A group of ochratoxin a degrading enzyme mutants and application thereof

Abstract

The application discloses a group of ochratoxin A degrading enzyme high-efficiency mutants and application thereof. The amino acid sequence of the ochratoxin A degrading enzyme high-efficiency mutant is shown in SEQ ID NO. 3 or SEQ ID NO. 5. The preferred nucleotide sequence is shown in SEQ ID NO. 4 or SEQ ID NO. 6. The kcat / Km value of the mutant reaches 26.8 times and 72.9 times of the wild type respectively, and is 0.061982 s ‑1 muM ‑1 and 0.168442 s ‑1 muM ‑1 , the catalytic efficiency to OTA is significantly enhanced, the degradation efficiency to OTA is improved, and the application has important significance for food safety problems and detoxification in food processing.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of enzyme engineering and food safety, and particularly relates to a group of ochratoxin A degrading enzyme ADH3 active high-efficiency mutants and application thereof. BACKGROUND

[0002] Ochratoxin A (OTA) is a strong carcinogenic, teratogenic and mutagenic toxin produced by Aspergillus and Penicillium fungi, which widely pollutes grains, feed and food, and seriously threatens human and animal health. Traditional OTA removal methods include physical adsorption, chemical degradation and biodegradation, among which the biodegradation method is concerned due to its high efficiency, green and specificity. Alcohol dehydrogenase 3 (ADH3) as an OTA degrading enzyme can generate non-toxic OTa by hydrolyzing the amide bond of OTA, and has broad application prospects.

[0003] In recent years, the mutation optimization of enzymes through directed evolution technology has become an effective strategy to improve enzyme activity. The construction of saturated mutation library combined with high-throughput screening method can quickly identify mutants with enhanced activity and improve the catalytic efficiency of enzymes. Although the role of ADH3 in OTA degradation has been reported, the activity optimization mutants still need to be further developed to achieve higher degradation rate and improve the application potential and range. Improving the degradation efficiency of ADH3 to OTA is of great significance for food safety detection and detoxification in food processing. SUMMARY

[0004] Based on the above problems, the present application provides a group of ochratoxin A degrading enzyme high-efficiency mutants and application thereof in efficiently degrading ochratoxin A, which provides new biological materials for the development of biological agriculture and related industries and other biological industries.

[0005] The first object of the present application is to provide a group of ochratoxin A degrading enzyme high-efficiency mutants, and the amino acid sequence of the mutant is shown in SEQ ID NO. 3 or SEQ ID NO. 5.

[0006] The second object of the present application is to provide a gene encoding the above-mentioned mutant.

[0007] Preferably, the nucleotide sequence is shown in SEQ ID NO. 4 or SEQ ID NO. 6.

[0008] The third object of the present application is to provide a recombinant expression vector or a recombinant engineering bacterium containing the above-mentioned gene.

[0009] The fourth object of the present application is to provide a preparation method of the above-mentioned mutant, which comprises using the above-mentioned recombinant expression vector or recombinant engineering bacterium.

[0010] A fifth object of the present invention is to provide a reagent containing the above-mentioned mutant as an active ingredient.

[0011] A sixth object of the present invention is to provide a method for degrading ochratoxin A for non-disease diagnosis and treatment purposes, comprising using the above-described mutant or the above-described reagent.

[0012] Preferably, the method includes the following steps: mixing the above-mentioned mutant or the above-mentioned reagent with a sample containing ochratoxin A and reacting it at pH 5-9 and 20-60°C.

[0013] Preferably, the concentration of the mutant is 1-40 μg / mL.

[0014] A seventh object of the present invention is to provide the use of the mutant described above, the gene described above, the recombinant expression vector or recombinant engineered bacteria described above, the reagent described above, or the method described above in the degradation of ochratoxin A for non-disease diagnosis and treatment purposes.

[0015] Advantages of this invention:

[0016] This invention provides a group of highly efficient mutants of ochratoxin A (OTA) degrading enzymes. The enzyme activities of the mutants ADH3-S88E-Q93R-A164G-F299Y-Q345S and ADH3-S88E-A164G-F299Y-Q345S are 26.8 times and 72.9 times that of the wild-type ADH3, respectively. The catalytic efficiency for OTA is significantly enhanced, thereby improving the degradation efficiency of OTA. This is of great significance for food safety issues and detoxification in food processing. Attached Figure Description

[0017] Figure 1 This is a comparison of the degradation curves and kinetic parameters of ADH3 wild-type and mutant at different initial OTA concentrations. Detailed Implementation

[0018] To achieve this invention, the present invention adopts the following technical solution:

[0019] DNA sequences were designed based on the protein sequences and synthesized by a gene synthesis company. Wild-type ADH3 protein (amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2), mutant ADH3-S88E-Q93R-A164G-F299Y-Q345S protein (amino acid sequence as shown in SEQ ID NO.3, nucleotide sequence as shown in SEQ ID NO.4), and mutant ADH3-S88E-A164G-F299Y-Q345S protein (amino acid sequence as shown in SEQ ID NO.5, nucleotide sequence as shown in SEQ ID NO.6) were generated using genetic engineering methods. Based on these proteins, a method for degrading OTA was established. This method can be applied to the detoxification of the common fungal toxin ochratoxin (OTA). SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5 all lack the signal peptide MPIRRRFASLLLLACAPAWA.

[0020] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0021] Example 1: Construction of expression vector

[0022] The wild-type nucleotide sequence of ADH3 is shown in SEQ ID NO.2, the nucleotide sequence of ADH3-S88E-Q93R-A164G-F299Y-Q345S is shown in SEQ ID NO.4, and the nucleotide sequence of ADH3-S88E-A164G-F299Y-Q345S is shown in SEQ ID NO.6.

[0023] All the above sequences were synthesized by a commercial company (Suzhou Silicon-based Biotechnology Co., Ltd.), and the genes were inserted into the Pet.M.3C vector (Suzhou Silicon-based Biotechnology Co., Ltd., catalog number Pet.M.3C: G080-2) using BamHI and EcoRI restriction sites to obtain recombinant plasmids. The recombinant plasmids were transformed into E. coli DH5α competent cells and plated on LB agar plates containing 100 μg / mL ampicillin (Amp) resistance. The cells were incubated at 37°C for 16 hours to obtain uniform single colonies. Single colonies were picked and incubated overnight at 37°C in 50 mL LB liquid medium. Colony PCR was performed using the universal primers T7 and T7-ter for the Pet.M.3C vector to verify the colonies. Positive clones were then extracted and sequenced for verification. The sequenced and verified recombinant plasmids were used in subsequent experiments.

[0024] Example 2: Expression of the target protein

[0025] S1. Transform E. coli BL21(DE3) competent cells with the recombinant plasmid that was correctly sequenced in Example 1. Add 1 μL of the recombinant plasmid to the competent cells, incubate on ice for about 30 minutes, then heat shock at 42°C for 90 seconds, and then incubate on ice for 5 minutes.

[0026] S2. Take 200 μL of competent cells transformed in step S1 into 200 μL of LB liquid medium resistant to Amp (100 μg / mL), and incubate at 220 rpm and 37℃ for about one hour. Spread the bacterial culture onto LB medium plates resistant to Amp (100 μg / mL) and incubate overnight at 37℃.

[0027] S3. Select a single colony in good condition from the resistance plate and inoculate it into 7 mL of LB liquid medium containing Amp (100 μg / mL) resistance. Incubate at 200 rpm and 37°C for 5 h. Transfer 4 mL of the cultured bacterial solution to 1 L of LB liquid medium containing Amp (100 μg / mL) resistance, add 2 mL of 100 mM zinc chloride solution, and incubate at 200 rpm and 37°C until OD reaches 0.5. 600 The concentration is 0.6-0.8. After standing at 4-8℃ for a period of time, the culture medium is cooled to 16℃, and IPTG is added to induce protein expression (the final concentration of IPTG in LB liquid medium is 0.3 mM). Expression is induced at 200 rpm and 16℃ for 16-18 hours.

[0028] Example 3: Purification of the Target Protein

[0029] S1. After induction, the bacterial culture was centrifuged at 4000 rpm for 15 min at 4℃. The supernatant was discarded, and the bacterial cells were resuspended in 25 mL of PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, the same below).

[0030] S2. Add 300 μL of protease inhibitor PMSF (100 mM dissolved in isopropanol) to the resuspended bacterial culture, and sonicate the bacterial cells (70% amplitude, sonicate for 5 s, pause for 5 s, for a total of 3 min; repeat the sonication 3 times).

[0031] S3. Centrifuge at 12000 rpm for 20 min at 4℃ to break up the bacterial cells.

[0032] S4. Pour the supernatant obtained in S3 into a Ni affinity column placed at 4℃, stir thoroughly to mix evenly, and stir again every 10 minutes to ensure that the protein is fully bound to the affinity column. The total binding time is about 1 hour.

[0033] S5. After binding for a period of time, the supernatant was drained and the affinity column was washed 5 times with 1×wash buffer (20 mM Tris-HCl, 500 mM NaCl, 15 mM Imidazole, pH 7.4).

[0034] S6. Add 3 mL of elution buffer (20 mM Tris-HCl, 500 mM NaCl, 500 mM Midazole, pH 7.4) to the column, stir to mix, and elute for 5 min. Collect the eluent on ice.

[0035] S7. Add 2.5 mL of elution buffer to the desalting column. After it has completely entered the column, add 3 mL of PBS buffer. The liquid flowing out at this point is the ADH3 protein in PBS buffer. Protein expression and purification were identified by SDS-PAGE, yielding wild-type ADH3 protein and mutant proteins ADH3-S88E-Q93R-A164G-F299Y-Q345S and ADH3-S88E-A164G-F299Y-Q345S.

[0036] Example 4: Comparison of methods and degradation kinetic parameters for OTA degradation between wild-type and mutant ADH3 cells

[0037] The proteins purified in Example 3 were diluted to working concentrations using PBS buffer at pH 7.4. To ensure the initial reaction rate remained within the linear range, the proteins were diluted to the following working concentrations and reaction times were set accordingly: ADH3 wild-type protein was diluted to 40 μg / mL, and the reaction time was set to 3 minutes; mutant ADH3-S88E-Q93R-A164G-F299Y-Q345S protein was diluted to 1 μg / mL, and the reaction time was set to 7 minutes; mutant ADH3-S88E-A164G-F299Y-Q345S protein was diluted to 1 μg / mL, and the reaction time was set to 3 minutes. OTA standard (1 mg / mL, methanol) was diluted using the same PBS buffer to prepare a series of substrate solutions with concentrations of 0.5, 1, 2, and 5 μM. The enzymatic reaction was carried out in a 150 μL system: 75 μL of the above-mentioned concentration of OTA substrate solution was added to the reaction tube, followed by a rapid addition of 75 μL of the corresponding concentration of protein working solution to initiate the reaction. The reaction system was immediately placed in a 40°C metal bath and incubated for the set time to ensure that the substrate consumption was in the linear phase. After the set time was reached, 150 μL of HPLC-grade methanol was added immediately to terminate the reaction, and the mixture was vortexed. The sample was filtered through a 0.22 μm filter membrane, aliquoted into sample vials, and the content of the degradation product Otα was detected by high-performance liquid chromatography (HPLC). HPLC conditions were as follows: C18 column (4.6 × 250 mm, 5 µm); mobile phase A was ultrapure water, and mobile phase B was acetonitrile:acetic acid:water (96:2:2, v / v / v); isocratic elution, with mobile phases A and B each accounting for 50% of the volume during elution; flow rate 1.0 mL / min; run time 25 min; fluorescence detector parameters were excitation wavelength 333 nm and emission wavelength 477 nm; injection volume 20 μL.

[0038] The initial reaction rate (v) at each substrate OTA concentration was calculated based on the concentration of the degradation product Otα. The maximum reaction rate (Vmax) and the Michaelis-Menten constant (Km) were obtained by nonlinear fitting using the Michaelis-Menten equation, with substrate concentration ([S], μM) as the x-axis and reaction rate (v, μmol·min⁻¹·mg⁻¹) as the y-axis. The catalytic constant kcat was calculated using the formula kcat = (Vmax × MW × 10⁻¹) / (Vmax × MW × 10⁻¹). -3 The formula is calculated as MW / 60, where MW is the molecular weight of the enzyme (g·mol⁻¹). This formula obtains the s⁻¹ unit based on the enzyme molar concentration by converting the time unit from min⁻¹ to s⁻¹ (i.e., dividing by 60). The catalytic efficiency kcat / Km value is ultimately used as the core evaluation index. The measurement results show ( Figure 1The kcat / Km value for OTA degradation by the wild-type ADH3 protein was 0.002311 s⁻¹μM⁻¹. In contrast, the kcat / Km values ​​of the mutants ADH3-S88E-Q93R-A164G-F299Y-Q345S and ADH3-S88E-A164G-F299Y-Q345S were 26.8 times and 72.9 times higher (0.061982 s⁻¹μM⁻¹ and 0.168442 s⁻¹μM⁻¹, respectively), demonstrating that the catalytic efficiency of the mutants ADH3-S88E-Q93R-A164G-F299Y-Q345S for OTA was significantly enhanced.

[0039] SEQ ID NO.1 (ADH3 wild-type amino acid sequence)

[0040] EPVAVQCGRLFDARSGQLKGPHTLLVADGRIRQVLPGTGADAAGARVVDLGDKVCLPGWTDLHVHLGSQSSPQSYSEDFRLDPVDHAFRAVGYAEKTLMAGFTSVRDLGGEVSPHLRDAINQGLVRGPRIFAAGKSIATTGGHADPTNGWNERLAHLVGAPGPAEGVVNSVDEARQAVRQRYKEGSDLIKITATGGVLSYARS GDAPQFTVDEIKAVVDTARDYGFRVAAHAHGTEGMKRAVQAGVTSIEHGTYMDDEVMRLMKQHGTWYVPTFYAGRFVTEKAAIDGYFPEVVRPKAARIGALI SQTAAKAYRNGVRIAFGTDQGVGPHGDNAREFVYMVEAGIPAAYALQAATVHAAQVLGVDDQGVLEPGKRADVIALAGNPLEDINAVLDVRFVMKDGVIYKQ

[0041] SEQ ID NO.2 (ADH3 wild-type nucleotide sequence)

[0042]

[0043] SEQ ID NO. 3 (Amino acid sequence of ADH3-S88E-Q93R-A164G-F299Y-Q345S mutant)

[0044] EPVAVQCGRLFDARSGQLKGPHTLLVADGRIRQVLPGTGADAAGARVVDLGDKVCLPGWTDLHVHLGEQSSPRSYSEDFRLDPVDHAFRAVGYAEKTLMAGFTSVRDLGGEVSPHLRDAINQGLVRGPRIFAAGKSIATTGGHGDPTNGWNERLAHLVGAPGPAEGVVNSVDEARQAVRQRYKEGSDLIKITATGGVLSYARSGDAPQFTVDEIKAVVDTARDYGFRVAAHAHGTEGMKRAVQAGVTSIEHGTYMDDEVMRLMKQHGTWYVPTFYAGRYVTEKAAIDGYFPEVVRPKAARIGALISQTAAKAYRNGVRIAFGTDSGVGPHGDNAREFVYMVEAGIPAAYALQAATVHAAQVLVDDQGVLEPGKRADVIALAGNPLEDINAVLDVRFVMKDGVIYKQ

[0045] SEQ ID NO. 4 (Nucleotide sequence of ADH3-S88E-Q93R-A164G-F299Y-Q345S mutant)

[0046]

[0047] SEQ ID NO.5 (Amino acid sequence of ADH3 - S88E - A164G - F299Y - Q345S mutant)

[0048] EPVAVQCGRLFDARSGQLKGPHTLLVADGRIRQVLPGTGADAAGARVVDLGDKVCLPGWTDLHVHLGEQSSPQSYSEDFRLDPVDHAFRAVGYAEKTLMAGFTSVRDLGGEVSPHLRDAINQGLVRGPRIFAAGKSIATTGGHGDPTNGWNERLAHLVGAPGPAEGVVNSVDEARQAVRQRYKEGSDLIKITATGGVLSYARSGDAPQFTVDEIKAVVDTARDYGFRVAAHAHGTEGMKRAVQAGVTSIEHGTYMDDEVMRLMKQHGTWYVPTFYAGRYVTEKAAIDGYFPEVVRPKAARIGALISQTAAKAYRNGVRIAFGTDSGVGPHGDNAREFVYMVEAGIPAAYALQAATVHAAQVLGVDDQGVLEPGKRADVIALAGNPLEDINAVLDVRFVMKDGVIYKQ

[0049] SEQ ID NO.6 (Nucleotide sequence of ADH3 - S88E - A164G - F299Y - Q345S mutant)

[0050]

Claims

1. A group of ochratoxin A degrading enzyme mutants, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO. 3 or SEQ ID NO.

5.

2. A gene encoding the mutant of claim 1.

3. The gene of claim 2, wherein, The nucleotide sequence is shown in SEQ ID NO. 4 or SEQ ID NO.

6.

4. A recombinant expression vector or a recombinant engineering bacterium containing the gene of claim 2.

5. A method of producing the mutant of claim 1, characterized by, 5. Use of the recombinant expression vector or the recombinant engineering bacterium of claim 4.

6. A reagent for degrading ochratoxin A for a purpose other than disease diagnosis and treatment, characterized by, 6. An agent containing the mutant of claim 1 as an active ingredient.

7. A method of degrading ochratoxin A for non-disease diagnostic and therapeutic purposes, characterized by, 7. Use of the mutant of claim 1 or the agent of claim 6.

8. The method of claim 7, wherein, 8. A method for degrading ochratoxin A, comprising the step of mixing the mutant of claim 1 or the agent of claim 6 with a sample containing ochratoxin A and reacting at pH 5-9 and 20-60°C.

9. The method of claim 8, wherein, 9. The concentration of the mutant before mixing with the sample is 1-40 μg / mL.

10. Use of the mutant of claim 1, the gene of claim 2, the recombinant expression vector or the recombinant engineering bacterium of claim 4, or the agent of claim 6 for degrading ochratoxin A for purposes other than disease diagnosis and treatment.

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