also provides a method for improving the degradation ability of dipeptidyl peptidase III against aflatoxins AFB1, AFB2, AFG1, and AFG2, wherein the method comprises mutating phenylalanine at position 375 of dipeptidyl peptidase III, as shown in SEQ ID NO.1, to leucine; or mutating glycine at position 391 of dipeptidyl peptidase III, as shown in SEQ ID NO.1, to phenylalanine.

[0016] This invention also provides a method for improving the degradation ability of dipeptidyl peptidase III against aflatoxins AFB1, AFB2, AFG1, and AFG2 under acidic conditions. The method involves mutating phenylalanine at position 375 of dipeptidyl peptidase III (as shown in SEQ ID NO.1) to leucine, and simultaneously mutating lysine at position 491 to asparagine; or mutating phenylalanine at position 375 of dipeptidyl peptidase III (as shown in SEQ ID NO.1) to leucine, and simultaneously mutating lysine at position 570 to glutamine; or mutating phenylalanine at position 375 of dipeptidyl peptidase III (as shown in SEQ ID NO.1) to leucine, and simultaneously mutating lysine at position 600 to asparagine; or mutating phenylalanine at position 375 of dipeptidyl peptidase III (as shown in SEQ ID NO.1) to leucine, and simultaneously mutating lysine at position 643 ...; or mutating phenylalanine at position 375 of dipeptidyl peptidase III (as shown in SEQ ID NO.1) to le The amino acid sequence of dipeptidyl peptidase III shown in SEQ ID NO.1 is modified by mutating phenylalanine at position 375 to leucine and lysine at position 644 to glutamine; or the amino acid sequence of dipeptidyl peptidase III shown in SEQ ID NO.1 is modified by mutating phenylalanine at position 375 to leucine and arginine at position 612 to glutamine; or the amino acid sequence of dipeptidyl peptidase III shown in SEQ ID NO.1 is modified by mutating phenylalanine at position 375 to leucine and arginine at position 620 to glutamine.

[0017] The present invention also provides an engineered Escherichia coli strain that expresses the above-mentioned dipeptidyl peptidase III mutant.

[0018] In one embodiment, the engineered Escherichia coli is constructed by cloning the gene encoding the dipeptidyl peptidase III mutant into an expression vector and transforming it into Escherichia coli host cells.

[0019] In one embodiment, the expression vector is selected from the pET series vectors or the pGEX series vectors.

[0020] In one embodiment, the Escherichia coli host is selected from... Escherichia coli BL21 (DE3) Escherichia coli BL21 Escherichia coli Rosetta (DE3) Escherichia coli C41 (DE3) Escherichia coli C43 (DE3) Escherichia coli JM109 Escherichia coli DH5α or its derivative strains.

[0021] The present invention also provides a method for degrading aflatoxins AFB1, AFB2, AFG1, and AFG2, the method comprising the following steps: adding the above-mentioned mutant or the above-mentioned recombinant enzyme catalyst or the recombinant enzyme prepared using the above-mentioned engineered Escherichia coli to a reaction system containing aflatoxins AFB1, AFB2, AFG1, and AFG2 for degradation.

[0022] In one embodiment, the degradation conditions are: pH 5.0~7.0 and temperature 25 ℃~45 ℃.

[0023] In one embodiment, the reaction system includes, but is not limited to, corn steep liquor.

[0024] The present invention also provides the application of the above-mentioned dipeptidyl peptidase III mutant, the above-mentioned recombinant cell, or the above-mentioned recombinant enzyme catalyst in the preparation of products that degrade aflatoxins AFB1, AFB2, AFG1, and AFG2.

[0025] The present invention also provides an Escherichia coli that produces the dipeptidyl peptidase III mutant F375L / K570Q.

[0026] In one embodiment, the recombinant genetically engineered bacteria uses pET-28a(+) as the expression vector, Escherichia coli BL21(DE3) as the expression strain, and expresses the mutant F375L / K570Q.

[0027] The present invention also provides a method for preparing the dipeptidyl peptidase III mutant F375L / K570Q, comprising the following steps: Using PCR-mediated site-directed mutagenesis, the F375L and K570Q mutation sites were introduced, respectively. The nucleotide sequence encoding the double mutant was inserted between the Sac I and Not I restriction sites of the pET-28a(+) vector to construct the recombinant plasmid pET-28a(+)-ADPPⅢ-F375L / K570Q. Sequencing confirmed that the sequence was correct.

[0028] Furthermore, the recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells to obtain recombinant genetically engineered bacteria.

[0029] Furthermore, the recombinant genetically engineered bacteria were induced to express the enzyme, and the bacterial cells were collected and broken to obtain crude enzyme solution.

[0030] Furthermore, the crude enzyme solution was purified by nickel column affinity chromatography, and after dialysis to remove imidazole, electrophoretically pure dipeptidyl peptidase III mutant F375L / K570Q was obtained.

[0031] In one embodiment, the induction is achieved when the bacterial culture reaches the OD value of the culture medium. 600When the concentration is 0.6~0.8, add the inducing agent IPTG to make the final concentration 0.2 mM, and induce at 20 ℃ and 220 rpm for 20 h.

[0032] The present invention also provides the application of the dipeptidyl peptidase mutant F375L / K570Q in aflatoxin degradation.

[0033] In one embodiment, the reaction pH is 5.0 to 9.0.

[0034] In one implementation, the reaction temperature is 25 ℃ to 45 ℃.

[0035] In one implementation, the reaction time is 24 hours.

[0036] In one implementation, the reaction vessel is a 24-well deep-well plate, and the oscillation speed is 600 rpm.

[0037] The present invention also provides the application of the dipeptidyl peptidase mutant F375L / K570Q in the degradation of aflatoxin in 10% (w / w) dilute corn steep liquor.

[0038] In one implementation method, the application specifically includes: obtaining corn pulp with an initial solids content of 40% (w / w) from a corn deep processing company; diluting the pulp with sterile deionized water at a volume ratio of 1:3 to obtain a corn pulp working solution with a solids content of 10% (w / w). The diluted corn pulp is centrifuged at 12,000 rpm for 10 min, the supernatant is collected, and the pH is adjusted to 5.0. Subsequently, it is autoclaved at 115 °C for 20 min and cooled to room temperature to serve as the sample to be degraded. Several portions of the prepared corn pulp supernatant, each with a volume of 6 mL, are taken, and purified double mutant F375L / K570Q enzyme solution with a final concentration of 20 µg / mL is added. An equal volume of citrate buffer is added to the blank control group. Samples are taken at fixed points, 1 mL each time, until 24 h.

[0039] In one embodiment, the double mutant F375L / K570Q enzyme solution is an enzyme solution purified by nickel column affinity chromatography.

[0040] In one implementation, the reaction temperature is 40 °C.

[0041] In one implementation, the reaction pH is 5.0.

[0042] In one implementation, the reaction vessel is a 24-well deep-well plate, and the oscillation speed is 600 rpm.

[0043] In one implementation, the reaction time is 0, 2, 4, 8, 12, or 24 hours.

[0044] Beneficial effects The present invention has the following advantages: 1. This invention utilizes substrate channel engineering and surface charge remodeling to obtain the double mutant F375L / K570Q, which exhibits approximately twice the catalytic efficiency against four aflatoxins compared to the wild type and successfully extends the enzyme's high-activity pH to a weakly acidic level of 5.0. The degradation rates of the four aflatoxins after 24 h reached 80.54±2.51%, 83.77±1.36%, 83.84±1.53%, and 76.58±2.22%, respectively, significantly superior to the wild-type enzyme.

[0045] 2. The catalytic efficiency constants of the double mutant provided by this invention against four aflatoxins under weakly acidic conditions (pH 5.0) using F375L / K570Q. k cat / K m The efficiency was 2.2 to 4.8 times higher than that of the wild type, with a Michaelis constant. K m By reducing the efficiency to 1 / 2 to 1 / 3 of that of the wild type, substrate affinity and catalytic turnover efficiency are simultaneously and significantly improved, breaking through the bottleneck of insufficient catalytic efficiency of wild-type enzymes.

[0046] The double mutant of this invention showed no significant substrate competition inhibition in a quaternary mixed contamination system of AFB1, AFB2, AFG1, and AFG2. The degradation rates of the four toxins in 24 h were 73.60±4.81%, 71.83±5.09%, 88.12±3.39%, and 85.06±3.25%, respectively. In a real corn steep liquor matrix, the degradation rate of AFB1 in 24 h was 84.32±2.23%, and the final residue met the limit requirements of my country's National Food Safety Standard for Limits of Mycotoxins in Food (GB 2761-2017). It can completely degrade low concentrations of AFG1, AFB2, and AFG2, showing excellent prospects for industrial application. Attached Figure Description

[0047] Figure 1 Agarose gel electrophoresis images of site-directed mutagenesis PCR amplification products; M: Marker; 1-4: ADPP Ⅲ-F375L amplification products; 5-8: ADPP Ⅲ-F375L / K570Q amplification products.

[0048] Figure 2Electrophoresis results of purified double mutant recombinase proteins; M: Marker; 1-7: F375L / K491N, F375L / K570Q, F375L / K600N, F375L / K643N, F375L / K644Q, F375L / R612Q, F375L / R620Q mutants.

[0049] Figure 3 The diagram shows the optimal temperature and pH of wild-type ADPP Ⅲ; (AD) represents the optimal temperature and pH of wild-type ADPP Ⅲ, where (A) is AFB1; (B) is AFB2; (C) is AFG1; and (D) is AFG2.

[0050] Figure 4 Steady-state kinetic analysis of wild-type ADPP III and double mutants against four aflatoxins; (AD) wild-type ADPP III, of which (A) AFB1; (B) AFB2; (C) AFG1; (D) AFG2; (EH) double mutant F375L / K570Q, of which (E) AFB1; (F) AFB2; (G) AFG1; (H) AFG2.

[0051] Figure 5 The degradation performance of wild-type ADPP III and F375L / K570Q double mutant under mixed aflatoxin contamination conditions was evaluated.

[0052] Figure 6 The degradation performance of F375L / K570Q mutant in corn steep liquor; (A) corn steep liquor with an initial solids content of 40% and pretreated diluted corn steep liquor; (B) the detection content of four aflatoxins in pretreated diluted corn steep liquor; (C) the change of the content of four aflatoxins with degradation time. Detailed Implementation

[0053] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0054] The aflatoxins AFB1, AFB2, AFG1, and AFG2 involved in the following examples were purchased from Qingdao Purybang Biotechnology Co., Ltd.

[0055] The culture media involved in the following examples are as follows: LB liquid medium: Each 1 L of medium contains 10.0 g peptone, 10.0 g sodium chloride, and 5.0 g yeast extract. It is autoclaved at 121 °C for 20 min before use.

[0056] TB liquid culture medium: Each 1 L of medium contains 12.0 g peptone, 24.0 g yeast extract, 4 mL glycerol, 2.31 g dipotassium hydrogen phosphate, and 12.54 g potassium dihydrogen phosphate. It is autoclaved at 121 °C for 20 min before use.

[0057] The detection methods involved in the following embodiments are as follows: Aflatoxin Degradation Reaction System and Detection Method The total reaction volume was 2 mL, consisting of: 50 mM buffer, purified enzyme solution with a final concentration of 20 µg / mL, 0.5 mM CuCl2, and single aflatoxins (AFB1 / AFB2 / AFG1 / AFG2) with a final concentration of 2.5 µg / mL. After thoroughly mixing the reaction system, it was placed in a 24-well plate and incubated with shaking at 600 rpm. Immediately after incubation, an equal volume of acetonitrile was added, and the reaction was terminated by vortexing. The plate was centrifuged at 12,000 rpm for 10 min, and the supernatant was filtered through a 0.22 µm organic phase filter membrane. The aflatoxin residue was then determined by UPLC-MS / MS.

[0058] UPLC-MS / MS detection conditions: The chromatographic column was a Waters BEHC18 column (2.1 mm × 100 mm, 1.7 µm), column temperature 40 ℃, injection volume 5 µL, mobile phase flow rate 0.3 mL / min; mobile phase A was acetonitrile, mobile phase B was 10 mM ammonium formate aqueous solution, gradient elution program was: 0.0–0.1 min, 10% A + 90% B; 0.1–6.0 min, 10% A linearly increased to 60% A; 6.1–7.1 min, 10% A + 90% B. Mass spectrometry conditions were: electrospray ionization positive ion mode, multiple reaction monitoring mode, nebulizer pressure 0.21 MPa, drying gas flow rate 8 L / min, gas temperature 550 ℃, capillary voltage 5500 V.

[0059] Calculation of aflatoxin degradation rate:

[0060] In the formula: C 0 represents the initial concentration of aflatoxin in the blank control group. C t This represents the residual concentration of aflatoxin in the reaction group.

[0061] Example 1: Site-directed mutagenesis of dipeptidyl peptidase III mutant and construction of recombinant strains.

[0062] 1. Site-directed mutagenesis primer design Using the wild-type ADPP Ⅲ gene (GenBank accession number: PP861154, its amino acid sequence is shown in SEQ ID NO.1) as a template, site-directed mutagenesis primers were designed targeting the F375L and G391F mutant sites. The primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and the primer sequences are as follows: F375L-F: 5'-gataaatttctgagcccggatctgacgagcctggaagtgctgagctttcagagc-3'; F375L-R: 5'-cagatccgggctcagaaatttatctttttcaaaatcttggccccacggcagttt-3'; G391F-F: 5'-gcagcggcattccggcgtttattaacctgccgaactatgatgacattcgt-3'; G391F-R: 5'-aaacgccggaatgccgctgctctgaaagctcagcacttccaggctcgtaa-3'.

[0063] The amino acid sequence of wild-type ADPP III is as follows (SEQ ID NO.1): MDDNIKQHYLADSPPTVVRLEIKSHFDNLSDPKLRKYAHFMSRAAFEGTRITLRQVSPESEPIYDLIVSLYRACNGNWNELAQNTNVSDEHLRFFLEYAAQFLGNCGNYKGFGDSKFIPRLPVESLEALASVTPETKAAFQKANSTGGGIYETKEQSLMHLGYSEAGHMT TYYPDSPSITKDEITAIGDLLEKKGLPLENTRLKKTPSGDFELLIASGISSPPSRDRRDLGDVHTLELEGNLKGKKLRLVFGDHIEEMAKIAHSIKLAEINAANDTQKRMLDAYAKSFGTGSIEAFKESQRVWVKDQKPVLETNIGFVETYRDPHGVRGEWEGFVALVNLER TRAFGKLVDSAESMIPKLPWGQDFEKDKFLSPDFTSLEVLSFQSSGIPAGINLPNYDDIRQNLGFKNVSLGNVLGAKAPDEPVPFIAEKDLEVYRRCRDPAFEVQVGIHELLGHGTGKLLQETSSGQYNFDISNPPVSPVTKKPVNSWYKPGQTWSSVFGAIASSYEECRA ECVAMVLGCDFGILKIFGFGDGTVDLNNEAGDVLFAAYLQMARAGLVALEFWDPKTQKWGQAHMQARYSILRTFLDAGDNFVKLAYTKEDLSDLEIHLDRSKILTHGRPAVEKYLQKLHVYKSTADVEAGKKLYDDITSVDEWWGTNVRDIVLKNKIPRKAAALEHHHHHH 2. Site-directed mutagenesis PCR amplification (1) Construction of recombinant plasmid pET-28a(+)-ADPP Ⅲ The wild-type ADPP Ⅲ gene (GenBank accession number: PP861154, SEQ ID NO.2) was chemically synthesized, and the wild-type ADPP Ⅲ gene was ligated to the Sac I and Not I sites of pET-28a(+) to prepare the recombinant plasmid pET-28a(+)-ADPP Ⅲ; (2) Using recombinant plasmid pET-28a(+)-ADPP Ⅲ as a template, site-directed mutagenesis amplification was performed using high-fidelity PCR. The PCR amplification system was as follows: plasmid template (10 ng / µL) 0.4 µL, forward primer (10 µM) 0.6 µL, reverse primer (10 µM) 0.6 µL, 2×Phanta Flash high-fidelity PCR premix 10.0 µL, ddH2O 8.4 µL, total system 20 µL. The PCR amplification program was: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, T m Anneal at +5℃ for 5 seconds, extend at 72℃ for 1 minute, for a total of 30 cycles; extend thoroughly at 72℃ for 1 minute; hold at 4℃.

[0064] 3. Purification and transformation of PCR products After verifying the correct band size by 1% agarose gel electrophoresis, the PCR products were purified using a PCR product purification kit. The purified products were transformed into E. coli DH5α competent cells, plated on LB solid medium containing 50 mg / L kanamycin, and incubated overnight at 37°C inverted. Single colonies were picked for liquid culture, and plasmids were extracted and sent to Suzhou Genewiz Biotechnology Co., Ltd. for DNA sequencing verification. Single mutant plasmids pET-28a(+)-F375L and pET-28a(+)-G391F with correct sequences were obtained.

[0065] 4. Construction of recombinant expression strains The recombinant plasmid containing the mutant, which was verified by sequencing, was chemically transformed into *E. coli* BL21(DE3) competent cells. The cells were plated on LB agar containing 50 mg / L kanamycin and incubated overnight at 37°C inverted. Single colonies were picked and sequenced to confirm the positive recombinant expression strain. E . coli BL21(DE3) / pET-28a(+)-F375L E . coli BL21(DE3) / pET-28a(+)-G391F, store at -80℃ for later use.

[0066] Recombinant expression strains of wild-type ADPP Ⅲ were constructed using the same method. E . coli BL21(DE3) / pET-28a(+)-ADPP Ⅲ was used as a control for subsequent experiments.

[0067] The results show: Electrophoresis images of mutants F375L and K570Q are shown below. Figure 1As shown, the PCR products of the mutants all showed a single clear band at approximately 2.3 kb, consistent with the expected product size, without any extraneous bands or tailing, indicating that the site-directed mutagenesis PCR amplification was successful.

[0068] Example 2: Induction, expression and purification of dipeptidyl peptidase III and its mutants.

[0069] 1. Induced expression The recombinant strain constructed in Example 1 ( E . coli BL21(DE3) / pET-28a(+)-F375L E . coli BL21(DE3) / pET-28a(+)-G391F were inoculated into 5 mL of LB liquid medium containing 50 mg / L kanamycin and cultured at 37°C with shaking at 220 rpm for 12 h to obtain seed culture. The seed culture was then transferred at a 1% (v / v) inoculation rate to 50 mL of TB liquid medium containing 50 mg / L kanamycin and cultured at 37°C with shaking at 220 rpm until the bacterial growth rate reached OD. 600 When the pH reaches 0.6-0.8, add IPTG to the culture medium to a final concentration of 0.2 mM and incubate at 20℃ and 220 rpm for 20 h. After induction, centrifuge the bacterial solution at 4℃ and 8000 rpm for 15 min, discard the supernatant and collect the bacterial cell pellet. Resuspend the bacterial cells twice with pre-chilled PBS buffer (pH 7.4), centrifuge again, and resuspend the bacterial cells with pre-chilled binding buffer. Place the pellet in an ice-water bath and use an ultrasonic cell disruptor to disrupt the cells under the following conditions: power 260 W, sonication for 2 s, interval 3 s, ice-water bath throughout, until the bacterial solution is clear and non-viscous. After disruption, centrifuge at 4℃ and 12000 rpm for 15 min and collect the supernatant as the crude enzyme solution.

[0070] 2. Protein purification and purity detection Prepare binding buffer (pH 8.0, 8.71 g / L K2HPO4, 17.53 g / L NaCl, 0.34 g / L imidazole, 10% (v / v) glycerol), wash buffer (pH 8.0, 8.71 g / L K2HPO4, 17.53 g / L NaCl, 3.40 g / L imidazole, 10% (v / v) glycerol), and elution buffer (pH 8.0, 8.71 g / L K2HPO4, 17.53 g / L NaCl, 17.02 g / L imidazole, 10% (v / v) glycerol) in advance). Filter all buffers through a 0.22 µm filter and degas. Connect the His-tag nickel column to the protein purification system and wash the nickel column with 5 column volumes of binding buffer at a flow rate of 5 mL / min until the UV absorbance A280 and conductivity stabilize. The crude enzyme solution was filtered through a 0.22 µm filter membrane and loaded onto a nickel column at a flow rate of 2 mL / min to ensure sufficient binding of the target protein to the nickel column packing material. After loading, the nickel column was washed with 5 column volumes of wash buffer at a flow rate of 5 mL / min to remove non-specifically bound contaminating proteins until the A280 value dropped to the baseline level. The column was then switched to elution buffer and linearly eluted at a flow rate of 5 mL / min, with real-time monitoring of the A280 absorption peak. The eluent showing a clear elution peak was collected as the target protein enrichment fraction. The collected target protein eluent was placed in a pre-treated dialysis bag, sealed, and placed in a container containing 50 mM Tris-HCl buffer (pH 7.0). Dialysis was performed at 4°C for 24 h, with the dialysis buffer changed 3 times to completely remove imidazole from the eluent. After dialysis, the purified protein was collected, aliquoted into sterile centrifuge tubes, flash-frozen in liquid nitrogen, and stored at -80°C for later use. The purity of the purified protein was determined by SDS-PAGE gel electrophoresis.

[0071] F375L pure enzyme solution and K570Q pure enzyme solution were prepared. At the same time, wild-type ADPPⅢ pure enzyme solution was prepared according to the above method.

[0072] Example 3: Construction of a double mutant The specific steps are as follows: Using the pET-28a(+)-F375L single-point mutant vector as a template, site-directed mutagenesis primers were designed for the K491N, K570Q, K600N, K643N, K644Q, R612Q, and R620Q mutant sites to construct double mutants. The primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0073] The primers involved are as follows: K491N-F:5'-cggtgaacagctggtataacccgggtcagacctggagcagcgtgtttggc-3'; K491N-R:5'-gttataccagctgttcaccggttttttggtcaccgggctcaccggcgggt-3'; K570Q-F:5'-gggatccgaaaacgcagcagtggggccaagcgcatatgcaagcgcgctat-3'; K570Q-R:5'-ctgctgcgttttcggatcccaaaattccagcgccaccaggcccgcgcgcg-3'; K600N-F:5'-gtgaaactggcgtataccaacgaagatctgagcgatctggaaattcatctg-3'; K600N-R:5'-gttggtatacgccagtttcacaaagttatcgcccgcatccagaaaggtgcg-3'; K643N-F:5'-cggatgtggaagcgggcaacaaactgtatgatgatattacgagcgtggat-3'; K643N-R:5'-gttgcccgcttccacatccgcggtgcttttatacacatgcagtttctgca-3'; K644Q-F:5'-gatgtggaagcgggcaaacagctgtatgatgatattacgagcgtggatgaa-3'; K644Q-R:5'-ctgtttgcccgcttccacatccgcggtgcttttatacacatgcagtttctg-3'; R612Q-F:5'-ctggaaattcatctggatcagagcaaaattctgacccatggccgcccggcg-3'; R612Q-R:5'-ctgatccagatgaatttccagatcgctcagatcttctttggtatacgccag-3'; R620Q-F: 5'-ctggaaattcatctggatcagagcaaaattctgacccatggccgcccggcg-3'; R620Q-R: 5'-ctgatccagatgaatttccagatcgctcagatcttctttggtatacgccag-3'.

[0074] The methods for PCR amplification, PCR product purification and transformation, and construction of recombinant expression strains were the same as in Example 1. The induction and purification of mutants were the same as in Example 2. F375L / K491N pure enzyme solution, F375L / K570Q pure enzyme solution, F375L / K600N pure enzyme solution, F375L / K643N pure enzyme solution, F375L / K644Q pure enzyme solution, F375L / R612Q pure enzyme solution, and F375L / R620Q pure enzyme solution were prepared respectively.

[0075] The results show: like Figure 2 As shown, the purified F375L / K491N, F375L / K570Q, F375L / K600N, F375L / K643N, F375L / K644Q, F375L / R612Q, and F375L / R620Q mutant proteins exhibit a single band at approximately 79 kDa, consistent with the theoretical molecular weight, with no obvious contaminating protein bands, and a purity exceeding 90%. Protein concentration was determined using the Bradford Protein Quantitative Kit, with bovine serum albumin (BSA) as the standard. The purified mutant protein concentration reached above 5.0 mg / mL, which met the requirements of subsequent experiments.

[0076] The purified protein concentrations of F375L / K491N, F375L / K570Q, F375L / K600N, F375L / K643N, F375L / K644Q, F375L / R612Q, and F375L / R620Q were 5.98 mg / mL, 6.35 mg / mL, 5.67 mg / mL, 6.12 mg / mL, 5.59 mg / mL, 5.41 mg / mL, and 5.23 mg / mL, respectively.

[0077] Example 4: Effects of temperature and pH on the activity of wild-type ADPP III in degrading four aflatoxins The specific steps of the reaction are as follows: (1) The reaction system is fixed as follows: The total reaction volume was 2 mL, and the system consisted of: 50 mM buffer, wild-type ADPP Ⅲ purified enzyme solution with a final concentration of 20 µg / mL, 0.5 mM CuCl2, and a single aflatoxin (AFB1 / AFB2 / AFG1 / AFG2) with a final concentration of 2.5 µg / mL. The buffer system was 50 mM Tris-HCl (pH 7.0). Five reaction temperature gradients were set at 25 ℃, 30 ℃, 35 ℃, 40 ℃, and 45 ℃. The degradation reaction was carried out according to the above method. After incubation at 600 rpm for 24 h, samples were taken to detect the aflatoxin residue. The degradation rate of the four aflatoxins by ADPP Ⅲ at different temperatures was calculated to determine the optimal reaction temperature.

[0078] (2) The fixed reaction system is: The total reaction volume was 2 mL, and the system consisted of: 50 mM buffer, wild-type ADPP Ⅲ purified enzyme solution with a final concentration of 20 µg / mL, 0.5 mM CuCl2, and a single aflatoxin (AFB1 / AFB2 / AFG1 / AFG2) with a final concentration of 2.5 µg / mL. The reaction temperature was 40 °C, and buffer systems with different pH values ​​were set up: 50 mM citrate buffer (pH 5.0, 6.0) and 50 mM Tris-HCl buffer (pH 7.0, 8.0, 9.0), covering the pH range of 5.0 to 9.0. The degradation reaction was carried out according to the above method, and samples were taken after incubation at 600 rpm for 24 h to detect the aflatoxin residue. The degradation rate of ADPP Ⅲ on the four aflatoxins under different pH conditions was calculated to determine the optimal reaction pH.

[0079] The results show: like Figure 3 As shown, within the temperature range of 25 ℃ to 40 ℃, the degradation rate of ADPP Ⅲ for the four aflatoxins gradually increased with increasing temperature, reaching the highest at 40 ℃. When the temperature rose to 45 ℃, the degradation rate decreased significantly, indicating that 40 ℃ is the optimal temperature for ADPP Ⅲ to degrade the four aflatoxins.

[0080] The optimal reaction pH for ADPP Ⅲ is 7.0. It can maintain good enzyme activity stability under weakly alkaline conditions, but its degradation activity is significantly reduced under weakly acidic conditions of pH 5.0~6.0, which clarifies the application defects of this enzyme due to insufficient acid resistance.

[0081] Example 5: Evaluation of the degradation effect of a single mutant on a single aflatoxin Using AFB1, AFB2, AFG1, and AFG2 as single substrates, the degradation rates of four aflatoxins by F375L and G391F single mutants were determined in the degradation reaction system of Example 4 at pH 7.0, 50 mM Tris-HCl buffer, and 40°C.

[0082] The specific steps are as follows: The fixed reaction system was as follows: the total reaction volume was 2 mL, and the system composition was: 50 mM Tris-HCl buffer (pH 7.0), purified enzyme solution with a final concentration of 20 µg / mL, 0.5 mM CuCl2, and a single aflatoxin (AFB1 / AFB2 / AFG1 / AFG2) with a final concentration of 2.5 µg / mL. The reaction temperature was 40 ℃, and the degradation reaction was carried out according to the above method. After incubation at 600 rpm for 24 h, samples were taken to detect the residual amount of aflatoxin and calculate the degradation rate of the single mutant for the four aflatoxins. The results are shown in Table 1: Table 1: Degradation effect of different ADPP III enzymes at pH 7.0

[0083] Example 6: Evaluation of the degradation effect of the double mutant on aflatoxin A Using AFB1, AFB2, AFG1, and AFG2 as single substrates, and in the degradation reaction system of Example 4 at 50 mM buffer and 40 °C, the 24-h degradation rates of four aflatoxins by double mutants F375L / K491N, F375L / K570Q, F375L / K600N, F375L / K643N, F375L / K644Q, F375L / R612Q, and F375L / R620Q were determined.

[0084] The specific steps are as follows: The fixed reaction system was as follows: the total reaction volume was 2 mL, and the system composition was: 50 mM buffer, purified enzyme solution with a final concentration of 20 µg / mL, 0.5 mM CuCl2, and a single aflatoxin (AFB1 / AFB2 / AFG1 / AFG2) with a final concentration of 2.5 µg / mL. The reaction temperature was 40 ℃, and different pH buffer systems were set up: 50 mM Tris-HCl buffer at pH 7.0, 50 mM citrate buffer at pH 6.0, or 50 mM citrate buffer at pH 5.0. The degradation reaction was carried out according to the above method, incubated at 600 rpm for 24 h, and samples were taken to detect the aflatoxin residue. The degradation rate of the double mutant for the four aflatoxins under different pH conditions was calculated. The results are shown in Tables 2-4. Table 2: Degradation effect of different ADPP III enzymes at pH 7.0

[0085] Table 3: Degradation effect of different ADPP III enzymes under pH 6.0 conditions

[0086] Table 4: Degradation effect of different ADPP III enzymes under pH 5.0 conditions

[0087] The results show: Previous experiments have determined the optimal reaction temperature and pH for wild-type dipeptidyl peptidase III (ADPP III). The standard reaction system is a 50 mM Tris-HCl buffer at pH 7.0 and a reaction temperature of 40 °C. Figure 4 As shown, under these conditions, after 24 hours of reaction, the degradation rates of wild-type ADPP Ⅲ on AFB1, AFB2, AFG1 and AFG2 were 21.57±1.92%, 20.32±2.98%, 42.62±4.53% and 40.80±2.14%, respectively.

[0088] Based on a substrate channel engineering strategy, a rational design was conducted. The principles were to reduce steric hindrance of the substrate channel, optimize hydrophobic interactions, and maintain the stability of the catalytic core structure. Fourteen single-point mutants were constructed targeting key hydrophobic residues within the substrate channel, aiming to improve the enzyme's catalytic efficiency by optimizing the substrate channel structure. Since this round of mutations only targeted amino acid residues within the substrate channel and did not involve modifications to the enzyme protein surface charge, the degradation activity evaluation of all mutants followed the standard reaction system of wild-type ADPP III.

[0089] Activity screening verified that the F375L and G391F mutants significantly improved the catalytic performance against four aflatoxins: after 24 h of reaction, the degradation rates of AFB1, AFB2, AFG1, and AFG2 by the F375L mutant increased to 41.25±3.07%, 39.13±1.75%, 85.60±3.58%, and 74.04±2.25%, respectively, while the degradation rates of AFB1, AFB2, AFG1, and AFG2 by the G391F mutant increased to 39.69±1.54%, 32.32±1.65%, 86.26±1.73%, and 79.34±0.78%, respectively, with the degradation efficiency increasing by approximately 2 times.

[0090] To further overcome the application bottleneck caused by the significant loss of enzyme activity under acidic conditions, based on the high activity of the F375L single mutant, and with the core objective of improving catalytic stability under acidic conditions, 50 single-point mutants were constructed using a rational design strategy of neutralizing surface charge. Subsequently, the degradation activities of each mutant against four aflatoxins were determined in 50 mM citrate buffer (weakly acidic conditions) at pH 5.0 and 6.0, and in a standard reaction system at pH 7.0. The screening results showed that the seven mutants K491N, K570Q, K600N, K643N, K644Q, R612Q, and R620Q maintained strong activity even under pH 5.0 conditions. Among them, the F375L / K570Q double mutant showed the best overall acid resistance. After reacting for 24 h under acidic conditions at pH 5.0, its degradation rates of AFB1, AFB2, AFG1, and AFG2 reached 80.54±2.51%, 83.77±1.36%, 83.84±1.53%, and 76.58±2.22%, respectively, achieving a balance between high catalytic activity and structural stability under acidic conditions.

[0091] Example 7: Enzyme kinetics determination of wild-type ADPP Ⅲ and double mutant F375L / K570Q The specific steps are as follows: Using four aflatoxins (AFB1, AFB2, AFG1, and AFG2) as substrates, and setting substrate concentrations of 0 µM, 1 µM, 2 µM, 5 µM, 10 µM, 20 µM, 50 µM, 100 µM, and 200 µM, the initial rate of the enzymatic reaction between wild-type ADPP Ⅲ and the F375L / K570Q double mutant was measured at 40℃ in 50 mM citrate buffer at pH 5.0. A nonlinear regression fitting was performed using Graphpad Prism software with substrate concentration as the x-axis and reaction rate as the y-axis. The Michaelis-Menten constant was calculated by substituting the substrate concentration into the equation. K m Maximum reaction rate V max The catalytic constant was calculated based on the enzyme concentration. k cat and catalytic efficiency k cat / K m All experiments were performed in triplicate. The results are shown in Table 5 below: Table 5: Enzyme kinetics determination of different ADPP III enzymes

[0092] The results are as follows: To quantitatively evaluate the catalytic performance of the enzyme, this study determined the steady-state kinetic parameters of wild-type ADPP III and the engineered mutant F375L / K570Q on four substrates: AFB1, AFB2, AFG1, and AFG2. Figure 4 As shown, the Michaelis constants for wild-type enzymes are related to AFB1, AFB2, AFG1, and AFG2. K m The values ​​were 57.73, 67.26, 52.92, and 57.81 µmol / L, respectively. Among the four substrates, AFG1 corresponds to... K m Lowest value (52.92 µmol / L), highest reaction rate V max The highest value (5.18 µmol / (L·h)) indicates that ADPP Ⅲ has the strongest substrate affinity and highest catalytic activity for AFG1; conversely, the value corresponding to AFB2 is the highest. K m The highest value indicates that the wild-type enzyme has a relatively weak affinity for its substrate.

[0093] In contrast, the F375L / K570Q mutant exhibited significantly optimized kinetic properties against all four aflatoxins. For example... Figure 5 As shown, it affects AFB1, AFB2, AFG1, and AFG2. K m The values ​​decreased to 28.59, 29.1, 14.67 and 18.79 µmol / L, respectively. This indicates a significant improvement in substrate affinity; simultaneously, for the four substrates... V max The values ​​rose to 4.20, 3.83, and 6.87 respectively. The concentration of 6.43 µmol / (L·h) demonstrates a significant improvement in the enzyme's catalytic turnover efficiency. .

[0094] It is worth noting that the performance improvement of AFG1 is the most significant, compared to the wild type. K m The value dropped by more than 3 times. V max The value also increased significantly.

[0095] The above results indicate a functional complementarity between the F375L and K570Q mutation sites. The amino acid substitution at the F375L site reduces the steric hindrance within the substrate channel by replacing the bulky aromatic side chain with smaller aliphatic residues, thereby promoting substrate entry into the channel and forming a catalytically favorable binding conformation without disrupting the hydrophobic microenvironment. The K570Q mutation does not directly participate in the catalytic reaction; its main function is to regulate the surface electrostatic potential of the enzyme protein, enhancing its structural stability under acidic conditions, and thus indirectly improving catalytic efficiency. In summary, the dual-mutation strategy simultaneously enhances substrate affinity. Km (Decrease in value) and enhancement of maximum catalytic rate ( V max (The value increased), ultimately significantly optimizing the overall catalytic performance of the enzyme.

[0096] Example 8: Evaluation of the degradation performance of the mutant in a mixed aflatoxin contamination system The specific steps are as follows: Constructing a mixed contamination system of four aflatoxins: In 50 mM citrate buffer at pH 5.0, the final concentrations of AFB1, AFB2, AFG1, and AFG2 were all 0.625 µg / mL (total AFs concentration was 2.5 µg / mL). 20 µg / mL of purified double mutant F375L / K570Q enzyme solution and 0.5 mM CuCl2 were added to the reaction system, with wild-type ADPP III as a control. The reaction was carried out at 40 ℃ and 600 rpm with shaking, and samples were taken at fixed points until 24 h. The residual amounts of the four aflatoxins in the system were detected by UPLC-MS / MS, and the degradation rate at the endpoint was calculated. Each treatment was performed in triplicate. The results are shown in Table 6. Table 6: Degradation performance of mutants in a mixed aflatoxin contamination system

[0097] The results are as follows: like Figure 5 As shown, F375L / K570Q maintained high degradation activity in a multi-substrate competitive system. After co-incubation for 24 h, its degradation rates for AFB1, AFB2, AFG1, and AFG2 reached 73.60±4.81%, 71.83±5.09%, 88.12±3.39%, and 85.06±3.25%, respectively. Among the four toxins, AFG1 showed the highest degradation rate, which is consistent with the degradation rate of the substrate in the kinetic analysis. K m lowest, V max The results were consistent. In contrast, the degradation efficiency of the wild-type enzyme was significantly lower under the same conditions, with degradation rates of less than 45% for all four toxins after 24 h. Throughout the incubation process, the F375L / K570Q mutant showed significantly better degradation performance than the wild-type at all time points, and its degradation curve showed a stable upward trend with reaction time, without any obvious substrate competition inhibition.

[0098] Example 9: Evaluation of the degradation performance of the double mutant in corn steep liquor matrix Corn steep liquor (CSL) is a core byproduct of wet corn processing and bioethanol production. Rich in nutrients such as protein, polysaccharides, and organic acids, it is an important raw material for the feed industry. However, during the wet corn steeping process, aflatoxin from corn contaminated with aflatoxin migrates with the water, remains in large quantities, and accumulates in the corn steep liquor, posing a potential biosafety risk to its subsequent resource utilization. To accurately evaluate the aflatoxin degradation capacity of the double mutant F375L / K570Q in corn steep liquor matrix, this study first pretreated the corn steep liquor.

[0099] 1. Pretreatment of corn steep liquor samples The corn pulp used in the experiment was provided by COFCO Group Co., Ltd., with an initial solids content of 40% (w / w). To facilitate subsequent processing and degradation reactions, the pulp was diluted with sterile deionized water at a volume ratio of 1:3 to obtain a corn pulp working solution with a solids content of 10% (w / w). The obtained corn pulp working solution was centrifuged at 12,000 rpm for 10 min, the supernatant was collected, and the pH was adjusted to 5.0. Subsequently, it was autoclaved at 115 ℃ for 20 min and cooled to room temperature, which served as the sample to be degraded. This supernatant was used both to determine the initial toxin background value (blank control) and as the degradation reaction system for the double mutant.

[0100] UPLC-MS / MS analysis revealed that all four major aflatoxins were detected in the pretreated corn steep liquor. The contamination was dominated by AFB1, accompanied by AFB2, AFG1, and AFG2. The initial average contents of AFB1, AFB2, AFG1, and AFG2 were 162.28 µg / L, 30.52 µg / L, 52.14 µg / L, and 9.06 µg / L, respectively.

[0101] After pretreatment, a large amount of insoluble solids and suspended impurities in corn steep liquor were removed, and a clear and transparent supernatant to be degraded was obtained. Pretreatment not only effectively reduced the interference of complex matrix on subsequent enzymatic reactions and quantitative detection of toxins, but also adjusted the pH of the system to 5.0, providing a suitable reaction environment for the activity of double mutant enzymes.

[0102] 2. Degradation of aflatoxin in corn steep liquor by the double mutant Take several 6 mL portions of the corn steep liquor supernatant prepared above, add purified double mutant F375L / K570Q enzyme solution (with wild-type ADPP Ⅲ as a control) to a final concentration of 20 µg / mL, and add an equal volume of citrate buffer (pH 5.0) to the blank control group. Incubate at 40 ℃ and 600 rpm with shaking. Take 1 mL samples at fixed points each time until 24 h. Immediately after sampling, add 4 mL of acetonitrile-water solution (84:16, v / v), vortex for 20 min, let stand for 5 min, centrifuge at 6000 rpm for 10 min, collect the supernatant, purify it using an aflatoxin solid-phase purification column, and determine the aflatoxin content using the above UPLC-MS / MS method.

[0103] The results are as follows Figure 6 And as shown in Table 7: Table 7: Degradation effect of double mutants on aflatoxin in corn steep liquor

[0104] The results show: Wild-type ADPP Ⅲ, possibly due to interference from complex components in corn steep liquor matrix, showed limited degradation rates only for AFB1 and AFB2 (28.06±1.76% and 12.38±1.47%, respectively), while exhibiting almost no degradation effect on the even lower concentrations of AFG1 and AFG2. In contrast, the double mutant maintained good degradation activity against all four aflatoxins in corn steep liquor matrix, but the degradation rates of different subtypes varied. AFB1 showed the fastest initial degradation rate, which may be related to the highest AFB1 substrate concentration in the system, leading to a higher probability of enzyme-substrate collision. After 24 h of reaction, the degradation rate of AFB1 by the double mutant reached 84.32±2.23%, and the final average residue was 25.50 µg / L, which is lower than the limit requirement of ≤50 µg / L for AFB1 in corn processed products stipulated in my country's National Food Safety Standard for Limits of Mycotoxins in Food (GB 2761-2017). Secondly, AFG1, the double mutant also showed good degradation performance. For AFB2 and AFG2, the double mutant exhibited faster degradation efficiency: AFB2 decreased to 8.5 µg / L after 4 h of reaction and was completely degraded by 8 h; AFG2 was below the method detection limit after 4 h of reaction, achieving complete degradation. These results confirm that the double mutant F375L / K570Q can maintain excellent catalytic activity and matrix tolerance in the complex matrix of corn steep liquor, demonstrating good prospects for industrial application.

[0105] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone 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. A dipeptidyl peptidase III mutant, characterized in that, The dipeptidyl peptidase III mutant is... It was obtained by mutating phenylalanine at position 375 of dipeptidyl peptidase III, as shown in SEQ ID NO.1, to leucine; Alternatively, it can be obtained by mutating glycine at position 391 of dipeptidyl peptidase III, as shown in SEQ ID NO.1, to phenylalanine; Alternatively, the amino acid sequence of dipeptidyl peptidase III, as shown in SEQ ID NO.1, can be obtained by mutating phenylalanine at position 375 to leucine and lysine at position 491 to asparagine. Alternatively, the amino acid sequence of dipeptidyl peptidase III, as shown in SEQ ID NO.1, can be obtained by mutating phenylalanine at position 375 to leucine and lysine at position 570 to glutamine. Alternatively, the amino acid sequence of dipeptidyl peptidase III, as shown in SEQ ID NO.1, can be obtained by mutating phenylalanine at position 375 to leucine and lysine at position 600 to asparagine. Alternatively, the amino acid sequence of dipeptidyl peptidase III, as shown in SEQ ID NO.1, can be obtained by mutating phenylalanine at position 375 to leucine and lysine at position 643 to asparagine. Alternatively, the amino acid sequence of dipeptidyl peptidase III, as shown in SEQ ID NO.1, can be obtained by mutating phenylalanine at position 375 to leucine and lysine at position 644 to glutamine. Alternatively, the amino acid sequence of dipeptidyl peptidase III, as shown in SEQ ID NO.1, can be obtained by mutating phenylalanine at position 375 to leucine and arginine at position 612 to glutamine. Alternatively, it can be obtained by mutating phenylalanine at position 375 of dipeptidyl peptidase III, as shown in SEQ ID NO.1, to leucine, and simultaneously mutating arginine at position 620 to glutamine.

2. The gene encoding the dipeptidyl peptidase III mutant of claim 1 or a recombinant vector carrying the gene.

3. A recombinant cell expressing the dipeptidyl peptidase III mutant of claim 1 or carrying the gene of claim 2 or the recombinant vector, preferably, the recombinant cell is a bacterial or fungal host cell.

4. A recombinant enzyme catalyst comprising the dipeptidyl peptidase III mutant of claim 1, wherein, It is any of the following forms: (1) Culture recombinant expression transformants containing dipeptidyl peptidase III mutant and isolate transformant cells containing recombinant dipeptidyl peptidase III mutant enzyme; (2) Culture recombinant expression transformants containing dipeptidyl peptidase III mutant, isolate transformant cells containing recombinant dipeptidyl peptidase III mutant enzyme, and break the transformant cells containing the recombinant dipeptidyl peptidase III mutant enzyme to obtain cell lysate. (3) Cultivate recombinant expression transformants containing dipeptidyl peptidase III mutants, isolate transformant cells containing recombinant dipeptidyl peptidase III mutant enzymes, break the transformant cells containing the recombinant dipeptidyl peptidase III mutant enzymes, obtain cell lysate, and freeze-dry the cell lysate of the recombinant dipeptidyl peptidase III mutant enzymes to obtain lyophilized enzyme powder.

5. A method of increasing the degradation capacity of dipeptidyl peptidase III on aflatoxins AFB1, AFB2, AFG1, AFG2, characterized in that, The method involves mutating phenylalanine at position 375 of dipeptidyl peptidase III, as shown in SEQ ID NO.1, to leucine; or mutating glycine at position 391 of dipeptidyl peptidase III, as shown in SEQ ID NO.1, to phenylalanine.

6. A method for improving the degradation ability of dipeptidyl peptidase III on aflatoxins AFB1, AFB2, AFG1, and AFG2 under acidic conditions, characterized in that, The method involves mutating phenylalanine at position 375 of dipeptidyl peptidase III (as shown in SEQ ID NO.1) to leucine, and simultaneously mutating lysine at position 491 to asparagine; or mutating phenylalanine at position 375 of dipeptidyl peptidase III (as shown in SEQ ID NO.1) to leucine, and simultaneously mutating lysine at position 570 to glutamine; or mutating phenylalanine at position 375 of dipeptidyl peptidase III (as shown in SEQ ID NO.1) to leucine, and simultaneously mutating lysine at position 600 to asparagine; or mutating phenylalanine at position 375 of dipeptidyl peptidase III (as shown in SEQ ID NO.1) to leucine, and simultaneously mutating lysine at position 643 to asparagine; or mutating phenylalanine at position 375 of dipeptidyl peptidase III (as shown in SEQ ID NO.1) to leucine, and simultaneously mutating lysine at position 644 to glutamine ...; or mutating phenylalanine at position 375 of dipeptidyl peptidase III (as shown in SEQ ID NO.1) to glutamine; or mutating phenylalanine at position 375 of dipeptidyl peptidase III (as shown in SEQ ID NO.1) to leucine, and simultaneously mutating lysine The amino acid sequence of dipeptidyl peptidase III shown in SEQ ID NO.1 is modified such that phenylalanine at position 375 is mutated to leucine, and arginine at position 612 is mutated to glutamine; or the amino acid sequence of dipeptidyl peptidase III shown in SEQ ID NO.1 is modified such that phenylalanine at position 375 is mutated to leucine, and arginine at position 620 is mutated to glutamine.

7. An engineered strain of *Escherichia coli*, characterized in that, The engineered Escherichia coli strain expressed the dipeptidyl peptidase III mutant as described in claim 1; Preferably, the engineered Escherichia coli strain is constructed by cloning the gene encoding the dipeptidyl peptidase III mutant into an expression vector and transforming it into Escherichia coli host cells. Preferably, the expression vector is selected from pET series vectors or pGEX series vectors; Preferably, the Escherichia coli host is selected from... Escherichia coli BL21 (DE3) Escherichia coli BL21 Escherichia coli Rosetta (DE3) Escherichia coli C41 (DE3) Escherichia coli C43 (DE3) Escherichia coli JM109 Escherichia coli DH5α or its derivative strains.

8. A method for degrading aflatoxins AFB1, AFB2, AFG1, and AFG2, characterized in that, The process includes the following steps: adding the mutant of claim 1, the recombinase catalyst of claim 4, or the recombinase prepared using the engineered Escherichia coli of claim 7 to a reaction system containing aflatoxins AFB1, AFB2, AFG1, and AFG2 for degradation.

9. The method according to claim 8, characterized in that, The degradation conditions are: pH 5.0~7.0, temperature 25 ℃~45 ℃; Preferably, the reaction system includes, but is not limited to, corn steep liquor.

10. The use of the dipeptidyl peptidase III mutant of claim 1, the recombinant cell of claim 3, or the recombinant enzyme catalyst of claim 4 in the preparation of products that degrade aflatoxins AFB1, AFB2, AFG1, and AFG2.

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