Preparation process of acid-adaptive zearalenone lactone hydrolase

Abstract

The invention relates to the technical field of enzyme engineering, and discloses a preparation process of acid-adaptive zearalenone lactone hydrolase, which comprises the following steps: providing recombinant escherichia coli containing a gene for coding mutant protein, the mutant protein mutates the 59th alanine into proline and / or mutates the 161st asparagine into phenylalanine based on a wild enzyme; carrying out enlarge culture on the strain, then carrying out induced expression, collecting thalli, breaking walls and separating supernate; and carrying out affinity chromatography purification and elution on the supernate to obtain the hydrolase. By introducing rigid and hydrophobic side chain mutation, the thermodynamic stability and catalytic efficiency of the enzyme are improved, so that the enzyme has excellent acid resistance; by combining optimized expression and purification conditions, efficient preparation of high-activity soluble protein is realized, the problems that an existing enzyme preparation is easy to inactivate and low in yield in an acid environment are solved, and the method is suitable for industrial production of feed additives.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, specifically to a preparation process for an acid-adapted zearalenone lactone hydrolase. Background Technology

[0002] Zearalenone is a fungal toxin widely found in moldy corn, wheat, and other grain feeds. It has estrogen-like effects, and ingestion by animals can cause reproductive system dysfunction, severely impacting the economic benefits of livestock farming. Among current methods for removing mycotoxins, biological detoxification using enzyme preparations has become an important means of solving feed toxin contamination due to its high specificity and lack of secondary pollution. Zearalenone lactone hydrolase, in particular, can specifically destroy the lactone ring structure of the toxin, converting it into a non-toxic product, making it a detoxification enzyme preparation with great application potential.

[0003] However, existing zearalenone lactone hydrolases still face significant technical bottlenecks in practical applications and industrial production. These enzyme preparations are typically used as feed additives and must withstand the highly acidic environment of the animal's stomach. Most wild-type lactone hydrolases exhibit poor acid resistance, readily undergoing protein structure unfolding or denaturation in the low pH environment of gastric juice, leading to enzyme inactivation before reaching the intestines and significantly reducing detoxification efficiency. Furthermore, wild-type enzymes often have low substrate affinity and catalytic efficiency, making it difficult to meet the demand for rapid degradation of high-concentration toxins within a short residence time in the animal's digestive tract.

[0004] Furthermore, in the production and preparation of enzyme preparations, the heterologous expression of such enzyme proteins using the *E. coli* expression system often faces the challenge of achieving soluble expression. Due to the protein's own folding characteristics or the influence of the intracellular environment, recombinant proteins tend to exist in the form of inactive inclusion bodies, resulting in low yields of active proteins in the fermentation supernatant. Although the protein can be recovered through denaturation and refolding processes, this increases the complexity and cost of the production process, and the refolding efficiency is usually unstable, limiting the large-scale production and application of this enzyme preparation. Therefore, developing a zearalenone lactone hydrolase with high acid resistance, high catalytic activity, and easy and efficient soluble expression, along with its preparation process, is a problem that needs to be solved. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a preparation process for an acid-adapted zearalenone lactone hydrolase. This process solves the problems of existing zearalenone lactone hydrolases being structurally unstable in acidic environments, prone to unfolding and inactivation, and difficult to adapt to the digestive environment of animal stomachs. Furthermore, wild-type enzymes have low catalytic efficiency for substrates and their derivatives, and are prone to forming inclusion bodies during preparation, resulting in low yields of soluble proteins.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A process for preparing an acid-tolerant zearalenone lactone hydrolase includes the following technical solution: Provides a recombinant Escherichia coli expression strain containing a gene encoding a mutant protein; the mutant protein is based on the wild-type enzyme shown in SEQ ID NO:2, with alanine at position 59 mutated to proline and / or asparagine at position 161 mutated to phenylalanine, and the mutant protein is expressed as a fusion protein with an affinity tag. The recombinant Escherichia coli expression strain was inoculated into liquid culture medium for expansion culture until the OD600 value of the bacterial solution reached 0.6-0.8; Add an inducer to the liquid culture medium and induce culture at 16-37℃ for 4-20 hours to obtain a fermentation broth containing the fusion protein; Collect the bacterial cells in the fermentation broth, and separate the supernatant after cell wall disruption treatment; The supernatant was purified by affinity chromatography, and the eluent containing the fusion protein was collected to obtain the acid-tolerant zearalenone lactone hydrolase.

[0007] By adopting the above technical solution and utilizing a specific site mutation strategy combined with optimized expression and purification processes, the stability and activity of the enzyme preparation were improved. The specific mechanism of action and effects are as follows: The conformational stabilization mechanism of the alanine-proline mutation at position 59 (A59P) is as follows: The pyrrolidine ring structure of the proline side chain restricts the rotational freedom of the N-Cα bond in the polypeptide backbone, thereby reducing the conformational entropy of the protein in its unfolded state. According to the thermodynamics of protein folding, the decrease in conformational entropy of the unfolded state increases the Gibbs free energy change of the protein folding reaction, making the native folded state of the protein more stable than the unfolded state. This rigid structural feature enhances the stability of the enzyme molecular backbone, making it less prone to unfolding or denaturation under acidic environments or thermal stress conditions, thus improving the enzyme's acid and heat resistance.

[0008] Regarding the catalytic enhancement mechanism of the mutation at position 161, from asparagine to phenylalanine (N161F): The wild-type enzyme has polar asparagine at position 161, while the substrate zearalenone has a hydrophobic macrocyclic lactone and benzene ring skeleton. After mutation to phenylalanine, a benzene ring side chain with a large hydrophobic surface area is introduced into the active site. This side chain can form specific hydrophobic interactions and π-π stacking interactions with the substrate molecule, enhancing the affinity between the enzyme and the substrate, manifested as a decrease in the Michaelis constant (Km). Simultaneously, the steric hindrance effect of phenylalanine optimizes the binding orientation of the substrate in the active pocket, making it more conducive to catalytic reactions, thereby improving catalytic efficiency (kcat / Km) and the broad-spectrum degradation ability of substrate derivatives.

[0009] Regarding the synergistic effect of the preparation process: Based on the high stability characteristics of the mutants mentioned above, this preparation process can use a wider range of induction temperatures (such as 37℃). The mutant protein can still maintain correct folding under high temperature induction, reducing the formation of inclusion bodies and improving the expression level and preparation efficiency of soluble proteins.

[0010] Preferably, the amino acid sequence of the mutant protein is selected from one of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0011] By adopting the above technical solution, the double-point mutant (A59P / N161F) shown in SEQ ID NO:5 combines the structural stability of A59P with the catalytic activity of N161F. While maintaining high catalytic activity, it also has excellent acid tolerance, making it suitable as a feed additive for use in the animal digestive tract environment.

[0012] Preferably, the step of providing a recombinant Escherichia coli expression strain containing a gene encoding a mutant protein includes: using a plasmid containing the gene shown in SEQ ID NO: 1 as a template, performing PCR amplification and site-directed mutagenesis using specific primers, transforming the amplification product into competent cells after DpnI digestion, and screening to obtain the recombinant Escherichia coli expression strain; the recombinant Escherichia coli expression strain is constructed by inserting the gene encoding the mutant protein into the pET-22b(+) expression vector and transforming it into Escherichia coli BL21(DE3), wherein the pET-22b(+) expression vector confers a C-terminal histidine affinity tag on the mutant protein.

[0013] By adopting the above technical solutions, DpnI digestion effectively degraded the methylated parental template plasmid, ensuring a high positive rate in the construction of mutant strains; the pET-22b(+) vector, in combination with BL21(DE3) host bacteria, achieved efficient transcriptional expression of exogenous proteins using the T7 strong promoter; the introduction of the C-terminal histidine tag simplified the subsequent purification operation using a nickel column.

[0014] Preferably, in the step of inoculating the recombinant Escherichia coli expression strain into a liquid culture medium for expansion culture, the inoculation amount is 0.5%-5% of the volume of the liquid culture medium, the culture temperature is 37°C, and the rotation speed is 180-220 rpm.

[0015] By adopting the above technical solution, the inoculum amount and culture conditions are controlled to keep the bacteria in the logarithmic growth phase. When the OD600 value reaches 0.6-0.8, the cell metabolic activity is vigorous, providing a sufficient material basis and energy reserve for the induced synthesis of exogenous proteins.

[0016] Preferably, the inducer is isopropyl-β-D-thiogalactoside, and its final concentration in the culture medium is 0.1 mM to 1.0 mM; the specific conditions for the induction culture are selected from any of the following: Condition A: The final concentration of isopropyl-β-D-thiogalactoside was 0.05mM-0.5mM, the induction temperature was 16℃, and the induction time was 20 hours; Condition B: The final concentration of isopropyl-β-D-thiogalactoside was 0.5mM-1.0mM, the induction temperature was 25℃, and the induction time was 6 hours; Condition C: The final concentration of isopropyl-β-D-thiogalactoside was 0.5mM-1.0mM, the induction temperature was 37℃, and the induction time was 4 hours.

[0017] By adopting the above technical solutions, induction strategies adapted to different production scenarios are provided. Low temperature combined with low concentration of inducer (condition A) is beneficial to reduce the protein synthesis rate and give the peptide chain sufficient folding time, which is suitable for protein expression with extremely high conformation requirements; high temperature combined with higher concentration of inducer (condition C) utilizes the high thermal stability of the mutant of this invention, shortens the fermentation cycle, and improves production efficiency; mesophilic induction (condition B) balances expression level and time cost.

[0018] Preferably, the cell wall disruption process employs ultrasonic disruption, and the lysis buffer used contains 20-100 mM tris(hydroxymethyl)aminomethane and 100-300 mM NaCl, with a pH value of 7.0-8.0.

[0019] By adopting the above technical solutions, specific salt concentration ranges and pH buffering systems help maintain protein solubility during cell disruption and prevent protein aggregation or precipitation caused by drastic environmental changes.

[0020] Preferably, the affinity tag is a histidine tag, the affinity chromatography purification uses a nickel ion affinity chromatography column, and the buffer system used contains 20-50 mM imidazole in the washing buffer, 300-600 mM imidazole in the elution buffer, and 300-600 mM NaCl in the buffer.

[0021] By adopting the above technical solution, the purity of the purified product is improved by competitively eluting non-specifically bound impurities with imidazole in the washing step at a concentration of 20-50 mM; the purification effect is improved by using a high concentration of imidazole (300-600 mM) to disrupt the coordination binding between histidine tags and nickel ions in the elution step; and the purification effect is further improved by using 300-600 mM NaCl in a high-salt environment to inhibit non-specific charge adsorption.

[0022] Preferably, the preparation process further includes a dialysis step after collecting the eluent containing the fusion protein: first, the eluent is dialyzed in a 20-100mM tris(hydroxymethyl)aminomethane buffer containing 5-20mM ethylenediaminetetraacetic acid, and then dialyzed in a 20-100mM tris(hydroxymethyl)aminomethane buffer without ethylenediaminetetraacetic acid; all dialysis steps are performed at 4°C.

[0023] By adopting the above technical solution, the first step of dialysis utilizes the chelating effect of 5-20mM ethylenediaminetetraacetic acid (EDTA) to remove residual nickel ions in the eluent, eliminating the potential inhibition and biotoxicity of heavy metal ions on enzyme activity; the second step of dialysis removes EDTA and high concentrations of imidazole, and replaces the enzyme solution with a physiological buffer system suitable for long-term storage; the low-temperature operation throughout the entire process effectively inhibits the activity of miscellaneous proteases, ensuring the stability of the finished enzyme.

[0024] This invention provides a preparation process for an acid-tolerant zearalenone lactone hydrolase. It has the following beneficial effects: 1. This invention reduces the conformational entropy of the unfolded state of the protein and improves the thermodynamic stability of the enzyme by mutating alanine at position 59 to proline and using the pyrrolidine ring structure of the proline side chain to restrict the rotation of the polypeptide backbone. This structural modification enables the enzyme molecule to maintain the conformation of the active site in an acidic environment, overcomes the defect of wild-type enzymes that are prone to unfolding and inactivation under the acidic conditions of the animal stomach, and improves the tolerance of enzyme preparations in the digestive tract environment.

[0025] 2. This invention introduces a hydrophobic benzene ring side chain into the active site by mutating asparagine at position 161 to phenylalanine, thereby forming hydrophobic interactions and π-π stacking interactions with the substrate. This modification optimizes the binding energy between the enzyme and the substrate, reduces the Michaelis constant, and improves the catalytic efficiency, resulting in an enzyme preparation with stronger degradation activity against zearalenone and its derivatives.

[0026] 3. The preparation process provided by this invention combines the enhanced heat resistance of mutant proteins. The high-temperature induction strategy can still maintain the correct folding of the protein, reduce the formation of inclusion bodies, and improve the yield of soluble proteins. With the aid of nickel affinity chromatography and stepwise dialysis, impurities and residual nickel ions are effectively removed, resulting in a high-purity enzyme preparation without heavy metal residues, which is suitable for industrial production. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the plasmid patterns of WT, A59P, N161F and the double-point mutant A59P / N161F of the present invention. Figure 2 The degradation rate of ZEN by WT, A59P, N161F and the double-point mutant A59P / N161F of the present invention under optimal conditions is shown in the figure. Figure 3 The graph shows the ZEN degradation concentration of WT, A59P, N161F and the double-point mutant A59P / N161F of this invention within 2 hours after incubation at 37°C and acidic pH 4.5 for 2 hours, followed by incubation at pH 7.0. Figure 4 The diagram shows the ZEN lactone hydrolase wild-type enzyme WT of the present invention, after incubation at 37°C for 2 hours under different acidic conditions (pH 4.0, 4.3, 4.5, 4.7 and 5.0) and then at pH 7.0 for 2 hours. Figure 5 The graph shows the ZEN degradation concentration of the ZEN lactone hydrolase mutant A59P of the present invention after incubation at 37°C for 2 hours under different acidic conditions (pH 4.0, 4.3, 4.5, 4.7 and 5.0) and then at pH 7.0 for 2 hours. Figure 6 The graph shows the ZEN degradation concentration of the ZEN lactone hydrolase mutant N161F of the present invention after incubation at 37°C for 2 hours under different acidic conditions (pH 4.0, 4.3, 4.5, 4.7 and 5.0) and then at pH 7.0 for 2 hours. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Preparation Examples 1-6: Preparation Example 1: This preparation example provides a method for preparing a recombinant expression strain containing the wild-type zearalenone lactone hydrolase gene, including the following steps: Using the zearalenone lactone hydrolase gene (nucleotide sequence shown in SEQ ID NO:1) from Monosporascus sp. GIB2 as a template, the recombinant plasmid pET-22b(+)-WT was constructed by ligating it into the pET-22b(+) expression vector via NdeI and XhoI restriction sites. The recombinant plasmid, verified by sequencing, was transformed into *E. coli* BL21(DE3) competent cells and plated on LB agar plates containing 50 μg / mL ampicillin. The plates were incubated upside down at 37°C for 12–16 hours. Single colonies were picked for colony PCR verification; positive clones were identified as recombinant strains expressing the wild-type enzyme (amino acid sequence shown in SEQ ID NO:2).

[0030] Preparation Example 2: This preparation example provides a method for preparing the single-point mutant A59P recombinant expression strain, including the following steps: Using the QuikChange site-directed mutagenesis principle, the recombinant plasmid pET-22b(+)-WT constructed in Example 1 was used as a template, and full plasmid PCR amplification was performed using high-fidelity DNA polymerase.

[0031] Primer design: Mutant primers were designed targeting amino acid position 59 to mutate the alanine (Ala) codon to the proline (Pro) codon. The primer sequences are as follows (lowercase letters indicate mutation sites): Forward primer A59P-F: 5'-TTCGATATGccgGGTATGTCGAGGTCATG-3'; Reverse primer A59P-R: 5'-ATACCcggCATATCGAAAGTGGTGCAAGTAA-3'.

[0032] PCR reaction system (50 μL): PrimeSTARMaxPremix(2×): 25μL; Upstream primer F (20 μM): 1 μL (final concentration 0.4 μM); Downstream primer R (20 μM): 1 μL (final concentration 0.4 μM); Template plasmid (10 ng / μL): 1 μL (total mass 10 ng); Sterile double-distilled water (ddH2O): 22 μL.

[0033] PCR reaction procedure: Pre-denaturation at 98℃ for 3 minutes; Denaturation at 98℃ for 10 seconds, annealing at 55℃ for 15 seconds, and extension at 72℃ for 4 minutes (calculated based on a plasmid size of approximately 6 kb) were performed for a total of 28 cycles. Extend the heat at 72℃ for 5 minutes.

[0034] Post-processing: After the reaction was completed, 1 μL of restriction endonuclease DpnI (10 U / μL) was added to the PCR product and digested at 37°C for 2 hours to degrade the methylated parental template plasmid.

[0035] Transformation and screening: 5 μL of the digestion product was transformed into Escherichia coli DH5α competent cells. After resistance screening and sequencing to verify that the mutation site was correct, the plasmid was extracted and transformed into Escherichia coli BL21(DE3) competent cells to obtain a recombinant strain expressing the single-point mutant A59P (amino acid sequence as shown in SEQ ID NO:3).

[0036] Preparation Example 3: This preparation example provides a method for preparing the single-point mutant N161F recombinant expression strain, including the following steps: Except for the primers used, the PCR amplification system, procedure and transformation screening steps were the same as those in Preparation Example 2.

[0037] Primer design: Mutant primers were designed to mutate the asparagine (Asn) codon to the phenylalanine (Phe) codon at amino acid position 161. The primer sequences are as follows: Forward primer N161F-F: 5'-CTCAGACGtttTTTGGTCACGACTTGACAG-3'; Reverse primer N161F-R: 5'-GACCAAAaaaCGTCTGAGGCATTTCCTCGCTCAGCA-3'.

[0038] Preparation results: A recombinant strain expressing the single-point mutant N161F (amino acid sequence as shown in SEQ ID NO:4) was obtained.

[0039] Preparation Example 4: This preparation example provides a method for preparing the recombinant expression strain A59P / N161F with two-point mutants, including the following steps: Using the plasmid pET-22b(+)-A59P containing the A59P mutation, which was verified by sequencing in Preparation Example 2, as a template, PCR amplification was performed using the primer pairs N161F-F and N161F-R from Preparation Example 3. The PCR reaction system, reaction procedure, DpnI digestion, and transformation screening steps were all the same as in Preparation Example 2. After sequencing confirmed that the expected mutations occurred simultaneously at positions 59 and 161, the plasmid was transformed into Escherichia coli BL21(DE3) competent cells to obtain a recombinant strain expressing the double-point mutant A59P / N161F (amino acid sequence shown in SEQ ID NO: 5).

[0040] Preparation Example 5: This preparation example provides a method for preparing the A59G mutant recombinant expression strain for comparison, including the following steps: Except for replacing the mutation primer with a primer that mutates alanine at position 59 to glycine (Gly), the remaining steps are the same as in Preparation Example 2.

[0041] The primer sequences are as follows (ggc encoding Gly): Forward primer A59G-F: 5'-TTCGATATGggcGGTATGTCGAGGTCATG-3'; Reverse primer A59G-R: 5'-ATACCgccCATATCGAAAGTGGTGCAAGTAA-3'; This preparation example aims to construct a control mutant with glycine at position 59 to verify the specific effect of the proline rigid structure in the A59P mutation.

[0042] Preparation Example 6: This preparation example provides a method for preparing the N161A mutant recombinant expression strain for comparison, including the following steps: Except for replacing the mutant primer with a primer that mutates asparagine at position 161 to alanine (Ala), the remaining steps are the same as in Preparation Example 3.

[0043] The primer sequences are as follows (GCG encodes Ala): Forward primer N161A-F: 5'-CTCAGACGgcgTTTGGTCACGACTTGACAG-3'; Reverse primer N161A-R: 5'-GACCAAAcgcCGTCTGAGGCATTTCCTCGCTCAGCA-3'; This preparation example aims to construct a control mutant with alanine at position 161 to verify the specific contribution of the hydrophobicity and steric effect of the phenylalanine side chain in the N161F mutation to the enhancement of enzyme activity.

[0044] Examples 1-3: Example 1: This example provides a preparation process for an acid-tolerant zearalenone lactone hydrolase, including the following steps: S1. Pick a single colony of the recombinant strain pET-22b(+)-A59P / N161F constructed in Preparation Example 4, inoculate it into 5 mL of LB liquid medium containing 50 μg / mL ampicillin, and culture at 37℃ and 200 rpm for 12 hours to obtain seed liquid; S2. Transfer the seed culture at an inoculation rate of 1% (volume ratio) to 200 mL of LB liquid medium containing 50 μg / mL ampicillin, and incubate at 37°C and 200 rpm for about 2-3 hours with shaking. Monitor the bacterial culture until the OD600 value reaches 0.6. S3. Add the inducer isopropyl-β-D-thiogalactoside (IPTG) to the culture medium to a final concentration of 0.1 mM, lower the culture temperature to 16℃, and induce culture at 180 rpm for 20 hours to promote soluble protein expression. S4. Centrifuge the fermentation broth at 4℃ and 8000 rpm for 10 minutes to collect the cell pellet. Resuspend the cells in 15 mL of lysis buffer (50 mM tris(hydroxymethyl)aminomethane, 200 mM NaCl, pH 7.5). Disrupt the cell walls using an ultrasonic cell disruptor under ice-water bath conditions (operating parameters: power 200 W, sonication for 3 seconds, interval for 5 seconds, total working time 10 minutes). Centrifuge the disrupted broth at 4℃ and 10000 rpm for 20 minutes and collect the supernatant. S5. Purification was performed using a nickel-NTA (Ni-NTA) affinity chromatography column. First, the column was equilibrated with 5 column volumes of equilibration buffer (50 mM tris(hydroxymethyl)aminomethane, 500 mM NaCl, pH 7.5). The supernatant was loaded at a flow rate of 0.5 mL / min. Then, 10 column volumes of wash buffer (containing 30 mM imidazole, 50 mM tris(hydroxymethyl)aminomethane, 500 mM NaCl, pH 7.5) were used to wash away non-specifically bound proteins until the UV absorbance returned to baseline. Finally, the target protein was eluted with elution buffer (containing 500 mM imidazole, 50 mM tris(hydroxymethyl)aminomethane, 500 mM NaCl, pH 7.5), and the elution peak was collected. S6. Transfer the eluted protein solution into a dialysis bag with a molecular weight cutoff of 14 kDa. Dialyze the solution at 4°C for 12 hours in 1 L of 50 mM Tris(hydroxymethyl)aminomethane buffer (pH 7.5) containing 10 mM EDTA to remove residual nickel ions. Then, transfer the solution to 1 L of 50 mM Tris(hydroxymethyl)aminomethane buffer (pH 7.0) and dialyze for 6 hours, changing the dialysis buffer once to remove imidazole and EDTA. Collect the dialyzed enzyme solution and perform SDS-PAGE electrophoresis to determine if the bands are single and the molecular weight is correct (approximately 29 kDa). Measure the protein concentration and store the solution at 4°C for later use.

[0045] Example 2: This example provides a preparation process for an acid-tolerant zearalenone lactone hydrolase, including the following steps: S1. Select single colonies of the recombinant strains constructed in Preparation Example 2, Preparation Example 3 and Preparation Example 4 respectively, and prepare seed liquid according to step S1 of Example 1. S2. Transfer the seed culture at a volume ratio of 1% to 200 mL of fresh LB medium (containing 50 μg / mL ampicillin) and incubate at 37℃ and 200 rpm until the OD600 value of the bacterial culture reaches 0.7. S3. Add IPTG to each culture medium to a final concentration of 1.0 mM, adjust the temperature to 25℃, and induce culture at 200 rpm for 6 hours. Steps S4, subsequent cell collection, disruption, purification, and dialysis are the same as steps S4 to S6 in Example 1. Finally, purified single-point mutant A59P protein, single-point mutant N161F protein, and double-point mutant A59P / N161F protein were obtained. The enzyme solutions obtained under these process conditions exhibit high activity and a suitable preparation cycle, making them suitable for subsequent testing of key enzymatic properties and stability.

[0046] Example 3: This example provides a preparation process for an acid-tolerant zearalenone lactone hydrolase, including the following steps: S1. Pick a single colony of the recombinant strain constructed in Example 4 and prepare seed liquid according to step S1 of Example 1. S2. Transfer the seed culture to fresh LB medium (containing 50 μg / mL ampicillin) at an inoculation rate of 1% (v / v) and incubate at 37°C and 200 rpm until the OD600 value of the bacterial culture reaches 0.8. S3. Add IPTG to the culture medium to a final concentration of 1.0 mM, maintain the temperature at 37°C, and rapidly induce culture at 220 rpm for 4 hours. Steps S4, subsequent cell collection, disruption, purification, and dialysis are the same as steps S4 to S6 in Example 1. This example aims to verify that the mutant protein of the present invention can still achieve detectable soluble expression levels under high temperature and high concentration inducer conditions that easily form inclusion bodies, demonstrating the robustness of its folding performance.

[0047] Comparative Examples 1-4: Comparative Example 1: This comparative example provides the preparation of wild-type zearalenone lactone hydrolase. The only difference from Example 2 is that the strain used in step S1 is the recombinant expression strain containing the wild-type gene (WT) constructed in Preparation Example 1, and the remaining induction expression and purification steps are the same.

[0048] Comparative Example 2: This comparative example provides the preparation of an empty vector control. The only difference from Example 2 is that the strain used in step S1 is Escherichia coli BL21(DE3) transformed with the empty vector pET-22b(+), and the other steps are the same.

[0049] Comparative Example 3: This comparative example provides a control enzyme preparation with the 59th position mutated to glycine (A59G). The only difference from Example 2 is that the strain used in step S1 is the recombinant strain expressing the A59G mutant constructed in Preparation Example 5; all other steps are the same.

[0050] Comparative Example 4: This comparative example provides a control enzyme preparation with a mutation at position 161 to alanine (N161A). The only difference from Example 2 is that the strain used in step S1 is the recombinant strain expressing the mutant N161A constructed in Preparation Example 6; all other steps are the same.

[0051] Test Examples 1-7: Test Example 1: Analysis of Recombinant Protein Expression Levels and Purity This test case quantitatively characterizes the soluble expression level of proteins prepared by each example and comparative example under different induction process conditions and the homogeneity of purified samples, verifying the feasibility of the mutant of the present invention in engineering preparation.

[0052] Experimental steps: S1. Take the final enzyme solution samples prepared in Examples 1 to 3 after nickel column affinity chromatography purification and dialysis treatment, and the wild-type enzyme solution sample prepared in Comparative Example 1 as the test objects. S2. Protein concentration was determined using a BCA protein concentration assay kit. A standard curve series of 0 to 2.0 mg / mL was prepared using bovine serum albumin as the standard. 20 μL of the sample to be tested was added to a 96-well plate, followed by 200 μL of BCA working solution, and incubated at 37°C for 30 minutes. The absorbance was measured at 562 nm using a microplate reader, and the protein concentration of each sample was calculated based on the regression equation of the standard curve. S3. Take 10 μg of protein sample and perform 12% SDS-PAGE gel electrophoresis for separation. After electrophoresis, stain with Coomassie Brilliant Blue R-250 for 2 hours, then treat with destaining solution until the background is clear. Scan the gel using a gel imaging analysis system, and calculate the gray values ​​of the target band with a molecular weight of approximately 29 kDa and the impurities in the lanes using optical density analysis software. S4. Calculate the purity of the target protein according to the formula. The purity is equal to the integral value of the optical density of the target band divided by the integral value of the total optical density of the lane, and then multiplied by 100%.

[0053] Experimental data: The results of expression level and purity determination for each sample are shown in Table 1.

[0054] Table 1. Results of concentration and purity determination of recombinant protein under different preparation processes. Results Analysis: According to the data in Table 1, under the standard induction process at a moderate temperature, the single-point mutants A59P, N161F, and the double-point mutant A59P / N161F involved in this invention all achieved soluble expression, with protein yields comparable to or slightly higher than the wild type. The protein concentration of the double-point mutant A59P / N161F was 2.89 mg / mL, with a purity of 96.3%, indicating that the introduction of proline at position 59 and phenylalanine at position 161 did not hinder the normal protein folding pathway, and that the hydrophobic core and rigid structure formed after the mutation facilitated protein accumulation in host cells.

[0055] Comparative results of Examples 1, 2, and 3 show that the double-point mutant A59P / N161F achieved detectable soluble expression under three induction conditions: low temperature for long duration, medium temperature for standard, and high temperature for short duration. Example 3, even under induction at 37℃, still yielded 1.74 mg / mL of soluble protein with a purity exceeding 90%. These results indicate that the mutant possesses strong structural stability. The introduction of proline restricts the conformational freedom of the polypeptide chain, reduces the conformational entropy of the unfolded state, and helps the protein maintain its correct folded structure under heat stress or rapid synthesis conditions, reducing inclusion body formation and making it suitable for various industrial fermentation processes.

[0056] Test Example 2: Determination of Enzyme-Catalyzed Reaction Kinetic Parameters This test case determined the kinetic constants of wild-type and mutant enzymes for the substrate zearalenone, and verified the mechanism by which the amino acid mutation at position 161 affects substrate affinity and catalytic efficiency.

[0057] Experimental steps: S1. Dissolve the zearalenone standard in methanol to prepare a series of substrate working solutions with different concentrations, so that the final concentrations of the substrate in the reaction system are 5 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM and 100 μM, respectively. S2. Establish a 1 mL reaction system, add 50 mM pH 7.5 tris(hydroxymethyl)aminomethane HCl buffer and the purified enzyme solution prepared in Example 2, control the final concentration of the enzyme solution at 0.5 μg / mL, and preheat in a 37°C water bath for 5 minutes; then add the corresponding concentration of substrate working solution to start the reaction, and control the reaction time within 5 minutes to ensure that the determination is in the initial rate stage of the reaction. S3. Immediately after the reaction is complete, add an equal volume of chromatographic grade acetonitrile to terminate the reaction. Filter the sample through a 0.22 μm filter membrane. Determine the concentration of the remaining substrate and product in the reaction solution using high performance liquid chromatography (HPLC) and calculate the initial reaction rate at different substrate concentrations. S4. Using substrate concentration as the x-axis and initial reaction rate as the y-axis, fit the data using the double reciprocal plotting method, calculate the Michaelis constant and maximum reaction rate, and calculate the catalytic constant and catalytic efficiency based on enzyme concentration.

[0058] Experimental data: The kinetic parameters of each enzyme sample are shown in Table 2.

[0059] Table 2. Results of kinetic parameter determination for wild-type and mutant enzymes. Enzyme sample source Michaelis constant (μM) Maximum reaction rate (μmol / min / mg) Catalytic constant (1 / s) Catalytic efficiency (I / s / mM) Comparative Example 1 (WT) 25.34 16.82 8.12 320.44 Example 2 (N161F) 12.15 29.47 14.23 1171.19 Example 2 (A59P / N161F) 13.89 27.65 13.35 961.12 Comparative Example 4 (N161A) 24.18 15.93 7.69 318.03 Results analysis: According to the data in Table 2, the Michaelis constant of the wild-type enzyme was 25.34 μM, while the Michaelis constant of the N161F mutant with the phenylalanine mutation decreased to 12.15 μM. A lower Michaelis constant indicates a stronger affinity of the enzyme for the substrate; the data show that the N161F mutant has approximately twice the affinity for the substrate zearalenone compared to the wild-type.

[0060] The wild-type enzyme has asparagine at position 161, with a polar amide group in its side chain, making it a hydrophilic amino acid. The substrate, zearalenone, has a macrocyclic lactone and aromatic ring structure, exhibiting strong hydrophobicity overall. Mutating position 161 to phenylalanine introduces a strongly hydrophobic group into the benzene ring structure of the phenylalanine side chain. This hydrophobic side chain interacts with the substrate molecule through hydrophobic interactions and π-π stacking. This non-covalent interaction alters the microenvironment of the enzyme's active site, allowing the enzyme molecule to bind to the substrate more effectively and reducing the Michaelis constant.

[0061] In Comparative Example 4, the 161 position was mutated to alanine, resulting in a Michaelis constant of 24.18 μM, which was not significantly different from the wild type, and no improvement in catalytic efficiency was observed. The alanine side chain is only a methyl group, which, although nonpolar, lacks the large-volume spatial structure and electron cloud distribution of the benzene ring, thus failing to form an effective and specific stacking interaction with the substrate. This result confirms that the performance improvement of N161F depends on the specific spatial structure and hydrophobic properties of the phenylalanine benzene ring side chain, rather than a simple change in polarity.

[0062] The two-point mutant A59P / N161F retained a low Michaelis constant (13.89 μM) and high catalytic efficiency, indicating that the proline mutation at position 59 did not interfere with the function at position 161. The catalytic constant of the N161F mutant increased from 8.12 to 14.231 s, suggesting that the introduction of phenylalanine may have restricted the orientation of the substrate in the active pocket, placing it in a conformation more favorable to nucleophilic attack reactions, thereby improving the catalytic conversion rate.

[0063] Test Example 3: Comparative Test of Enzyme Activity under Optimal Conditions This test example measures the degradation ability of enzyme solutions prepared in each example and comparative example on the substrate zearalenone under optimal reaction conditions of high temperature and alkaline pH. By comparing the differences in specific enzyme activity of different mutants, the contribution of key site mutations to catalytic efficiency is verified.

[0064] Experimental steps: S1. A 50 mM, pH 9.0 tris(hydroxymethyl)aminomethane-HCl buffer solution was used as the reaction medium. Zearalenone standard was accurately weighed and dissolved in methanol to prepare a substrate stock solution, which was then diluted with the reaction buffer to achieve a final substrate concentration of 20 μg / mL in the reaction system. S2. Take the purified enzyme solutions of A59P, N161F, and A59P / N161F prepared in Example 2, as well as the wild-type enzyme solution prepared in Comparative Example 1 and the N161A enzyme solution prepared in Comparative Example 4. Dilute each enzyme solution to a suitable concentration, add the enzyme solution to 1 mL of the reaction system, control the protein amount between 0.1 μg and 0.5 μg, and react in a 60°C constant temperature water bath for 3 minutes; S3. Immediately after the reaction is complete, add an equal volume of chromatographic grade acetonitrile to terminate the reaction. After mixing thoroughly, filter through a 0.22 μm filter membrane. Determine the residual amount of zearalenone in the reaction solution using high performance liquid chromatography (HPLC) and calculate the substrate reduction. S4. An enzyme activity unit is defined as the amount of enzyme required to degrade 1 μg of zearalenone per minute under the above-mentioned test conditions. The specific enzyme activity is calculated by dividing the number of enzyme activity units by the number of milligrams of enzyme protein involved in the reaction.

[0065] Experimental data: The results of the specific enzyme activity assay for each enzyme sample are shown in Table 3.

[0066] Table 3 Results of specific enzyme activity assays for different enzyme samples Results Analysis: According to the data in Table 3, the specific enzyme activity of the wild-type enzyme was 38.42 U / mg. After introducing the A59P single-point mutation, the enzyme activity was 30.20 U / mg, which was lower than that of the wild type. This indicates that although the introduction of proline at position 59 enhanced rigidity, it affected the catalytic rate under optimal conditions to some extent.

[0067] After introducing the N161F single-point mutation, the specific enzyme activity increased to 42.65 U / mg, approximately 1.11 times that of the wild type. Combined with data analysis from Comparative Example 4, mutating asparagine at position 161 to alanine resulted in a specific enzyme activity of only 36.91 U / mg, showing no significant change compared to the wild type. This comparative result confirms that the increased activity of the N161F mutant is not due to the removal of the original asparagine residue, but rather depends on the newly introduced phenylalanine residue. The benzene ring structure of the phenylalanine side chain has a large hydrophobic surface area and spatial volume, forming a stronger hydrophobic interaction with the hydrophobic zearalenone substrate. It may also optimize the binding posture of the substrate in the active pocket through electron cloud overlap, thereby lowering the reaction energy barrier and improving catalytic conversion efficiency.

[0068] The specific enzyme activity of the double-point mutant A59P / N161F was 138.76 U / mg, maintaining the high activity characteristics of the N161F mutant. Data indicate that the surface-located A59P structural mutation and the active site-located N161F functional mutation are spatially compatible, and their combination can simultaneously confer high catalytic activity and potential structural stability to the enzyme molecule without functional cancellation.

[0069] Test Example 4: Comparative Test of Tolerance under Acidic Environment This test case determined the survival rate of wild-type enzymes and various mutants under different acidic pH conditions, and verified the effect of the proline mutation at position 59 on the rigidity of enzyme molecular structure and acid stability.

[0070] Experimental steps: S1. Prepare citric acid and sodium citrate buffer solutions at pH 4.0, pH 4.5, and pH 5.0 as acidic treatment media. The purified enzyme solutions A59P, N161F, and A59P / N161F prepared in Example 2, as well as the wild-type enzyme solution prepared in Comparative Example 1 and the A59G enzyme solution prepared in Comparative Example 3, were selected as test subjects.

[0071] S2. Mix each enzyme solution with the above-mentioned buffer solutions of different pH values ​​at a volume ratio of 1:9, and incubate in a constant temperature water bath at 37°C for 2 hours; at the same time, set up an enzyme solution without acid treatment as a control group, and set its activity to 100%.

[0072] S3. After treatment, a high concentration of pH 9.0 tris(hydroxymethyl)aminomethane (HCl) buffer was added to the mixture to restore the system to the optimal reaction pH. Subsequently, the residual enzyme activity of each treatment group was measured at 60°C according to the method in Test Example 3.

[0073] S4. Calculate the residual enzyme activity rate, which is equal to the enzyme activity measured after acid treatment divided by the enzyme activity of the untreated control group. The result is expressed as a percentage.

[0074] Experimental data: The residual enzyme activity of each enzyme sample after 2 hours of treatment under different pH conditions is shown in Table 4.

[0075] Table 4. Residual enzyme activity after 2 hours of treatment under different pH conditions. Results Analysis: According to the data in Table 4, the wild-type enzyme is more sensitive to acidic environments. After treatment at pH 4.0 for 2 hours, the residual activity of the wild-type enzyme was only 14.5%, indicating that the low pH environment caused the wild-type enzyme protein structure to unfold or denature, and the conformation of the active site was disrupted.

[0076] The introduction of the A59P mutation significantly improved the stability of the enzyme under acidic conditions. The A59P mutant retained 68.2% of its residual activity at pH 4.0 and 92.4% of its activity at pH 5.0. The proline introduced at position 59 has a pyrrolidine ring side chain structure, which restricts the rotation of the N-Cα bond in the polypeptide backbone and reduces the conformational entropy of the unfolded protein. This entropy effect increases the thermodynamic stability of the folded state relative to the unfolded state, enhances the rigidity of the enzyme molecule backbone, and enables it to resist structural damage induced by acidic environments.

[0077] Comparative Example 3 involved mutating alanine at position 59 to glycine (A59G). Glycine has the smallest side chain and the highest degree of conformational freedom. Data showed that the residual activity of the A59G mutant at pH 4.0 decreased to 8.4%, lower than that of the wild type. This reverse control experiment confirmed that the improved stability at position 59 depended on the rigid restriction effect of proline introduction, rather than the removal of alanine. The introduction of the more flexible glycine accelerated the inactivation of the enzyme under acidic conditions.

[0078] The residual activity of the double-site mutant A59P / N161F was close to that of the single-site mutant A59P under various pH conditions, at 65.9% (pH 4.0), 79.8% (pH 4.5), and 91.1% (pH 5.0), respectively. The data indicate that although the phenylalanine mutation at position 161 altered the hydrophobic environment of the active site, it did not disrupt the overall structural stability brought about by the A59P mutation. The single-site mutant N161F also maintained extremely high residual activity after treatment at pH 4.0-5.0, comparable to A59P. At pH 4.5, the residual activity of N161F was 80.2%, a significant increase compared to the wild type (2.3%), indicating that the phenylalanine mutation at the N161F site not only enhanced enzyme activity but also made a significant contribution to the enzyme's acid stability. The double-site mutant combines the acid resistance of A59P with the high catalytic activity of N161F.

[0079] Test Example 5: Enzyme activity stability test under different induction processes This test case compares and measures the enzymatic properties of the two-point mutant A59P / N161F prepared under different temperature induction conditions. By examining the specific enzyme activity and the activity retention rate after heat stress, the adaptability of the mutant molecular structure to fluctuations in fermentation process parameters is verified.

[0080] Experimental Procedure: S1. The purified double-point mutant A59P / N161F protein solutions prepared in Example 1 (induced at 16℃), Example 2 (induced at 25℃), and Example 3 (induced at 37℃) were selected as test samples. The protein concentration of each group of samples was uniformly adjusted to 0.2 mg / mL using 50 mM pH 7.5 tris(hydroxymethyl)aminomethane (HCl) buffer.

[0081] S2. Following the method in Test Example 3, the initial specific enzyme activity of the three groups of samples was measured at 60℃ and pH 9.0. Each group of samples was measured in parallel three times and the average value was taken to evaluate the effect of different induction temperatures on protein folding efficiency and active site formation.

[0082] S3. Conduct a heat stress stability test. Place the three groups of enzyme solutions with the same concentration in a 65°C constant temperature water bath for 60 minutes. During the treatment, seal the tube opening to prevent water evaporation and concentration changes. After the heat treatment, immediately place the tubes in an ice water bath for 5 minutes to stop the heat effect.

[0083] S4. Determine the residual enzyme activity of each group of samples after heat stress treatment and calculate the activity retention rate; the activity retention rate is equal to the specific enzyme activity after heat treatment divided by the initial specific enzyme activity before treatment, and the result is expressed as a percentage.

[0084] Experimental data: The performance test results of enzyme samples prepared under different induction processes are shown in Table 5.

[0085] Table 5. Data on enzyme activity and thermal stability of mutants prepared at different induction temperatures. Results Analysis: According to the data in Table 5, the initial specific enzyme activities of the enzyme samples prepared in Examples 1, 2, and 3 were 142.15 U / mg, 138.76 U / mg, and 133.92 U / mg, respectively. The data indicate that the specific enzyme activity of the double-point mutant A59P / N161F fluctuated less when the induction temperature changed from 16℃ to 37℃. Normally, wild-type enzymes are prone to misfolding or inclusion body formation under high-temperature induction at 37℃, leading to a decrease in the specific enzyme activity of soluble proteins. The results of this test show that this mutant maintained high catalytic activity under 37℃ induction conditions, with its specific enzyme activity reaching over 94% of that of the low-temperature induction group.

[0086] Under heat stress at 65°C for 60 minutes, the activity retention rates of the three groups of samples were 87.3%, 85.8%, and 82.1%, respectively. The low-temperature induced group showed slightly higher stability, but the high-temperature induced group (Example 3) did not show a drastic decline in stability and still retained more than 80% of its activity. This result confirms that the rigid structure effect introduced by the A59P mutation is universal and unaffected by the thermal environment during protein synthesis.

[0087] Mechanistic analysis suggests that the introduction of proline at position 59 restricts the rotation angle of the polypeptide backbone, reducing the conformational entropy of the unfolded state and increasing the Gibbs free energy change of the protein folding reaction, thus making the native folded state more thermodynamically stable. This endogenous structural rigidity enables enzyme molecules to resist hydrophobic surface exposure and intermolecular aggregation caused by thermal perturbations during rapid high-temperature synthesis, allowing them to fold rapidly and accurately into the active native conformation. This characteristic provides flexibility to the production process, allowing the use of lower-energy-consumption, shorter-cycle high-temperature induction processes in industrial fermentation without significantly reducing product quality and stability.

[0088] Test Example 6: Broad-spectrum substrate test. This test example examines the degradation ability of wild-type enzyme and the two-point mutant A59P / N161F on zearalenone and its main derivatives, and verifies the broad-spectrum catalytic performance of the mutant of this invention in treating a variety of structurally similar toxins.

[0089] Experimental steps: S1. Weigh out zearalenone, α-zearalenol, β-zearalenol and zearalenone standards respectively, and dissolve them in methanol to prepare substrate stock solutions with consistent concentrations; dilute with glycine sodium hydroxide buffer at pH 9.0 to make the final concentration of the substrate in each reaction system 20 μg / mL.

[0090] S2. Take the enzyme solution of the double-point mutant A59P / N161F prepared in Example 2 and the wild-type enzyme solution prepared in Comparative Example 1, and adjust the enzyme solution concentration to keep the amount of protein added to the reaction system consistent, both being 0.5 μg.

[0091] S3. Add the enzyme solution to the reaction system containing different substrates, with a total volume of 1 mL, and react at a constant temperature of 45℃ for 30 minutes.

[0092] S4. After the reaction is complete, add an equal volume of chromatographic grade acetonitrile to terminate the reaction. After mixing evenly, filter through a 0.22μm filter membrane.

[0093] S5. The residual concentration of each substrate in the reaction solution is determined by high performance liquid chromatography, and the relative degradation rate is calculated. The relative degradation rate is equal to the difference between the initial substrate concentration and the residual substrate concentration, divided by the initial substrate concentration. The result is expressed as a percentage.

[0094] Experimental data: The degradation effects of the two enzymes on different substrates are shown in Table 6.

[0095] Table 6. Relative degradation rates of zearalenone derivatives by wild type and mutant. Results analysis: According to the data in Table 6, under the same reaction conditions, the degradation abilities of wild-type enzymes for different substrates varied significantly. Wild-type enzymes exhibited some degradation ability for ZEN and ZAN containing ketone groups, with degradation rates of 32.4% and 28.7%, respectively; however, for derivatives whose ketone groups were reduced to hydroxyl groups, especially the β-configuration β-ZOL, the degradation rate decreased to 6.3%. The data indicate that the active site of the wild-type enzyme is highly sensitive to structural changes in the substrate side chain, thus limiting its catalytic range.

[0096] The two-point mutant A59P / N161F exhibited high degradation activity against all four substrates. For ZEN and ZAN, the mutant increased the degradation rates to 96.8% and 94.1%, respectively. Simultaneously, the mutant improved the recognition ability of hydroxylated derivatives, achieving degradation rates of 83.5% and 71.2% for α-ZOL and β-ZOL, respectively.

[0097] Mechanistic analysis revealed that zearalenone and its derivatives share a hydrophobic macrolide backbone and a benzene ring structure. The wild-type asparagine side chain at position 161 is short and highly polar, primarily relying on hydrogen bonds or polar interactions to recognize specific functional groups of the substrate. When the substrate ketone group is replaced with a hydroxyl group and its spatial orientation changes, the binding interaction weakens. The phenylalanine (N161F) side chain introduced in the mutant contains a benzene ring, providing a hydrophobic interaction platform and a larger spatial occupancy. This hydrophobic interaction mainly targets the common hydrophobic backbone and benzene ring moiety of the substrate, reducing the enzyme's dependence on side chain-specific polar groups. Therefore, regardless of whether the substrate side chain is a ketone group or a hydroxyl group with different configurations, the N161F mutant can bind to the substrate through hydrophobic adsorption and π-π stacking interactions, broadening the enzyme's substrate spectrum.

[0098] Test Example 7: Tolerance Test to Gastrointestinal Proteases This test case simulates the physiological environment of the animal digestive tract. Wild-type enzymes and mutant enzymes were treated with pepsin and trypsin, and their survival rate under protease hydrolysis was measured to verify the effect of rigidification of molecular structure on improving resistance to protease degradation.

[0099] Experimental Procedure: S1. Prepare a pH 3.0 glycine hydrochloride buffer containing 0.5 mg / mL pepsin and a pH 7.5 tris(hydroxymethyl)aminomethane (HCl) buffer containing 0.5 mg / mL trypsin. The purified enzyme solutions A59P, N161F, and A59P / N161F prepared in Example 2, and the wild-type enzyme solution prepared in Comparative Example 1 were selected as test subjects.

[0100] S2. Mix each enzyme solution with the pepsin treatment solution at a volume ratio of 1:1 and incubate in a constant temperature water bath at 37°C for 60 minutes; separately mix each enzyme solution with the trypsin treatment solution at a volume ratio of 1:1 and incubate at the same temperature for 60 minutes.

[0101] S3. Set up a control group by mixing each enzyme solution with the corresponding pH buffer that does not contain protease in the same proportion and incubating them under the same conditions.

[0102] S4. After incubation, pH 9.0 buffer was added to the pepsin treatment group to adjust the pH value to inactivate pepsin, and the protease inhibitor PMSF was added to the trypsin treatment group to terminate the hydrolysis reaction; the residual specific enzyme activity of each group of samples was determined according to the method in Test Example 3.

[0103] S5. Calculate the residual enzyme activity rate, which is equal to the enzyme activity of the protease-treated group divided by the enzyme activity of the corresponding control group. The result is expressed as a percentage.

[0104] Experimental data: The residual enzyme activity of each enzyme sample after trypsin treatment is shown in Table 7.

[0105] Table 7 Residual enzyme activity after treatment with pepsin and trypsin Results analysis: According to the data in Table 7, wild-type enzymes exhibit weak resistance to digestive tract proteases. After 60 minutes of pepsin treatment, the residual activity of wild-type enzymes was 18.3%, while after trypsin treatment, the residual activity was 29.5%. These data indicate that wild-type enzymes possess numerous flexible regions on their surface that are easily recognized by proteases, leading to the hydrolysis and breakage of the enzyme protein backbone, resulting in inactivation.

[0106] The introduction of the A59P mutation enhanced the enzyme's tolerance to both proteases. The A59P mutant exhibited a residual rate of 72.4% after pepsin treatment and 81.2% after trypsin treatment, representing increases of nearly 4-fold and 2.75-fold, respectively, compared to the wild type. The introduction of proline at position 59 increased the rigidity of the polypeptide chain, restricting conformational freedom in local regions and reducing thermal fluctuations. This rigidity effect reduced the exposure probability of potential cleavage sites, making it difficult for them to enter the active pocket of the protease, thereby blocking the hydrolysis reaction.

[0107] The residual rates of the single-point mutant N161F were 21.6% and 33.7%, respectively, showing no significant improvement compared to the wild type. This indicates that the N161F site is mainly involved in the substrate binding function of the active site, and its internal hydrophobic environment has little impact on the surface attack behavior of the protease.

[0108] The residual rates of the two-point mutant A59P / N161F after treatment with pepsin and trypsin were 76.8% and 85.4%, respectively. This result confirms that the structural rigidity conferred by the A59P mutation and the catalytic core optimization brought about by the N161F mutation can be superimposed. High tolerance indicates that when this enzyme preparation is used as a feed additive, it can withstand the enzymatic environment in the animal's digestive tract, ensuring that the active enzyme reaches the small intestine to exert its detoxification effect.

[0109] Appendix: SEQIDNO:1: ATGCGGACAAGGTCGACCCTCACCGACAGGAATGGGATCACCTGGTACTATGAGCAAGAAGGATCAGGTCCTCATGTGGTTCTCATCCCTGACGGATTGGGAGAGTGCCACATGATGGACAAGCCCATGTCACTGATAGCAGCCAAGGGATTTACTTGCACCACTTTCGATATGGCTGGTATGTCGAGGTCATGGGATGCCCCGCCGGAGACATACCAAGATGTCACGGCCCAGAAGCTAGCTAGCTACGTCATCAGTATCCTCGACGAGCTGCATATCGACTATGCTACGTTCTGGGGTTGTAGCTCGGGAGGTGCGACCGTGCTGGCGTTGGCCGCTGACTACCCCGAACGTATGCGGAACGGGCTACCGCATGAAGTTCCGACGGCCGCTAGCCCTCTGTTCGGTCAGCTCTTAAAACTGGCCGAGATGGAAGACGAGGCTATCGTGAAGATGCTGAGCGAGGAAATGCCTCAGACGAACTTTGGTCACGACTTGACAGCATGGCATGAGCTAGGTGAGGAGGTTCACGCAAGGCTGCGGAAGAACTATCCTCGCTGGGCCCGCGGCTACCCTCATACTCTGCCGCTCTCTTCCCCTACTAGTAAAGAGGACCTGACGAAGCGACCTCTGGACTGGACAGTTGGTGGGGATACACCGACTCGTGTGTTCTTCGATAATATAGTCACGGCCAGCAAAGCTGGTATCCCCATAGGAACGCTCCCAGGCATGCACTTCCCGTATCTATCGCACCCGGAGGTTCTGGCTGAGCATATCGTCGATACAACTCGAAAGTACCTGCACCACCACCACCACCACTAA SEQIDNO:2: MRTRSTLTDRNGITWYYEQEGSGPHVVLIPDGLGECHMMDKPMSLIAAKGFTCTTFDMAGMSRSWDAPPETYQDVTAQKLASYVISILDELHIDYATFWGCSSGGATVLALAADYPERMRNGLPHEVPTAASPLFGQLLKLAEMEDEAIVKMLSEEMPQTNFGHDLTAWHELGEEVHARLRKNYPRWARGYPHTLPLSSPTSKEDLTKRPLDWTVGGDTPTRVFFDNIVTASKAGIPIGTLPGMHFPYLSHPEVLAEHIVDTTRKYLHHHHHH* SEQIDNO:3: MRTRSTLTDRNGITWYYEQEGSGPHVVLIPDGLGECHMMDKPMSLIAAKGFTCTTFDMPGMSRSWDAPPETYQDVTAQKLASYVISILDELHIDYATFWGCSSGGATVLALAADYPERMRNGLPHEVPTAASPLFGQLLKLAEMEDEAIVKMLSEEMPQTNFGHDLTAWHELGEEVHARLRKNYPRWARGYPHTLPLSSPTSKEDLTKRPLDWTVGGDTPTRVFFDNIVTASKAGIPIGTLPGMHFPYLSHPEVLAEHIVDTTRKYLHHHHHH* SEQIDNO:4: MRTRSTLTDRNGITWYYEQEGSGPHVVLIPDGLGECHMMDKPMSLIAAKGFTCTTFDMAGMSRSWDAPPETYQDVTAQKLASYVISILDELHIDYATFWGCSSGGATVLALAADYPERMRNGLPHEVPTAASPLFGQLLKLAEMEDEAIVKMLSEEMPQTFFGHDLTAWHELGEEVHARLRKNYPRWARGYPHTLPLSSPTSKEDLTKRPLDWTVGGDTPTRVFFDNIVTASKAGIPIGTLPGMHFPYLSHPEVLAEHIVDTTRKYLHHHHHH* SEQIDNO:5: MRTRSTLTDRNGITWYYEQEGSGPHVVLIPDGLGECHMMDKPMSLIAAKGFTCTTFDMPGMSRSWDAPPETYQDVTAQKLASYVISILDELHIDYATFWGCSSGGATVLALAADYPERMRNGLPHEVPTAASPLFGQLLKLAEMEDEAIVKMLSEEMPQTFFGHDLTAWHELGEEVHARLRKNYPRWARGYPHTLPLSSPTSKEDLTKRPLDWTVGGDTPTRVFFDNIVTASKAGIPIGTLPGMHFPYLSHPEVLAEHIVDTTRKYLHHHHHH*

Claims

1. A preparation process for an acid-tolerant zearalenone lactone hydrolase, characterized in that, Includes the following steps: Provides a recombinant Escherichia coli expression strain containing a gene encoding a mutant protein, wherein the mutant protein is based on the wild-type enzyme shown in SEQ ID NO:2, by mutating alanine at position 59 to proline and / or mutating asparagine at position 161 to phenylalanine, and wherein the mutant protein is expressed as a fusion protein with an affinity tag. The recombinant Escherichia coli expression strain was inoculated into liquid culture medium for expansion culture until the OD600 value of the bacterial solution reached 0.6-0.8; Add an inducer to the liquid culture medium and induce culture at 16-37℃ for 4-20 hours to obtain a fermentation broth containing the fusion protein; Collect the bacterial cells in the fermentation broth, and separate the supernatant after cell wall disruption treatment; The supernatant was purified by affinity chromatography, and the eluent containing the fusion protein was collected to obtain the acid-tolerant zearalenone lactone hydrolase.

2. The preparation process of an acid-adapted zearalenone lactone hydrolase according to claim 1, characterized in that, The amino acid sequence of the mutant protein is selected from one of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:

5.

3. The preparation process of an acid-adapted zearalenone lactone hydrolase according to claim 1, characterized in that, The step of providing a recombinant Escherichia coli expression strain containing a gene encoding a mutant protein includes: Using a plasmid containing the gene shown in SEQ ID NO:1 as a template, PCR amplification and site-directed mutagenesis were performed using specific primers. The amplification product was digested with DpnI and then transformed into competent cells. The recombinant Escherichia coli expression strain was obtained by screening. The recombinant Escherichia coli expression strain was constructed by inserting the gene encoding the mutant protein into the pET-22b(+) expression vector and transforming it into Escherichia coli BL21(DE3). The pET-22b(+) expression vector confers a C-terminal histidine affinity tag to the mutant protein.

4. The preparation process of an acid-adapted zearalenone lactone hydrolase according to claim 1, characterized in that, In the step of inoculating the recombinant Escherichia coli expression strain into liquid culture medium for expansion culture, the inoculation amount is 0.5%-5% of the volume of liquid culture medium, the culture temperature is 37℃, and the rotation speed is 180-220 rpm.

5. The preparation process of an acid-adapted zearalenone lactone hydrolase according to claim 1, characterized in that, The inducer is isopropyl-β-D-thiogalactoside, with a final concentration in the culture medium of 0.1 mM to 1.0 mM.

6. The preparation process of an acid-adapted zearalenone lactone hydrolase according to claim 5, characterized in that, The specific conditions for the induction culture are selected from any of the following: Condition A: The final concentration of isopropyl-β-D-thiogalactoside was 0.05mM-0.5mM, the induction temperature was 16℃, and the induction time was 20 hours; Condition B: The final concentration of isopropyl-β-D-thiogalactoside was 0.5mM-1.0mM, the induction temperature was 25℃, and the induction time was 6 hours; Condition C: The final concentration of isopropyl-β-D-thiogalactoside was 0.5mM-1.0mM, the induction temperature was 37℃, and the induction time was 4 hours.

7. The preparation process of an acid-adapted zearalenone lactone hydrolase according to claim 1, characterized in that, The cell disruption process employs ultrasonic disruption, and the lysis buffer used contains 20-100 mM tris(hydroxymethyl)aminomethane and 100-300 mM NaCl, with a pH of 7.0-8.

0.

8. The preparation process of an acid-adapted zearalenone lactone hydrolase according to claim 1, characterized in that, The affinity tag is a histidine tag, and the affinity chromatography purification uses a nickel ion affinity chromatography column. The buffer system used is as follows: Equilibration buffer: Contains 50 mM tris(hydroxymethyl)aminomethane and 300-600 mM NaCl, pH 7.0-8.0; Washing buffer: contains 20-50 mM imidazole, 50 mM tris(hydroxymethyl)aminomethane and 300-600 mM NaCl, pH 7.0-8.0; Elution buffer: Contains 300-600mM imidazole, 50mM tris(hydroxymethyl)aminomethane and 300-600mM NaCl, pH 7.0-8.

0.

9. The preparation process of an acid-adapted zearalenone lactone hydrolase according to claim 1, characterized in that, The preparation process, after collecting the eluent containing the fusion protein, also includes a dialysis step: first, the eluent is dialyzed in a 20-100mM tris(hydroxymethyl)aminomethane buffer containing 5-20mM ethylenediaminetetraacetic acid, and then dialyzed in a 20-100mM tris(hydroxymethyl)aminomethane buffer without ethylenediaminetetraacetic acid.

10. The preparation process of an acid-adapted zearalenone lactone hydrolase according to claim 9, characterized in that, All dialysis steps were performed at 4°C. The dialysis time with ethylenediaminetetraacetic acid (EDTA) was 8-16 hours, and the dialysis time without EDTA was 4-8 hours, with the dialysis solution being changed once during the process.

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