High-efficiency mutant of ochratoxin A degrading enzyme and application of high-efficiency mutant

Abstract

The invention discloses a group of ochratoxin A degrading enzyme efficient mutants and application thereof. The amino acid sequence of the high-efficiency mutant of the ochratoxin A degrading enzyme is as shown in SEQ ID NO. 3 or SEQ ID NO. 5. And the preferable nucleotide sequence is as 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 kcat / Km value of a wild type respectively and is 0.061982 s <-1 > mu M <-1 > and 0.168442 s <-1 > mu M <-1 > respectively, the OTA catalytic efficiency is remarkably enhanced, the OTA degradation efficiency is improved, and the mutant has important significance on 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 high-activity mutants of ochratoxin A degrading enzyme ADH3 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, greenness 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 a saturated mutant 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, further development of its activity optimization mutants is still needed to achieve higher degradation rate and improve application potential and range. Improving the degradation efficiency of ADH3 on 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 high-activity mutants of ochratoxin A degrading enzyme and application thereof in efficient degradation of 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 high-activity mutants of ochratoxin A degrading enzyme, 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 application is to provide an agent containing the mutant described above as an active ingredient.

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

[0012] Preferably, the method comprises the step of mixing the mutant described above or the agent described above with a sample containing ochratoxin A and reacting 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 application is to provide the use of the mutant described above, the gene described above, the recombinant expression vector or the recombinant engineering bacteria described above, the agent described above or the method described above in degrading ochratoxin A for non-disease diagnosis and treatment purposes.

[0015] Advantages of the present application: The present application provides a group of ochratoxin A degrading enzyme mutants with high efficiency, the enzyme activity of the mutants ADH3-S88E-Q93R-A164G-F299Y-Q345S and ADH3-S88E-A164G-F299Y-Q345S is 26.8 times and 72.9 times that of wild-type ADH3, the catalytic efficiency of OTA is significantly enhanced, the degradation efficiency of OTA is improved, which is of great significance for food safety problems and detoxification in food processing. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is the degradation curve and kinetic parameter comparison of ADH3 wild type and mutant under different initial concentrations of OTA. DETAILED DESCRIPTION

[0017] To achieve the present application, the present application adopts the following technical solutions: The DNA sequence was designed according to the protein sequence and was synthesized by a gene synthesis company. The ADH3 wild-type protein (amino acid sequence as shown in SEQ ID NO. 1, nucleotide sequence as shown in SEQ ID NO. 2), ADH3-S88E-Q93R-A164G-F299Y-Q345S mutant protein (amino acid sequence as shown in SEQ ID NO. 3, nucleotide sequence as shown in SEQ ID NO. 4) and ADH3-S88E-A164G-F299Y-Q345S mutant protein (amino acid sequence as shown in SEQ ID NO. 5, nucleotide sequence as shown in SEQ ID NO. 6) were generated by genetic engineering method. Based on the above-mentioned proteins, a method for degrading OTA was established. The method can be applied to the detoxification of common fungal toxin ochratoxin (OTA). SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO. 5 do not contain the signal peptide MPIRRRFASLLLLACAPAWA.

[0018] The following examples are further illustrations of the application and are not intended to limit the application.

[0019] Example 1: Construction of expression vector The ADH3 wild-type nucleotide sequence is shown in SEQ ID NO. 2, the ADH3-S88E-Q93R-A164G-F299Y-Q345S nucleotide sequence is shown in SEQ ID NO. 4, and the ADH3-S88E-A164G-F299Y-Q345S nucleotide sequence is shown in SEQ ID NO. 6.

[0020] The above-mentioned sequences were synthesized by a commercial company (Suzhou Silic Biotech Co., Ltd.), and the genes were inserted into the Pet.M.3C vector (Suzhou Silic Biotech Co., Ltd., item number Pet.M.3C:G080-2) using BamHI and EcoRI enzyme cutting sites to obtain recombinant plasmids. The recombinant plasmids were transformed into E. coli DH5α competent cells, spread on LB medium plates containing 100 μg / mL ampicillin (Amp) resistance, and incubated at 37°C for 16 hours to obtain uniform growth of single colonies. Single colonies were picked and cultured in 50 mL LB liquid medium at 37°C overnight. The single colonies were verified by bacterial liquid PCR using the Pet.M.3C vector universal primers T7 and T7-ter. After obtaining positive clones, the plasmids were extracted and sequenced. The recombinant plasmids verified by sequencing were used for subsequent experiments.

[0021] Example 2: Expression of target protein S1. Transform the recombinant plasmid sequenced correctly in Example 1 into E. coli BL21 (DE3) competent cells respectively. Take 1 μL of the recombinant plasmid and add it to the competent cells, and then place them in an ice bath for about 30 minutes. Then, heat shock them at 42°C for 90 seconds, and then place them in an ice bath for 5 minutes.

[0022] S2. Take 200 μL of the competent cells transformed in step S1 and add them to 200 μL of LB liquid medium resistant to Amp (100 μg / mL), and then incubate them at 220 rpm and 37°C for about one hour. Then, spread the bacterial liquid on an LB medium plate resistant to Amp (100 μg / mL), and then incubate it in a 37°C constant temperature incubator overnight.

[0023] S3. Pick a single colony with a good state on the resistant plate and inoculate it in 7 mL of LB liquid medium containing Amp (100 μg / mL), and then incubate it at 200 rpm and 37°C for 5 hours. Then, transfer 4 mL of the cultured bacterial liquid to 1 L of LB liquid medium containing Amp (100 μg / mL), and then add 2 mL of 100 mM zinc chloride solution, and then incubate it at 200 rpm and 37°C until the OD 600 of the bacterial liquid is 0.6-0.8. Then, cool the medium to 16°C by placing it at 4-8°C for a period of time, and then add IPTG to induce protein expression (the final concentration of IPTG in the LB liquid medium is 0.3 mM), and then incubate it at 200 rpm and 16°C for 16-18 hours.

[0024] Example 3: Purification of the target protein S1. Centrifuge the induced bacterial liquid at 4000 rpm and 4°C for 15 minutes, discard the supernatant, and then resuspend the bacterial cells with 25 mL of PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, same below).

[0025] S2. Add 300 μL of protease inhibitor PMSF (100 mM dissolved in isopropyl alcohol) to the resuspended bacterial liquid, and then break the bacterial cells by ultrasonic waves (70% amplitude, 5 seconds of ultrasonic wave and 5 seconds of pause, for a total of 3 minutes; repeat the breaking 3 times).

[0026] S3. Centrifuge the broken bacterial cells at 12000 rpm and 4°C for 20 minutes.

[0027] S4. Pour the supernatant obtained in S3 into a Ni affinity column placed at 4°C, and then mix it well by stirring every 10 minutes to make the protein bind to the affinity column fully, and then the total binding time is about 1 hour.

[0028] S5. After a period of time, the supernatant was collected and the column was washed with 1 x wash buffer (20 mM Tris-HCl, 500 mM NaCl, 15 mM Imidazole, pH 7.4) for 5 times.

[0029] S6. 3 mL elution buffer (20 mM Tris-HCl, 500 mM NaCl, 500 mM Imidazole, pH 7.4) was added to the column, mixed well and eluted for 5 min, and the eluate was collected on ice.

[0030] S7. 2.5 mL eluate was added to the desalting column, and then 3 mL PBS buffer was added. The liquid flowing out below was ADH3 protein in PBS buffer. The protein expression and purification were identified by SDS-PAGE, and ADH3 wild-type protein and mutant proteins ADH3-S88E-Q93R-A164G-F299Y-Q345S and ADH3-S88E-A164G-F299Y-Q345S were obtained.

[0031] Example 4: Method and comparison of degradation kinetics parameters of ADH3 wild-type and mutant for degrading OTA The proteins obtained from the purification of Example 3 were diluted to working concentrations using PBS buffer at pH 7.4. To ensure that the initial reaction rate was in the linear range, each protein was diluted to the following working concentrations and the corresponding reaction time was set: 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. The OTA standard (1 mg / mL, solvent: methanol) was diluted using the same PBS buffer to prepare a series of substrate solutions at concentrations of 0.5, 1, 2, and 5 μM. The enzymatic reaction was carried out in a total volume of 150 μL: 75 μL of the OTA substrate solution at the above-mentioned concentration was added to a reaction tube, and then 75 μL of the protein working solution at the corresponding concentration was quickly added to start the reaction. The reaction system was immediately placed in a 40°C metal bath, and incubated for the time set as above to ensure that the substrate consumption was in the linear phase. After reaching the set time, the reaction was terminated by immediately adding 150 μL of HPLC-grade methanol and vortexing to mix. The sample was filtered through a 0.22 μm filter membrane, then aliquoted into a sample loading bottle, and the content of degradation product Otα was detected using high-performance liquid chromatography (HPLC). The HPLC conditions were as follows: C18 chromatographic column (4.6 × 250 mm, 5 μm); mobile phase A: ultrapure water, mobile phase B: acetonitrile:acetic acid:water (96:2:2, v / v / v); isocratic elution, mobile phase A and B each accounted for 50% by volume during elution, flow rate 1.0 mL / min; run time 25 min; fluorescence detector parameters: excitation wavelength 333 nm, emission wavelength 477 nm; injection volume 20 μL.

[0032] The initial reaction rate (v) at each substrate OTA concentration was calculated based on the concentration of degradation product Otα. With the substrate concentration ([S], μM) as the abscissa and the reaction rate (v, μmol·min -1 ·mg -1 ) as the ordinate, the Michaelis-Menten equation was used for nonlinear fitting to obtain the maximum reaction rate (Vmax) and Michaelis constant (Km). The catalytic constant kcat was calculated according to the formula kcat = (Vmax × MW × 10 -3 ) / 60, where MW is the molecular weight of the enzyme (g·mol -1 ), and the formula is obtained by converting the time unit from min -1 to s -1 (i.e., dividing by 60) to obtain the s -1unit. Finally, the catalytic efficiency kcat / Km value was taken as the core evaluation index. The determination results showed that Figure 1 ), the kcat / Km value of ADH3 wild-type protein degrading OTA was 0.002311 s -1 μM -1 . In comparison, 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 (0.061982 s -1 μM -1 and 0.168442 s -1 μM -1 respectively) of that of the wild-type ADH3, proving that the catalytic efficiency of the mutants ADH3-S88E-Q93R-A164G-F299Y-Q345S and ADH3-S88E-A164G-F299Y-Q345S on OTA was significantly enhanced.

[0033] SEQ ID NO. 1 (ADH3 wild-type amino acid sequence) EPVAVQCGRLFDARSGQLKGPHTLLVADGRIRQVLPGTGADAAGARVVDLGDKVCLPGWTDLHVHLGSQSSPQSYSEDFRLDPVDHAFRAVGYAEKTLMAGFTSVRDLGGEVSPHLRDAINQGLVRGPRIFAAGKSIATTGGHADPTNGWNERLAHLVGAPGPAEGVVNSVDEARQAVRQRYKEGSDLIKITATGGVLSYARSGDAPQFTVDEIKAVVDTARDYGFRVAAHAHGTEGMKRAVQAGVTSIEHGTYMDDEVMRLMKQHGTWYVPTFYAGRFVTEKAAIDGYFPEVVRPKAARIGALISQTAAKAYRNGVRIAFGTDQGVGPHGDNAREFVYMVEAGIPAAYALQAATVHAAQVLGVDDQGVLEPGKRADVIALAGNPLEDINAVLDVRFVMKDGVIYKQ SEQ ID NO. 2 (ADH3 wild-type nucleotide sequence) SEQ ID NO. 3 (Amino acid sequence of ADH3 - S88E - Q93R - A164G - F299Y - Q345S mutant) EPVAVQCGRLFDARSGQLKGPHTLLVADGRIRQVLPGTGADAAGARVVDLGDKVCLPGWTDLHVHLGEQSSPRSYSEDFRLDPVDHAFRAVGYAEKTLMAGFTSVRDLGGEVSPHLRDAINQGLVRGPRIFAAGKSIATTGGHGDPTNGWNERLAHLVGAPGPAEGVVNSVDEARQAVRQRYKEGSDLIKITATGGVLSYARSGDAPQFTVDEIKAVVDTARDYGFRVAAHAHGTEGMKRAVQAGVTSIEHGTYMDDEVMRLMKQHGTWYVPTFYAGRYVTEKAAIDGYFPEVVRPKAARIGALISQTAAKAYRNGVRIAFGTDSGVGPHGDNAREFVYMVEAGIPAAYALQAATVHAAQVLGVDDQGVLEPGKRADVIALAGNPLEDINAVLDVRFVMKDGVIYKQ SEQ ID NO. 4 (Nucleotide sequence of ADH3 - S88E - Q93R - A164G - F299Y - Q345S mutant) SEQ ID NO. 5 (ADH3-S88E-A164G-F299Y-Q345S mutant amino acid sequence) EPVAVQCGRLFDARSGQLKGPHTLLVADGRIRQVLPGTGADAAGARVVDLGDKVCLPGWTDLHVHLGEQSSPQSYSEDFRLDPVDHAFRAVGYAEKTLMAGFTSVRDLGGEVSPHLRDAINQGLVRGPRIFAAGKSIATTGGHGDPTNGWNERLAHLVGAPGPAEGVVNSVDEARQAVRQRYKEGSDLIKITATGGVLSYARSGDAPQFTVDEIKAVVDTARDYGFRVAAHAHGTEGMKRAVQAGVTSIEHGTYMDDEVMRLMKQHGTWYVPTFYAGRYVTEKAAIDGYFPEVVRPKAARIGALISQTAAKAYRNGVRIAFGTDSGVGPHGDNAREFVYMVEAGIPAAYALQAATVHAAQVLGVDDQGVLEPGKRADVIALAGNPLEDINAVLDVRFVMKDGVIYKQ SEQ ID NO. 6 (ADH3-S88E-A164G-F299Y-Q345S mutant nucleotide sequence)

Claims

1. A group of highly efficient mutants of ochratoxin A degrading enzyme 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. An agent, characterized in that, 6. An agent containing the mutant of claim 1 as an active ingredient.

7. A method of degrading ochratoxin A for non-disease diagnosis and treatment 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, The concentration of the mutant 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 diagnosis and treatment of diseases.

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