Use of an amide hydrolase otaH in degrading ochratoxin a

Abstract

The application discloses application of an amide hydrolase OtaH in degrading ochratoxin A, and relates to the technical field of enzyme engineering and food safety biotechnology. The amino acid sequence of the amide hydrolase OtaH is shown as SEQ ID NO. 2, the coding gene sequence of the amide hydrolase OtaH is shown as SEQ ID NO. 1, and the application provides application of the amide hydrolase OtaH in degrading ochratoxin A. The amide hydrolase OtaH can specifically hydrolyze the amide bond of ochratoxin A (OTA) to generate almost non-toxic ochratoxin alpha, the enzyme has high catalytic efficiency, 5.0 mu g / mL of the enzyme amount can completely degrade 12.4 mu M OTA in 5 minutes, and more than 50% activity is still maintained after incubation at 65 DEG C for 1 hour, thereby showing excellent thermal stability. The amide hydrolase OtaH can be widely used in detoxification treatment of OTA in feed and food, and the harm of OTA to human beings and animals is reduced.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and food safety biotechnology, specifically to the application of an amide hydrolase OtaH in the degradation of ochratoxin A. Background Technology

[0002] Ochratoxin A (OTA) is produced by Aspergillus spp. ( Aspergillus ) and Penicillium ( Penicillium Secondary metabolites produced by fungi widely contaminate agricultural products and processed foods such as grains, legumes, grapes, coffee, spices, and nuts. Otamine sulfate (OTA) exhibits strong nephrotoxicity, hepatotoxicity, immunotoxicity, and carcinogenicity, and is classified as a Group 2B carcinogen by the International Agency for Research on Cancer (IARC). OTA enters the human and animal body through the food chain, posing a serious threat to health and causing enormous economic losses to the global agricultural and food industries.

[0003] Currently, the main detoxification methods for OTA (Adsorption-Oriented Food) include physical, chemical, and biological methods. Physical adsorption methods suffer from incomplete detoxification and nutrient loss; chemical methods may produce harmful residues and affect food flavor; biological methods, especially enzymatic detoxification, have become the most promising technology due to their advantages of high efficiency, specificity, safety, and environmental friendliness.

[0004] Amide hydrolases specifically hydrolyze the amide bonds in OTA molecules, breaking them down into nearly non-toxic ochratoxin α (OTα). This pathway is widely recognized as the safest and most effective biological detoxification route for OTA. Currently reported OTA amidolytic enzymes include those derived from... Aspergillus niger OTase, Stenotrophomonas acidaminiphila ADH3, Lysobacter ADH2 sp., etc. However, existing enzymes suffer from poor thermostability and high preparation costs, limiting their industrial application. For example, commercially available bovine carboxypeptidase A (CPA) has extremely low catalytic efficiency; 67 mg / mL of commercially available CPA can only degrade 10% of OTA within 1 h (starting concentration 50 mg / L); and most reported enzymes have optimal temperatures below 50°C, making them prone to inactivation in high-temperature processes during food processing and feed preparation. Therefore, there is an urgent need to explore and develop OTA amidolytic enzymes with higher catalytic efficiency and better thermostability. Summary of the Invention

[0005] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide an application of amide hydrolase OtaH in the degradation of ochratoxin A, specifically an application of amide hydrolase OtaH in the degradation of ochratoxin A and a method for removing ochratoxin A from food, feed or agricultural products.

[0006] The technical solution of the present invention is as follows:

[0007] The first aspect of the present invention provides the application of amide hydrolase OtaH in the degradation of ochratoxin A, wherein the amino acid sequence of amide hydrolase OtaH is shown in SEQ ID NO.2.

[0008] The amide hydrolase OtaH of the present invention consists of 417 amino acids and has a theoretical molecular weight of 44.9 kDa.

[0009] SEQ ID NO.2:

[0010] MDKLIRNGVVIDGTGNVIEDAVIAIKGDRIAAIGPASDFIGIDAKEVIDAQGGYILPGLIDTHVHMMMEIKDVRESLITPFSMRFYEALTYMKRTLDAGITTVR DAGYADLGVKQAIENGLISGPRMQISVNPLTITGGHGDNWMVSDMDLTNKAYPGFPSGVCDGPEQVRQKVREMLRAGADIIKVHATGGVMSPTDHPEFTQFSMEE LQIIVEEAAYRKGRKVMAHAQGAEGVKQAVRAGIHSIEHGIFLDDEAIELMLKHGTYLVPTLLAPVSVVEASEANESMPAYAVDKAKEVMEIHQQSVARAYEAG VKIAMGTDAGVMAHGTNLRELALMCDIGMSPMEAILASTKIAAECLGWEDRIGTIAEGKLADLVIVQENPLENIASLADTNNIKTVMQGGKLKKQLSIQTGIPS.

[0011] In one specific embodiment of the present invention, the nucleotide sequence of the gene encoding the amide hydrolase OtaH is shown in SEQ ID NO.1.

[0012] The gene encoding the amide hydrolase OtaH of this invention is 1254 bp in length.

[0013] SEQ ID NO.1:

[0014]

[0015] In one specific embodiment of the present invention, the application includes using the amide hydrolase OtaH to degrade ochratoxin A in food, feed, or agricultural products.

[0016] In one specific embodiment of the present invention, the food includes corn flour, wheat flour, milk, tea, beer, almond powder, or fruit juice.

[0017] In one specific embodiment of the present invention, the application method includes:

[0018] The amide hydrolase OtaH was mixed with a sample containing ochratoxin A to obtain a mixture. The pH of the mixture was adjusted to 6-8 and the temperature to 40℃-60℃, so that the amide hydrolase OtaH specifically hydrolyzed the amide bond of ochratoxin A to generate ochratoxin α.

[0019] In one specific embodiment of the present invention, 5.0 μg / mL of the amide hydrolase OtaH can completely degrade 12.4 μM ochratoxin A within 5 minutes, with a catalytic efficiency k cat / K m The value is 4028.57 mM -1 min -1 .

[0020] A second aspect of the present invention provides a method for removing ochratoxin A from food, feed, or agricultural products, comprising the following steps:

[0021] S1. The solution containing the amide hydrolase OtaH is mixed with the sample containing ochratoxin A to obtain a mixture; wherein the amino acid sequence of the amide hydrolase OtaH is shown in SEQ ID NO.2;

[0022] S2. Adjust the pH of the mixture to 6-8 and the temperature to 40℃-60℃. The amide hydrolase OtaH in the mixture reacts with ochratoxin A until the content of ochratoxin A in the mixture is detected to be lower than the threshold.

[0023] This invention has at least one of the following beneficial effects:

[0024] The amide hydrolase OtaH of this invention can specifically hydrolyze the amide bond of ochratoxin A, thereby catalyzing the formation of non-toxic ochratoxin α (OTα) from OTA. It exhibits high catalytic activity; 5.0 μg / mL of OtaH can completely degrade 12.4 μM OTA within 5 minutes, with a catalytic efficiency k... cat / K m The value is 4028.57 mM -1 min-1 Furthermore, the amide hydrolase OtaH of this invention also exhibits excellent thermal stability. Experiments have shown that the amide hydrolase OtaH of this invention retains more than 50% of its activity after incubation at 65°C for 1 hour. Therefore, the amide hydrolase OtaH of this invention is not easily inactivated in high-temperature processes of food processing and feed preparation, possessing both high catalytic efficiency and thermal stability, and has excellent application prospects in removing residual OTA from food and feed. Attached Figure Description

[0025] Figure 1 This is an SDS-PAGE electrophoresis image of the purified OtaH protein from Example 2.

[0026] Figure 2 This is the HPLC chromatogram of OTA degradation by OtaH in Example 3.

[0027] Figure 3 The curves show the time progression of OTA degradation and OTα generation by OtaH in Example 3.

[0028] Figure 4 The image shows the enzymatic kinetics curve of OtaH on OTA in Example 3.

[0029] Figure 5 This is a study on the enzymatic properties of OtaH in Example 4. In the figure, A represents the enzyme activity of OtaH protein at different temperatures, B represents the enzyme activity of OtaH protein at different pH values, C represents the enzyme activity of OtaH protein at 60℃ and 65℃, and D represents the stability of OtaH protein under different pH conditions.

[0030] Figure 6 This is a diagram showing the effect of OtaH on the degradation of OTA in corn flour in Example 5. Detailed Implementation

[0031] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and are not intended to limit the invention.

[0032] Example 1: Synthesis of otaH gene and construction of recombinant expression vector

[0033] 1.1 Screening, Identification and Preservation of Strains JX-8

[0034] Screening: 5 mL of sauerkraut juice sample was inoculated into 50 mL of MSM medium containing 12.4 μM OTA and cultured at 30℃ with shaking at 180 r / min for 3 days. 5 mL of the culture was then transferred to fresh MSM medium, and the enrichment was repeated 5 times. The final enriched solution was subjected to 10...-1 ~10 -6 Gradient dilution, take 10 -4 ~10 -6 0.1 mL of each dilution was spread onto LB agar plates containing 12.4 μM OTA and incubated at 30°C for 5 days. Single colonies with different morphologies were picked and inoculated into LB liquid medium and incubated at 37°C and 180 rpm for 24 h. The strain was purified three times using the streak plate method to obtain a pure strain. The pure strain was inoculated into LB liquid medium containing 12.4 μM OTA and incubated at 37°C and 180 rpm for 24 h. The OTA residue was detected by HPLC, and the strain with the highest degradation rate was screened and named JX-8.

[0035] Identification: Molecular biological identification was performed on strain JX-8. Based on the identification results, the strain was determined to belong to *Bacillus saccharophilus*. Terribacillus saccharophilus ), named Terribacillus saccharophilus The nucleotide sequence of the 16S rRNA gene of strain JX-8 is shown in SEQ ID NO. 5.

[0036] Preservation: Strain JX-8 was deposited on May 8, 2025 at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 2025996.

[0037] 1.2 Synthesis and Amplification of the otaH Gene

[0038] Genome sequencing analysis, alignment and functional verification of strain JX-8 revealed an amide hydrolase gene named otaH, whose nucleotide sequence is shown in SEQ ID NO.1, is 1254 bp in size and encodes 417 amino acids.

[0039] Based on the nucleotide sequence shown in SEQ ID NO. 1, the whole genome was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The synthesized otaH was cloned into the pUC57 vector, the recombinant vector was named pUC-otaH, and then transformed into Escherichia coli DH5α.

[0040] Forward primer: 5'-TAAGAAGGAGATATACATATGGACAAGCTTATTCGCAATGGTGTCG-3' (SEQ ID NO. 3) and reverse primer: 5'-GTGGTGGTGGTGCTCGAGACTTGGAATACCTGTCTGGATAGAAA-3' (SEQ ID NO. 4). Using pUC-otaH as a template, the otaH gene was amplified by PCR. The PCR amplification system is shown in Table 1.

[0041] Table 1

[0042]

[0043] PCR amplification program: 95℃ pre-denaturation for 30 s → 95℃ denaturation for 15 s, 65℃ annealing for 15 s, 72℃ extension for 30 s, for 30 cycles → 72℃ extension for 10 min, cool to room temperature.

[0044] After PCR amplification, the amplification products were detected by agarose gel electrophoresis, and the corresponding DNA fragments were purified and recovered using a gel purification and recovery kit.

[0045] 1.3 Double digestion of plasmid pET-29a(+)

[0046] The plasmid pET-29a(+) was double-digested using rapid restriction endonucleases (QuickCut™ NdeI and QuickCut™ XhoI) manufactured by Takara. The 50 μL digestion system is shown in Table 2.

[0047] Table 2

[0048]

[0049] The enzyme digestion system was placed in a 37°C water bath and reacted for 1 h. The reaction was then terminated with DNA loading buffer (10× Loading Buffer). The enzyme digestion efficiency was detected by agarose gel electrophoresis, and the linearized vector was purified and recovered using a gel purification and recovery kit.

[0050] 1.4 Recombinant transformation of otaH and pET-29a(+) vectors and screening of positive transformants

[0051] The amplified otaH fragment and the linearized vector pET-29a(+) were recombinated using a homologous recombination kit. The recombination system is shown in Table 3.

[0052] Table 3

[0053]

[0054] The recombination reaction was carried out at 37°C for 30 min, and then immediately placed on ice. The recombination product was converted to [a specific product] using a chemical conversion method. E. coli The recombinant expression strain BL21(otaH) was obtained from BL21(DE3) competent cells.

[0055] Example 2: Induction and purification of OtaH

[0056] Single colonies of the recombinant expression strain BL21(otaH) were picked and inoculated into 5 mL of LB liquid medium (containing 50 μg / mL kanamycin) and cultured overnight at 37°C and 180 rpm.

[0057] Transfer the culture to 500 mL LB medium at a volume ratio of 1:100 and incubate until OD500. 600 ≈0.6, add IPTG to a final concentration of 0.1 mM, and induce expression at 16°C for 12 hours.

[0058] Collect bacterial cells by centrifugation, resuspend in PBS buffer (50 mM, pH 7.4), and sonicate (200 W power, 2 seconds operation, 3 seconds interval, 20 minutes in total).

[0059] The lysate was centrifuged at 12,000 × g for 30 minutes, and the supernatant was passed through Ni 2+ Purification using NTA affinity chromatography column. Elute contaminating proteins with equilibration buffer containing 20 mM imidazole, and then elute the target protein with elution buffer containing 300 mM imidazole.

[0060] The eluent was dialyzed to remove imidazole, yielding OtaH protein with a purity >95% and a size of approximately 45 kDa, consistent with the theoretical prediction. Figure 1 Protein concentration was determined using the BCA method.

[0061] The amino acid sequence of the OtaH protein was determined to be as shown in SEQ ID NO. 2.

[0062] Example 3 OtaH enzyme activity and kinetics determination

[0063] The enzyme activity and kinetics of the OtaH protein in Example 2 were measured. The methods and results are as follows:

[0064] A standard 1 mL enzymatic reaction system was established, containing 50 mM PBS buffer (pH 7.4), 12.4 μM OTA, and 5.0 μg / mL enzyme (OtaH protein from Example 2).

[0065] The reaction was carried out at 37°C for 5 minutes, and then an equal volume of acetonitrile was added to terminate the reaction. After centrifugation at 12000 rpm for 10 minutes, the supernatant was collected for HPLC analysis.

[0066] HPLC conditions: C18 column, mobile phase acetonitrile-water-glacial acetic acid (45:54:1, v / v), flow rate 1.0 mL / min, detection wavelength 330 nm.

[0067] A system without enzyme solution was used as a control. Enzyme activity was calculated by measuring the reduction in the OTA peak area.

[0068] HPLC analysis showed that the peak area of ​​OTA treated with OtaH was significantly reduced, and a product was generated that had the same retention time as the ochratoxin α (OTα) standard. Figure 2 ).

[0069] A 5.0 μg / mL enzyme can completely degrade 12.4 μM OTA within 5 minutes, generating an equal amount of OTα ( Figure 3 ).

[0070] One unit of enzyme activity (U) is defined as the amount of enzyme required to degrade 1 μg OTA per minute under the above conditions, and the specific enzyme activity is calculated to be 160 U / mg.

[0071] With a fixed enzyme amount, the OTA substrate concentration was varied (1.24 μM–49.5 μM), and the reaction time was 5 minutes. The initial reaction rate was determined by HPLC. K was calculated using GraphPad Prism 8 software based on nonlinear fitting of the Michaelis-Menten equation. m The value is 7.0 μM, V max The value is 3.1 μM / min ( Figure 4 Further calculation yields k cat / K m The value is 4028.57 mM -1 min -1 .

[0072] Example 4 Characterization of the enzymatic properties of OtaH

[0073] The OtaH protein in Example 2 was subjected to enzymatic characterization. The methods and results are as follows:

[0074] 1. Optimal reaction temperature:

[0075] 12.4 μM OTA and 5.0 μg / mL enzyme were added to 50 mM PBS buffer (pH 7.4), with a total volume of 1 mL. The enzyme reaction sample was incubated at temperatures ranging from 20℃ to 80℃ to determine enzyme activity. The relative enzyme activity was calculated using the enzyme activity at the optimal reaction temperature as 100%, thus determining the optimal reaction temperature for OtaH to be 50℃. Figure 5 (A in the middle).

[0076] 2. Optimal pH:

[0077] Enzyme activity was measured at 50°C after adding 12.4 μM OTA and 5.0 μg / mL enzyme to different pH buffer systems (pH 4.0–pH 10.0). The relative enzyme activity was calculated using the enzyme activity at the optimal pH as 100%, thus determining the optimal pH to be 7.0. Figure 5 (B in the middle).

[0078] 3. Thermal stability

[0079] 5.0 μg / mL of enzyme was added to 50 mM PBS buffer (pH 7.4) and incubated at 60℃ and 65℃ for different times. After rapid cooling in an ice bath, 12.4 μM OTA was added to the reaction system, and the residual enzyme activity was measured at 50℃. The results showed that after incubation at 65℃ for 1 hour, the enzyme activity still retained more than 50% ( Figure 5 (C in the middle).

[0080] 4. pH stability:

[0081] The enzyme solution was incubated in a pH buffer system (pH 4.0–pH 10.0) at 4°C for 2 hours, and the residual enzyme activity was measured at 50°C and pH 7.0. The results showed that OtaH was very stable under neutral conditions, retaining more than 90% of its initial activity at pH 6.0–pH 8.0. Figure 5 (D in the middle).

[0082] Example 5: OtaH degrades ochratoxin A in real samples.

[0083] OTA was added to corn flour at a final concentration of 1000 μg / kg to prepare a contaminated sample. 0.5 g of the contaminated sample was weighed and 0.5 mL of OtaH enzyme solution was added. The mixture was reacted at 50°C for 1 hour, with a sample without enzyme solution used as a control. OTA was extracted according to the national standard method, and the OTA residue was detected by HPLC according to the method described in Example 3. The results showed that the OTA degradation rate reached over 99% after 1 hour of reaction. Figure 6 This indicates that OtaH can efficiently remove OTA from corn flour.

[0084] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of an amide hydrolase OtaH in the degradation of ochratoxin A, characterized in that, The amino acid sequence of the amide hydrolase OtaH is shown in SEQ ID NO.2; The application method includes: The amide hydrolase OtaH was mixed with a sample containing ochratoxin A to obtain a mixture. The pH of the mixture was adjusted to 6-8 and the temperature to 40℃-60℃, so that the amide hydrolase OtaH specifically hydrolyzed the amide bond of ochratoxin A to generate ochratoxin α.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the amide hydrolase OtaH is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, The application includes using the amide hydrolase OtaH to degrade ochratoxin A in food, feed, or agricultural products.

4. The application according to claim 3, characterized in that, The food products include one of milk, beer, and fruit juice.

5. The application according to claim 3, characterized in that, The agricultural products include one of the following: corn flour, wheat flour, tea, and almond flour.

6. The application according to claim 1, characterized in that, The amide hydrolase OtaH at a concentration of 5.0 μg / mL was able to completely degrade 12.4 μM ochratoxin A within 5 minutes, with a catalytic efficiency k. cat / K m The value is 4028.57 mM -1 min -1 .

7. A method for removing ochratoxin A from food, feed, or agricultural products, characterized in that, Includes the following steps: S1. A solution containing amide hydrolase OtaH is mixed with a sample containing ochratoxin A to obtain a mixture; wherein the amino acid sequence of amide hydrolase OtaH is shown in SEQ ID NO.2; S2. Adjust the pH of the mixture to 6-8 and the temperature to 40℃-60℃. The amide hydrolase OtaH in the mixture reacts with ochratoxin A until the content of ochratoxin A in the mixture is detected to be lower than the threshold.

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