Use of an amide bond-hydrolyzing enzyme in degrading ochratoxin a

Abstract

The application provides application of an amide bond hydrolase in degrading ochratoxin A, and the amide bond hydrolase has any one of the amino acid sequences shown in (I), (II) or (III): (I) an amino acid sequence shown in SEQ ID NO:1; (II) an amino acid sequence with homology of greater than or equal to 90% to the amino acid sequence shown in SEQ ID NO:1, and an amino acid sequence with the same function or similar function as the sequence shown in (I); (III) an amino acid sequence obtained by modifying, substituting, deleting or adding at least one amino acid of the amino acid sequence shown in SEQ ID NO:1. The amide bond hydrolase has the function of degrading ochratoxin A, and the degradation rate is greater than or equal to 99.09%, and can be used as a detoxicant of ochratoxin A, and has important application value in food processing and food safety guarantee.

Description

Technical Field

[0001] This invention belongs to the field of biodegradation technology, specifically relating to the application of an amide bond hydrolase in the degradation of ochratoxin A. Background Technology

[0002] Ochratoxin A (OTA) is a secondary metabolite produced by various Aspergillus and Penicillium fungi. It can contaminate grains, fruits and vegetables, and is commonly detected in legumes, coffee, fruits, wine, nuts and spices.

[0003] There is an urgent need to develop efficient and sustainable methods to remove toxins from food and feed. Currently, physical, chemical, and biological methods and various degradation mechanisms have been explored and applied. For example, physical removal strategies include irradiation, light treatment, ultrasonic treatment, thermal treatment, and adsorption. Chemical control methods include treatment with chlorine dioxide, alkalis, and ozone. However, these methods still have some drawbacks in practical application, such as incomplete toxin degradation after physical treatment and secondary pollution caused by the use of chemical reagents. Furthermore, these methods generally suffer from environmental unfriendliness, lack of specificity, serious resource waste, and high safety risks, making it difficult to meet the actual needs of food processing and ensuring food security.

[0004] Microbial biocatalysis offers advantages such as mild reaction conditions, environmental friendliness, and sustainability. However, microbial detoxification presents numerous uncontrollable challenges in practice, including the introduction of toxic microbial metabolites, decreased activity due to mutations, and the depletion of nutrients in food and grains during detoxification. Developing specific enzyme elements for OTA biodegradation has become a more ideal approach. Currently, some carboxypeptidases, amidases, and peptidases are known to hydrolyze OTA into less toxic products. However, these peptidases exhibit low OTA degradation rates, typically 30-40%, which is insufficient for practical applications.

[0005] Classical theory posits that enzymes possess strict substrate and reaction specificity. However, recent studies have revealed that some enzymes can catalyze reactions with substances different from their natural substrates; this phenomenon reflects the substrate generalization of enzymes. Previous studies reported that over 30% of enzymes in *E. coli* exhibit generalization, and these enzymes catalyze more than 60% of endogenous metabolic reactions in *E. coli*. This characteristic is also fully reflected in the ProtMiscuity database established by Rueda et al. ProtMiscuity contains 57 enzymes and 88 primary and mixed reactions they catalyze, with approximately 12% of the enzymes catalyzing three or more mixed reactions.

[0006] Therefore, by using methods such as toxin degradation pathway prediction and enzyme element mining, we can explore OTA-degrading enzymes and discover novel amide bond hydrolases that can efficiently degrade OTA, which has important application value in food processing and ensuring food security. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide an amide bond hydrolase for the degradation of ochratoxin A. This invention is the first to discover an amide bond hydrolase derived from *Rhodanobacter thiooxydans* that possesses the function of degrading ochratoxin A. This amide bond hydrolase exhibits a degradation rate of over 99.09% for ochratoxin A and can serve as an antidote for ochratoxin A, possessing significant application value in food processing and ensuring food security.

[0008] To achieve this objective, the present invention employs the following technical solution:

[0009] In a first aspect, the present invention provides the application of an amide bond hydrolase in the degradation of ochratoxin A, said amide bond hydrolase having any one of the amino acid sequences shown in (I), (II) or (III):

[0010] (I) The amino acid sequence as shown in SEQ ID NO:1;

[0011] (II) An amino acid sequence that has ≥90% homology with the amino acid sequence shown in SEQ ID NO:1 and has the same or similar function as the sequence described in (I);

[0012] (III) An amino acid sequence obtained by modifying, substituting, deleting or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO:1, and which has the same or similar function as the sequence described in (I).

[0013] SEQ ID NO:1:

[0014] MSMPRLLLVVAVSCLPLAAGADAPARDDAGSQRINLIECARLLDPAAGRMLGQTTLVIEDGRIKEIHPGAIDIEPYRKAAGTLRVVRLPDATCMPGLIDSHTHLTMQFSRDS YSDKFRLNPADHAIRGTVYAKRTLLAGFTTVRNLGDDDNASIALRNAVNAGLVPGPHIFTTGKPLGTTGGHADPSNGFRWDLQGDPGPKDGIIDSPAEAWKAVRQHYKDGADL IKIMPSGGVLDESSSSGNPQMTLEEIEAVVAAAHDYGFTVAAHAHGAEAIRRAVLGGVDSIEHGTFMDAPDMKLMKEHGTWYVPTIIAGQYVMEKAKQGWYPPQVARKAEEVG PVILDTAGKAYKAGVKIAFGTDAGVYPHGDNAREFVYMVQAGMPSMFVLQAATTHAAELLHKSDQLGRIAVGRGADVIAVPGNPLDDITVMQHVSFVMKDGVVYKQDGKPAI.

[0015] Preferably, the amide bond hydrolase is derived from Rhodanobacter thiooxydans.

[0016] Preferably, the amide bond hydrolase catalyzes the cleavage of amide bonds in ochratoxin A.

[0017] This invention marks the first discovery of an amide bond hydrolase (amino acid sequence shown in SEQ ID NO:1) derived from *Rhodanobacter thiooxydans*, with a length of 450 amino acids. Under experimental conditions, this enzyme degraded 99.09% of ochratoxin A, demonstrating excellent degradation efficiency. The amide bond hydrolase catalyzes the degradation of ochratoxin A into non-toxic substances (such as...). Figure 1 As shown, ochratoxin A contains amide bonds, which are good hydrolysis sites. The amide bond hydrolase hydrolyzes the amide bonds of ochratoxin A to generate ochratoxin α and L-phenylalanine, which have been reported to have significantly reduced toxicity.

[0018] It is understood that this application has discovered that an amide bond hydrolase (amino acid sequence shown in SEQ ID NO:1) derived from *Rhodanobacter thiooxydans* possesses the function of degrading ochratoxin A. Therefore, enzymes from *Rhodanobacter thiooxydans* or similar strains with sequences and functions identical or similar to the sequence shown in SEQ ID NO:1 are also expected to possess the function of degrading ochratoxin A. Furthermore, enzymes obtained by modifying the sequence shown in SEQ ID NO:1 using conventional techniques in the art by substituting, deleting, or adding one or more amino acid residues, while possessing the same or similar function as the original protein, are also expected to possess the function of degrading ochratoxin A.

[0019] Preferably, the amide bond hydrolase comprises either naturally derived or artificially synthesized enzymes.

[0020] Preferably, the naturally derived amide bond hydrolase is an amide bond hydrolase isolated and purified from the culture medium of Rhodanobacter thiooxydans.

[0021] Preferably, the artificially synthesized amide bond hydrolase is an amide bond hydrolase expressed through genetic engineering.

[0022] Preferably, the genetically engineered amide bond hydrolase is prepared using a method comprising the following steps:

[0023] (1) Synthesize the nucleic acid encoding the amide bond hydrolase, and link the nucleic acid sequence to the protein expression vector plasmid to obtain the reaction template plasmid;

[0024] (2) The reaction template plasmid was added to a cell-free reaction system and incubated to obtain an amide bond hydrolase expressed by genetic engineering.

[0025] Preferably, in step (1), the nucleic acid encoding the amide bond hydrolase includes the nucleotide sequence shown in SEQ ID NO:2.

[0026] SEQ ID NO:2:

[0027]

[0028] Preferably, in step (1), the protein expression vector plasmid includes the D2P expression plasmid.

[0029] Preferably, in step (2), the incubation conditions are 35-37℃ (e.g., 35℃, 36℃, or 37℃, etc.) for 2.5-3.5h (e.g., 2.5h, 3h, or 3.5h, etc.).

[0030] In a second aspect, the present invention provides a method for degrading ochratoxin A, the method comprising: mixing the amide bond hydrolase described in the first aspect with a sample containing ochratoxin A to achieve degradation of ochratoxin A.

[0031] Thirdly, the present invention provides the application of the amide bond hydrolase described in the first aspect in the preparation of products that degrade ochratoxin A.

[0032] Fourthly, the present invention provides an ochratoxin A biodegrading agent, wherein the biodegrading agent contains the amide bond hydrolase described in the first aspect.

[0033] Preferably, the biodegradable agent further includes any one or a combination of at least two of a carrier, a preservative, or a protein protectant.

[0034] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] This invention is the first to discover an amide bond hydrolase derived from Rhodanobacter thiooxydans that has the function of degrading ochratoxin A. The degradation rate of ochratoxin A by the amide bond hydrolase is above 99.09%, and it can be used as an antidote for ochratoxin A, which has important application value in food processing and ensuring food security. Attached Figure Description

[0037] Figure 1 This is a schematic diagram illustrating the degradation reaction principle of ochratoxin A;

[0038] Figure 2 This is a graph showing the degradation activity results of ochratoxin A;

[0039] Figure 3 This is a chromatogram of the secondary mass spectrometry results for ochratoxin A;

[0040] Figure 4This is a secondary mass spectrometry result of the degradation products of ochratoxin A;

[0041] Figure 5 This is a diagram showing the degradation results of toxins in corn flour;

[0042] Figure 6 This is a diagram showing the degradation results of toxins in wheat flour. Detailed Implementation

[0043] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0044] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0045] Example 1

[0046] This embodiment provides an amide bond hydrolase derived from *Rhodanobacter thiooxydans*, which is 450 amino acids long. Ochratoxin A contains amide bonds, which are excellent hydrolysis sites. Therefore, an amide bond hydrolase was designed to catalyze the degradation of ochratoxin A. The reaction principle is as follows: Figure 1 As shown.

[0047] The amino acid sequence of the amide bond hydrolase is shown in SEQ ID NO:1, SEQ ID NO:1:

[0048] MSMPRLLLVVAVSCLPLAAGADAPARDDAGSQRINLIECARLLDPAAGRMLGQTTLVIEDGRIKEIHPGAIDIEPYRKAAGTLRVVRLPDATCMPGLIDSHTHLTMQFSRDSYSDKFRLNPADHAIRGTVYAKRTLLAGFTTVRNLGDDDNASIALRNAVNAGLVPGPHIFTTGKPLGTTGGHADPSNGFRWDLQGDPGPKDGIIDSPAEAWKAVRQHYKDGADLIKIMPSGGVLDESSSSGNPQMTLEEIEAVVAAAHDYGFTVAAHAHGAEAIRRAVLGGVDSIEHGTFMDAPDMKLMKEHGTWYVPTIIAGQYVMEKAKQGWYPPQVARKAEEVGPVILDTAGKAYKAGVKIAFGTDAGVYPHGDNAREFVYMVQAGMPSMFVLQAATTHAAELLHKSDQLGRIAVGRGADVIAVPGNPLDDITVMQHVSFVMKDGVVYKQDGKPAI。

[0049] The nucleotide sequence encoding the amide bond hydrolase is shown in SEQ ID NO:2, SEQ ID NO:2:

[0050]

[0051] Example 2

[0052] In this embodiment, an amide bond hydrolase was expressed using a cell-free culture system. The gene sequence was synthesized by Shanghai Sangon Biotech, and the D2P expression plasmid and cell-free expression reaction system were both from Shanghai Kangma Biotechnology Co., Ltd.

[0053] (1) Synthesize the sequence of SEQ ID NO:2 and ligate the sequence to the protein expression vector plasmid D2P by homologous recombination technology;

[0054] (2) The recombinant plasmid containing the target gene was used as a reaction template and added to a cell-free reaction system. The reaction was carried out at 37°C for 3 hours to obtain the target protein. The reaction volume was 1 mL, and the final concentration of the reaction template plasmid was 1 ng / μL. A blank control was set up, i.e., the reaction solution without the template plasmid, to eliminate the influence of enzymes and reaction conditions present in the reaction system.

[0055] Example 3

[0056] This embodiment uses the amide bond hydrolase expressed in Example 2 for degradation experiments.

[0057] After the reaction in Example 2 was completed, the reaction solution was centrifuged at low speed, and the supernatant of the reaction system was transferred to a new centrifuge tube. Ochratoxin A (final concentration of 2 μg / mL) was added, and the mixture was incubated at 37°C for 3 hours. After incubation, the mixture was centrifuged at high speed. The supernatant was then collected, an equal volume of methanol was added, and the mixture was vigorously shaken and centrifuged at high speed again. The supernatant was filtered through a 0.22 μm organic filter membrane, and the content of ochratoxin A after the reaction was detected using liquid chromatography-mass spectrometry developed by Azam et al. (Azam, MS et al. (2016) Degrading ochratoxin A and zearalenone mycotoxins using a multifunctional recombinant enzyme. Toxins (Basel) 10.3390 / toxins11050301).

[0058] The supernatant of the reaction system containing the candidate enzyme was incubated with ochratoxin A. The catalytic activity of the enzyme was determined by measuring the amount of ochratoxin A before and after incubation. Ochratoxin A standard used in the assay was purchased from Sigma-Aldrich, USA. Methanol and acetonitrile used in the assay were high-performance liquid chromatography (HPLC) grade, manufactured by Merck, Germany. Other HPLC analytical grade solvents and chemicals were provided by Aladdin. Ultrapure water (18.2 MΩ·cm) was obtained from Millipore, USA. The method developed by Tian et al. was used for the detection of ochratoxin A.

[0059] The liquid chromatography system was a Thermo Scientific Accela 1250 ultra-high performance liquid chromatography system, and the chromatographic column was an Agilent Extend-C18 column (100 mm × 4.6 mm, 3.5 μm). The column temperature was set to 30 °C, the sample tray temperature was set to 4 °C, and the sample injection volume was set to 10 μL. The mobile phase A was 5 mM ammonium acetate aqueous solution, and the mobile phase B was pure methanol, with a flow rate of 0.35 mL / min. The gradient elution program was as follows: 0 min, 15% B; 1 min, 15% B; 6.5 min, 90% B; 8.5 min, 90% B; 9 min, 15% B; 12 min, 15% B.

[0060] Mass spectrometry analysis was performed using a TSQ Vantage™ triple quadrupole mass spectrometer (Thermo Fisher Scientific, USA) with alternating positive and negative electrospray ionization modes. The nebulizer (N2) and dry gas (N2) pressures were 30 psi and 20 psi, respectively. The ion source parameters were as follows: collision voltage: 3.5 kV (ESI+) and 3.0 kV (ESI-), transport capillary temperature: 250 °C, and nebulizer temperature: 350 °C.

[0061] The content of each component in a liquid sample (μg / mL) = C*F / 1000.

[0062] Where C is the concentration read by the instrument, in ng / mL; V is the volume of the sample extract, in mL; M is the total amount of sample weighed, in mg; and F is the dilution factor.

[0063] Degradation rate = (M1-M2) / M1×100%.

[0064] Where M1 is the toxin concentration before the reaction, and M2 is the toxin concentration after the enzyme reaction.

[0065] Degradation experiment results are as follows Figure 2 As shown, under the experimental conditions, 99.09% of ochratoxin A was degraded, demonstrating good degradation efficiency. Secondary mass spectrometry verification confirmed that the enzyme degradation products were the target products (e.g., ...). Figure 3 and Figure 4 (As shown).

[0066] Example 4

[0067] This example investigated the effect of amide bond hydrolase on the treatment of feed containing ochratoxin A. The experimental steps are as follows:

[0068] In this embodiment, the enzyme-containing reaction system after the reaction in Example 2 was completed was used as the experimental group, and the reaction system in Example 1 without the addition of the template plasmid was used as the blank control. The degradation of OTA in wheat flour (Fulinmen wheat core general wheat flour, purchased from JD.com) and corn flour (Beichun organic corn flour, purchased from JD.com) was carried out respectively.

[0069] The specific experimental procedure includes:

[0070] OTA standard was added to wheat flour and corn flour at a final concentration of 1000 ng / g. 500 μL of enzyme-containing reaction system was mixed with 500 mg of wheat flour and corn flour with added OTA, respectively. The same mixing operation was performed on the reaction system without template plasmid as a control. After incubation at 37°C for 3 hours, 500 μL of methanol was added to stop the reaction.

[0071] OTA detection and degradation rate calculation are based on Example 3.

[0072] The results are as follows Figure 5 and Figure 6 As shown, the degradation rate of OTA in wheat flour was 69.25%, and the degradation rate of OTA in corn flour was 38.16%, indicating that the enzyme discovered in this invention can directly degrade OTA in grain crops when used alone, and has broad application prospects in the development of biodegradable agents.

[0073] In summary, this invention is the first to discover an amide bond hydrolase derived from Rhodanobacter thiooxydans that has the function of degrading ochratoxin A. The degradation rate of the amide bond hydrolase is above 99.09% under experimental conditions, and it can be used as an antidote for ochratoxin A, which has important application value in food processing and ensuring food security.

[0074] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. The application of an amide bond hydrolase in the degradation of ochratoxin A, characterized in that, The amino acid sequence of the amide bond hydrolase is as follows: The amino acid sequence is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The amide bond hydrolase catalyzes the cleavage of amide bonds in ochratoxin A.

3. The application according to claim 1, characterized in that, The amide bond hydrolases include those of natural origin or those synthesized artificially.

4. The application according to claim 3, characterized in that, The naturally derived amide bond hydrolase is from Rhodanobacter thiooxydans The amide bond hydrolase was isolated and purified from the culture medium.

5. The application according to claim 3, characterized in that, The artificially synthesized amide bond hydrolase is an amide bond hydrolase expressed through genetic engineering.

6. The application according to claim 5, characterized in that, The genetically engineered amide bond hydrolase was prepared using a method comprising the following steps: (1) Synthesize the nucleic acid encoding the amide bond hydrolase, and link the nucleic acid sequence to the protein expression vector plasmid to obtain the reaction template plasmid; (2) The reaction template plasmid was added to a cell-free reaction system and incubated to obtain the amide bond hydrolase expressed by genetic engineering.

7. The application according to claim 6, characterized in that, In step (1), the nucleic acid encoding the amide bond hydrolase includes the nucleotide sequence shown in SEQ ID NO:

2.

8. The application according to claim 6, characterized in that, In step (1), the protein expression vector plasmid includes the D2P expression plasmid.

9. The application according to claim 6, characterized in that, In step (2), the incubation conditions are 35-37℃ for 2.5-3.5 h.

10. A method for degrading ochratoxin A, characterized in that, The method includes: mixing the amide bond hydrolase according to any one of claims 1-9 with a sample containing ochratoxin A to achieve degradation of ochratoxin A.

11. The application of an enzyme in the preparation of an ochratoxin A biodegrading agent, characterized in that, The biodegrading agent contains an amide bond hydrolase as described in any one of claims 1-9.

12. The application according to claim 11, characterized in that, The biodegradable agent also includes any one or a combination of at least two of the following: a carrier, a preservative, or a protein protectant.

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