Use of an enzyme in the degradation of ochratoxin a

Abstract

The application discloses application of an enzyme in degrading ochratoxin A. The amino acid sequence of the enzyme comprises: (1) a sequence shown in SEQ ID NO. 1, or (2) an amino acid sequence obtained by substituting, deleting or adding one or at least two amino acid residues from the sequence in (1) and being identical or similar in function to the sequence in (1), or (3) an amino acid sequence having at least 90% sequence homology with the sequence in (1) or (2) and being identical or similar in function to the sequence in (1). The application first discovers that an enzyme derived from Thermomonas fusca has the function of degrading ochratoxin A, and catalyzes ochratoxin A to degrade into non-toxic substances, so as to be expected to be applied in degrading ochratoxin A, and the degradation rate is more than 90%, and the application has substantial application value and great significance in the aspect of biological detoxification of food and feed.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of an enzyme in the degradation of ochratoxin A. Background Technology

[0002] Ochratoxin A (OTA) is a secondary metabolite produced by various Aspergillus and Penicillium fungi. It mainly contaminates cereal crops and has toxic side effects on humans and animals, including nephrotoxicity, immunotoxicity, and carcinogenicity, affecting the safety of food and feed. There is an urgent need to develop efficient and sustainable methods to remove it from food and feed.

[0003] 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 applications, 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, significant resource waste, and high safety risks, making it difficult to meet the actual needs of food processing and ensuring food security. This urges researchers to find alternative methods that have less negative impact on human health, are more sustainable, and can maintain the original nutrition and flavor of food.

[0004] Microbial biocatalysis offers advantages such as mild reaction conditions, environmental friendliness, and sustainability. For example, CN113604409A discloses a strain of *Bacillus shortwave diffusa* ML17 that degrades ochratoxin A and B, and its applications. ML17 and its metabolites can efficiently and stably degrade OTA and OTB. However, microbial detoxification involves many uncontrollable factors in practical operations, such as the introduction of toxic microbial metabolites, decreased activity due to mutations, and the consumption of nutrients in food and grains during detoxification. Meanwhile, the discovery of specific enzyme elements for OTA biodegradation has become a more ideal approach. For instance, CN109666664A discloses a method for preparing carboxypeptidase A with ochratoxin A degradation function and its applications. The prepared carboxypeptidase A possesses biological catalytic activity without further treatment, efficiently and stably degrading ochratoxin A, and maintaining stable degradation activity in various environments. Nevertheless, due to insufficient activity, stability, and / or recombinant reduction capacity, OTA hydrolases reported to date have rarely been applied in practical production.

[0005] In conclusion, the discovery of novel enzymes that degrade ochratoxin A is of great significance for the field of biological detoxification of food and feed. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides an application of an enzyme in the degradation of ochratoxin A. This invention discovers a novel enzyme for degrading ochratoxin A, which is expected to be applied to biological detoxification in the food and feed industries, and will play a very important role in the healthy development of agriculture and animal husbandry.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides the application of an enzyme in the degradation of ochratoxin A, wherein the amino acid sequence of the enzyme comprises:

[0009] (1) The sequence shown in SEQ ID NO.1, or,

[0010] (2) An amino acid sequence obtained by substituting, deleting, or adding one or at least two amino acid residues to the sequence described in (1), and having the same or similar function to the sequence described in (1), or,

[0011] (3) An amino acid sequence that has at least 90% sequence homology with the sequence described in (1) or (2) and has the same or similar function as the sequence described in (1).

[0012] In this invention, an enzyme derived from Thermomonasfusca (amino acid sequence as shown in SEQ ID NO. 1) was discovered for the first time to possess the function of degrading ochratoxin A, catalyzing the degradation of ochratoxin A into non-toxic substances (reaction as shown in Formula I), thus holding promise for application in the degradation of ochratoxin A, such as biological detoxification in the food and feed industries.

[0013] It is understood that this application has discovered that an enzyme derived from *Thermomonasfusca* (amino acid sequence as shown in SEQ ID NO. 1) has the function of degrading ochratoxin A. Therefore, enzymes in *Thermomonasfusca* or similar strains with sequences and functions identical or similar to those shown in SEQ ID NO. 1 are also expected to have the function of degrading ochratoxin A. In addition, enzymes obtained by modifying the sequence shown in SEQ ID NO. 1 by substituting, deleting, or adding one or more amino acid residues using common techniques in the art, while having the same or similar functions as the original protein, are also expected to have the function of degrading ochratoxin A.

[0014]

[0015] SEQ ID NO.1:

[0016] MRTRLALAASLLAAVAPTASAATSGDAVLACGRLFDSRSGALLGPHTVVVRGGRIAEVVPGRSPDAAGAIDLSDRTCLPGFTDLHVHMGSQSSPQSYSEGFRLDPVDYAYRSVGYAEKTLSAGFTSVRDLGGEVALHLRDAINQGLVKGPRIFAAGKSIATTGGHADPTNGWNDALSHLVGPPGPTDGVVNSVDDARQAVRQRYKDGSDVIKITAT GGVLSYAASGDAPQFTVDEVKAIVDTAKDYGYRVAAHAHGKEGMTRAILGGVTSIEHGTYMDEEVMRLMKARGTWYVPTIYAGRFVAEKAKIDGYFPDVVRPKAARIG ALIQDTAGKAYRNGVKIAFGTDMGVGPHGDNAREFLYMVEAGIPAAQALQAATIRAAEVLGVDDQGVLAPGKRADIVALPGDPLADIGNVLKVDFVMKDGVVHREPAR.

[0017] In a second aspect, the present invention provides a method for degrading ochratoxin A, the method comprising:

[0018] The enzyme described in the first aspect is mixed with a sample containing ochratoxin A degradation agent to achieve the degradation of ochratoxin A.

[0019] In this invention, after co-incubating the enzyme described in the first aspect with ochratoxin A, a significant decrease in the concentration of ochratoxin A can be detected, resulting in the production of a corresponding non-toxic product. Ultimately, the degradation rate of ochratoxin A can reach over 90%.

[0020] Thirdly, the present invention provides the application of the enzyme described in the first aspect in the preparation of ochratoxin A degradation products.

[0021] Fourthly, the present invention provides an ochratoxin A biodegrading agent, wherein the biodegrading agent contains the enzyme described in the first aspect.

[0022] Based on the discovered ability to degrade ochratoxin A, ochratoxin A degradation products can be further prepared.

[0023] Preferably, the biodegradable agent further includes any one or a combination of at least two of a carrier, a preservative, or a protein protectant.

[0024] It is understood that carriers, preservatives, or protein protectants commonly used in the field for biodegradable agents are also applicable to this invention.

[0025] Preferably, the carrier comprises a physiologically acceptable compatible carrier.

[0026] Preferably, the physiologically acceptable carrier includes any one or a combination of at least two of the following: rice husk, rice bran, maltodextrin, cyclodextrin, wheat bran, starch, bentonite, oligosaccharides, or yeast cell wall.

[0027] Preferably, the preservative includes any one or a combination of at least two of potassium sorbate, ethylparaben, methylparaben, or antibiotics.

[0028] Preferably, the antibiotic includes any one or a combination of two of penicillin, gentamicin, or vancomycin.

[0029] Preferably, the protein protectant comprises any one or a combination of at least two of bovine serum albumin, mannitol, glycerol, butylene glycol, sodium chloride, or sodium benzoate.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] This invention is the first to discover an enzyme derived from Thermomonasfusca (amino acid sequence shown in SEQ ID NO.1) that has the function of degrading ochratoxin A, catalyzing the degradation of ochratoxin A into non-toxic substances, and thus has the potential to be applied to the degradation of ochratoxin A, such as biological detoxification in the food and feed industries. Attached Figure Description

[0032] Figure 1 The graph shows the degradation rate of ochratoxin A.

[0033] Figure 2A Image showing the results of secondary mass spectrometry detection of ochratoxin A;

[0034] Figure 2B Image of secondary mass spectrometry results of degradation products of ochratoxin A;

[0035] Figure 3 The graph shows the degradation rate of ochratoxin A in wheat flour.

[0036] Figure 4 The graph shows the degradation rate of ochratoxin A in corn flour. Detailed Implementation

[0037] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0038] 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.

[0039] Example 1

[0040] This embodiment demonstrates the expression of an enzyme derived from Thermomonasfusca (amino acid sequence shown in SEQ ID NO.1).

[0041] The enzyme gene sequence (SEQ ID NO.2) was synthesized by Shanghai Sangon Biotech Co., Ltd., and the D2P expression plasmid and cell-free expression reaction system (reference: Zhang, K. et al. (2021) Systemic Expression, Purification, and Initial Structural Characterization of Bacteriophage T4 Proteins Without Known Structure Homologs. Front Microbiol 12, 674415.10.3389 / fmicb.2021.674415) were obtained from Shanghai Kangma Biotechnology Co., Ltd. After the above enzyme sequence was synthesized, it was ligated into the protein expression vector plasmid using homologous recombination technology. The recombinant plasmid containing the target gene was used as a reaction template and added to the 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.

[0042] SEQ ID NO.2:

[0043]

[0044] Example 2

[0045] This embodiment performs a degradation test on ochratoxin A (OTA).

[0046] The enzyme-containing reaction system from Example 1 was used as the experimental group, and the reaction system without template plasmid in Example 1 was used as the blank control. The supernatant of the reaction system was collected by low-speed centrifugation and placed in a new centrifuge tube. OTA (final reaction concentration of 2 μg / mL) was added and incubated at 37°C for 3 hours. After incubation, the system was centrifuged at high speed, and the supernatant was collected. An equal volume of methanol was added, and the system 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 OTA after the reaction was detected based on the liquid chromatography-mass spectrometry method developed by Tian et al. (refer to Tian, ​​Y. et al. (2016) Detoxification of Deoxynivalenol via Glycosylation Represents Novel Insights on Antagonistic Activities of Trichoderma when Confronted with Fusarium graminearum. Toxins (Basel) 8.10.3390 / toxins8110335).

[0047] The OTA standards used in the detection experiments were purchased from Sigma-Aldrich, USA. Methanol and acetonitrile used in the experiments 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 M·cm⁻¹) was obtained from Millipore, USA. The method developed by Tian et al. was used for OTA detection. The HPLC system was a Thermo Scientific Accela 1250 HPLC system, with an Agilent Extend-C18 column (100 mm × 4.6 mm, 3.5 μm). The column temperature was set to 30 °C, the sample pan temperature to 4 °C, and the sample injection volume to 10 μL. The mobile phase A was 5 mM ammonium acetate aqueous solution, and phase B was pure methanol; the mobile phase flow rate was 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. 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. Ion source parameters were as follows: collision voltage: 3.5 kV (ESI+) and 3.0 kV (ESI-), transport capillary temperature: 250 °C, nebulizer temperature: 350 °C. The concentration of each component in the liquid sample (μg / mL) was calculated as C*F / 1000. Where C is the instrument reading concentration in ng / mL; V is the sample extract volume in mL; M is the total sample weight in mg; and F is the dilution factor. Degradation rate = (M1-M2) / M1*100%. M1 is the OTA concentration before the reaction, and M2 is the OTA concentration after the enzyme reaction.

[0048] like Figure 1 As shown, the enzyme derived from *Thermomonas fusca*, with a length of 432 amino acids, exhibited a degradation rate of 96.03% for OTA in the experimental strip, demonstrating good degradation efficiency. Further verification by secondary mass spectrometry confirmed that the enzyme-catalyzed degradation product of OTA was the target product. Figure 2A and Figure 2B ).

[0049] Example 3

[0050] In this embodiment, the enzyme-containing reaction system after the reaction in Example 1 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.

[0051] The specific experimental procedure includes:

[0052] 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.

[0053] OTA detection and degradation rate calculation are based on Example 2.

[0054] The results are as follows Figure 3 and Figure 4 As shown, the degradation rate of OTA in wheat flour was 66.79%, and the degradation rate in corn flour was 33.62%, 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.

[0055] In summary, this invention is the first to discover an enzyme derived from Thermomonasfusca (amino acid sequence shown in SEQ ID NO.1) that possesses the function of degrading ochratoxin A, catalyzing the degradation of ochratoxin A into non-toxic substances. Therefore, it is expected to be applied to the degradation of ochratoxin A with a degradation rate of over 90%, which has substantial application value and great significance in the biological detoxification of food and feed.

[0056] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. The application of an enzyme in the degradation of ochratoxin A, characterized in that, The amino acid sequence of the enzyme is as follows: The sequence shown in SEQ ID NO.1; The enzyme is derived from Thermomonas fusca .

2. A method for degrading ochratoxin A, characterized in that, The method includes: The enzyme described in claim 1 is mixed with a sample containing ochratoxin A to achieve the degradation of ochratoxin A.

3. The application of an enzyme in the preparation of an ochratoxin A biodegrading agent, characterized in that, The biodegradable agent contains the enzyme described in claim 1.

4. The application according to claim 3, 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.

5. The application according to claim 4, characterized in that, The carriers include physiologically acceptable compatible carriers.

6. The application according to claim 5, characterized in that, Physiologically acceptable compatible carriers include any one or a combination of at least two of the following: rice husk, rice bran, maltodextrin, cyclodextrin, wheat bran, starch, bentonite, oligosaccharides, or yeast cell walls.

7. The application according to claim 4, characterized in that, The preservative includes any one or a combination of at least two of potassium sorbate, ethylparaben, or methylparaben.

8. The application according to claim 4, characterized in that, The protein protectant includes any one or a combination of at least two of bovine serum albumin, mannitol, glycerol, butylene glycol, sodium chloride, or sodium benzoate.

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