Preparation and application of bacillus subtilis sourced salt-tolerant amine oxidase KCYOBN capable of degrading biogenic amine

Abstract

The invention discloses preparation and application of salt-tolerant amine oxidase KCYOBN sourced from bacillus subtilis and capable of degrading biogenic amine, and belongs to the technical field of molecular biology. The invention provides the amine oxidase from bacillus subtilis, and the expression of the amine oxidase in escherichia coli is realized. The invention also provides an application of the amine oxidase in degradation of biogenic amines, the amine oxidase is added into fermented food, the biogenic amines can be effectively degraded, an enzyme library for degrading the biogenic amines can be expanded, and the safety of the fermented food is further improved.

Description

Technical Field

[0001] The present invention relates to the preparation and application of a salt-tolerant amine oxidase KCYOBN that degrades biogenic amines and is derived from Bacillus subtilis, belonging to the technical field of molecular biology. Background Art

[0002] Biogenic amines are a class of low-molecular-weight nitrogen-containing organic compounds with significant biological activities, widely present in various fermented foods such as dairy products, fish products, meat products, fermented vegetables, and alcoholic beverages. In highly salted fermented foods such as soy sauce, broad bean paste, and certain pickled foods, the content of these biogenic amines is often high. The main types include putrescine, tyramine, histamine, cadaverine, etc. Intake of excessive biogenic amines can lead to health risks, from mild allergic symptoms to severe poisoning, and may even be fatal. Therefore, strictly controlling the content of biogenic amines in fermented foods is particularly crucial for improving their quality.

[0003] Current research indicates that biogenic amines cannot be removed by heating and cooking, and their generation needs to be controlled during the fermentation process. Amine oxidase derived from microorganisms is an effective way to degrade biogenic amines in foods and plays an important role in ensuring food safety. Amine oxidase can oxidize and remove the amino group of biogenic amines, converting them into corresponding aldehydes, thereby reducing their content. Flavin-dependent monoamine oxidase uses FAD as a cofactor to be responsible for oxidizing biogenic amines to generate imino groups, which are further hydrolyzed to form aldehyde compounds.

[0004] However, a high-salt environment will inhibit the activity of amine oxidase. Excessive salt concentration may affect the structure and function of the enzyme, becoming a challenge in its application in highly salted fermented foods. Currently, there is a lack of research on amine oxidase suitable for high-salt conditions and its enzymatic properties. Therefore, obtaining salt-tolerant amine oxidase is crucial for improving the quality of fermented foods. Summary of the Invention

[0005] The purpose of the present invention is to solve the problem that the content of biogenic amines in existing traditional fermented foods is generally high, but the salinity will inhibit the catalytic effect of amine oxidase. To provide a salt-tolerant amine oxidase KCYOBN that degrades biogenic amines and is derived from Bacillus subtilis, and use the method of degrading biogenic amines with the recombinant enzyme to reduce the content of biogenic amines in fermented foods and improve the quality of traditional fermented foods.

[0006] The first object of the present invention is to provide an amine oxidase, which is (a) or (b):

[0007] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.1;

[0008] (b) A protein derived from (a) in which one or several amino acids in the amino acid sequence are substituted, deleted, or added and has amine oxidase activity.

[0009] The present invention also provides a gene encoding the amine oxidase.

[0010] In one embodiment, the gene contains the nucleotide sequence shown in SEQ ID NO.2.

[0011] The present invention also provides a recombinant expression plasmid carrying the gene.

[0012] In one embodiment, the plasmid includes, but is not limited to, pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K, pPIC9K or pTrc series vectors.

[0013] In one embodiment, the pET series vectors include pET24a(+), pET28a(+), pET29a(+), pET30a(+); the Duet series vectors include pRSFDuet-1, pCDFDuet-1; the pTrc series vectors include pTrc99a.

[0014] In one embodiment, the recombinant expression plasmid is pET28a(+).

[0015] The present invention also provides a recombinant microbial cell expressing the amine oxidase.

[0016] In one embodiment, the recombinant microbial cell includes, but is not limited to, Escherichia coli, Bacillus or yeast.

[0017] The present invention also provides a genetically engineered bacterium that uses Escherichia coli as a host and expresses the amine oxidase shown in SEQ ID NO.1.

[0018] In one embodiment, the genetically engineered bacterium uses Escherichia coli BL21(DE3) as a host.

[0019] In one embodiment, the genetically engineered bacterium uses a pET series plasmid as an expression vector.

[0020] In one embodiment, the genetically engineered bacterium uses pET28a(+) as an expression vector and expresses the amine oxidase shown in SEQ ID NO.1.

[0021] The present invention also provides a method for constructing the genetically engineered bacterium, which is to ligate the gene sequence shown in SEQ ID NO.2 with a vector and transform it into Escherichia coli cells.

[0022] In one embodiment, the vector is pET28a(+).

[0023] In one embodiment, the gene sequence is ligated between the HindIII and BamHI sites of pET28a(+).

[0024] The present invention also provides a method for producing amine oxidase, which comprises inoculating the genetically engineered bacterium into a culture medium for culturing and collecting the amine oxidase.

[0025] In one embodiment, the method is to collect the bacterial cells from the cell culture broth, break the cells to obtain a crude enzyme solution containing amine oxidase.

[0026] In one embodiment, the method further comprises purifying the crude enzyme solution.

[0027] In one embodiment, the purification includes, but is not limited to, purification methods well known in the art such as affinity chromatography, gel filtration chromatography / molecular sieve, ion exchange chromatography, ammonium sulfate precipitation / polyethylene glycol (PEG) precipitation, etc.

[0028] In one embodiment, the affinity chromatography includes metal chelate affinity chromatography (such as purifying proteins using His tags), immunoaffinity chromatography, etc.

[0029] In one embodiment, the purification is carried out by nickel column affinity chromatography.

[0030] In one embodiment, the culturing is to inoculate the genetically engineered bacterium into LB medium and culture until the OD600 reaches 0.6 - 0.8, and then induce enzyme production with IPTG.

[0031] In one embodiment, the induction is carried out at 16 - 37 °C.

[0032] In one embodiment, the final concentration of IPTG is 0.4 - 0.6 mmol·L -1 。

[0033] In one embodiment, the induction is carried out at 25 °C and 200 rpm for 10 h.

[0034] The present invention also provides the application of the amine oxidase in reducing biogenic amines.

[0035] In one embodiment, the application is to contact the amine oxidase with biogenic amines in the environment.

[0036] In one embodiment, the environment includes a liquid environment, a semi-solid environment or a solid environment.

[0037] In one embodiment, the application includes reducing the content of biogenic amines in fermented foods.

[0038] In one embodiment, the fermented food includes fermented dairy products, fermented bean products, fermented meat products, fermented cereal products, fermented vegetable products, fermented condiments or fermented alcoholic beverages.

[0039] In one embodiment, the fermented dairy products include, but are not limited to, yogurt or cheese; the cheese includes, but is not limited to, cheddar cheese, mozzarella cheese, feta cheese, etc.

[0040] In one embodiment, the fermented meat products include, but are not limited to, sausages (such as German sausages, Italian salami, etc.), hams, bacon, dried meats, fish sauce.

[0041] In one embodiment, the fermented vegetable products include, but are not limited to, pickles, sauerkraut, kimchi, pickled vegetables, etc.

[0042] In one embodiment, the fermented soy products include, but are not limited to, fermented bean curd or natto.

[0043] In one embodiment, the fermented condiments include, but are not limited to, soy sauce, miso, vinegar, broad bean paste.

[0044] In one embodiment, the fermented alcoholic beverages include, but are not limited to, beer, wine, rice wine.

[0045] In one embodiment, the application is to add the amine oxidase to yellow rice wine to reduce the content of the main biogenic amines therein.

[0046] In one embodiment, the application is to add the amine oxidase to yellow rice wine and react at 25 - 28 °C for at least 7 days.

[0047] In one embodiment, the application is to add the amine oxidase to cooking wine to reduce the content of the main biogenic amines therein.

[0048] In one embodiment, the application is to add the amine oxidase to cooking wine and react at 25 - 28 °C for at least 7 days.

[0049] In one embodiment, the application is to add the amine oxidase to soy sauce to reduce the content of the main biogenic amines therein.

[0050] In one embodiment, the application is to add the amine oxidase to soy sauce and react at 25 - 28 °C for at least 7 days.

[0051] In one embodiment, the application is to add the amine oxidase to fish sauce to reduce the content of the main biogenic amines therein.

[0052] In one embodiment, the application is to add the amine oxidase to fish sauce and react at 25 - 28 °C for at least 7 days.

[0053] In one embodiment, the application is to add the amine oxidase to red wine to reduce the content of major biogenic amines therein.

[0054] In one embodiment, the application is to add the amine oxidase to red wine and react at 25-28 °C for at least 7 days.

[0055] In one embodiment, the biogenic amines include, but are not limited to, one or more of tryptamine, phenethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine.

[0056] The present invention provides an enzyme preparation for degrading biogenic amines, and the enzyme preparation contains an amine oxidase with an amino acid sequence as shown in SEQ ID NO.1.

[0057] In one embodiment, the enzyme preparation further contains a stabilizer that protects the stability of the enzyme during production, storage, and use, and prevents the enzyme from being inactivated or degraded.

[0058] In one embodiment, the stabilizer includes, but is not limited to, saccharides, polyols, proteins, polymers, metal ions, etc.

[0059] The present invention provides a method for degrading biogenic amines in an environmental system. The method is to use the amine oxidase or a genetically engineered bacterium expressing the amine oxidase to degrade biogenic amines. The amino acid sequence of the amine oxidase is as shown in SEQ ID NO.1, and the environmental system is a non-in vivo environment.

[0060] In one embodiment, the environmental system includes, but is not limited to, dairy products, fish products, meat products, and fermented foods.

[0061] In one embodiment, the environmental system includes, but is not limited to, fermented foods such as yellow rice wine, soy sauce, low-salt soy sauce, cooking wine, fruit wine, and fish sauce.

[0062] In one embodiment, the genetically engineered bacterium uses a bacterium or a fungus as a host cell.

[0063] In one embodiment, the biogenic amines include, but are not limited to, one or more of tryptamine, phenethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine.

[0064] The present invention also provides the application of the above enzyme preparation, or the above amine oxidase with an amino acid sequence as shown in SEQ ID NO.1, or a genetically engineered bacterium expressing an amine oxidase with an amino acid sequence as shown in SEQ ID NO.1 in the preparation of a product for degrading biogenic amines in an environmental system.

[0065] The present invention also provides the use of the above enzyme preparation, or the amine oxidase with the amino acid sequence as shown in SEQ ID NO.1, or the genetically engineered bacterium expressing the amine oxidase with the amino acid sequence as shown in SEQ ID NO.1 in the preparation of fermented foods.

[0066] In one embodiment, the fermented foods include but are not limited to fermented vegetables and alcoholic beverages.

[0067] In one embodiment, the fermented foods include but are not limited to yellow rice wine, soy sauce, low-salt soy sauce, cooking wine, fruit wine and fish sauce.

[0068] In one embodiment, the host cell of the genetically engineered bacterium is a bacterium or a fungus.

[0069] The present invention also provides the use of the above enzyme preparation, or the amine oxidase with the amino acid sequence as shown in SEQ ID NO.1, or the genetically engineered bacterium expressing the amine oxidase with the amino acid sequence as shown in SEQ ID NO.1 in the degradation of biogenic amines in the food field.

[0070] Beneficial effects:

[0071] (1) The recombinant amine oxidase KCYOBN from Bacillus subtilis provided by the present invention has strong performance in degrading biogenic amines. This enzyme also has certain pH stability, temperature stability and can tolerate a certain concentration of NaCl. When the concentration of NaCl is 20%, KCYOBN can maintain a relative enzyme activity of more than 84% for biogenic amines, which helps to achieve the degradation of biogenic amines in fermented foods, especially fermented foods with high salinity.

[0072] (2) The present invention also provides the use of the amine oxidase KCYOBN in the degradation of biogenic amines in fermented foods. This enzyme can degrade the biogenic amines in commercially available soy sauce 1 within 7 days, and the degradation rates of phenethylamine, putrescine, cadaverine, histamine, tyramine and spermidine reach 17.70%, 33.10%, 50.14%, 56.11%, 44.63% and 15.73% respectively. The total degradation rate reaches 29.02%.

[0073] (3) The present invention also provides the use of the amine oxidase KCYOBN in the degradation of biogenic amines in fermented foods. This enzyme can degrade the biogenic amines in commercially available yellow rice wine with an alcohol content of 18% vol within 7 days, and the degradation rates of phenethylamine, putrescine, cadaverine, histamine, tyramine and spermidine reach 3.16%, 16.97%, 2.74%, 22.30%, 3.65% and 38.74% respectively. The total degradation rate reaches 15.24%.

[0074] (4) The present invention also provides the application of the amine oxidase KCYOBN in degrading biogenic amines in fermented foods. This enzyme can degrade biogenic amines in commercially available cooking wine within 7 days, and the degradation rates of tryptamine, phenethylamine, putrescine, cadaverine, histamine, tyramine and spermidine are 72.70%, 26.01%, 38.76%, 52.11%, 40.05%, 16.46% and 0.6% respectively. The total degradation rate reaches 45.97%.

[0075] (5) The present invention also provides the application of the amine oxidase KCYOBN in degrading biogenic amines in fermented foods. This enzyme can degrade biogenic amines in commercially available fish sauce within 7 days, and the degradation rates of tryptamine, phenethylamine, putrescine, cadaverine, histamine, tyramine and spermidine are 17.60%, 100%, 24.36%, 8.52%, 66.94%, 12.49% and 47.21% respectively. The total degradation rate reaches 38.33%.

[0076] (6) The present invention also provides the application of the amine oxidase KCYOBN in degrading biogenic amines in fermented foods. This enzyme can degrade biogenic amines in commercially available red wine within 7 days, and the degradation rates of tryptamine, phenethylamine, putrescine, cadaverine, histamine, tyramine and spermidine are 99.81%, 1.99%, 2.67%, 35.55%, 6.56%, 32.02% and 4.82% respectively. The total degradation rate reaches 27.80%.

[0077] (7) The present invention also provides the application of the amine oxidase KCYOBN in degrading biogenic amines in fermented foods. This enzyme can degrade biogenic amines in commercially available soy sauce 2 within 7 days, and the degradation rates of tryptamine, phenethylamine, putrescine, cadaverine, histamine, tyramine and spermidine are 4.07%, 16.89%, 12.00%, 24.87%, 7.85%, 8.86% and 22.56% respectively. The total degradation rate reaches 10.76%.

[0078] (8) The present invention also constructs recombinant Escherichia coli expressing amine oxidase KCYOBN. After inducing culture at 25 °C and 200 r·min -1 for 10 h and then performing ultrasonic disruption, the protein content of the collected crude enzyme solution is measured. The results show that 100 mL of bacterial liquid with an OD of 1 contains 2000 μg of KCYOBN enzyme protein. Description of the Drawings

[0079] Figure 1 Agarose gel electrophoresis verification diagram of the amine oxidase gene: M: DNA Maker; 1: PCR amplification product (1434 bp).

[0080] Figure 2 Plasmid map of the amine oxidase pET28a-KCYOBN expression vector.

[0081] Figure 3 The degradation ability of amine oxidase KCYOBN to different biogenic amines under different induction conditions. Putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenethylamine (PHE), tryptamine (TRY), spermine (SPE) and spermidine (SPD); (A): M: PageRuler TM Prestained protein molecular weight standard, 10 to 180 kDa; 1: pET28a empty vector; 2 - 3: Supernatant and precipitate of crude enzyme solution under the induction conditions of 0.2 mM IPTG, 16 °C, 10 h; 4 - 5: Supernatant and precipitate of crude enzyme solution under the induction conditions of 0.5 mM IPTG, 20 °C, 14 h; 6 - 7: Supernatant and precipitate of crude enzyme solution under the induction conditions of 0.4 mM IPTG, 25 °C, 10 h; 8 - 9: Supernatant and precipitate of crude enzyme solution under the induction conditions of 0.2 mM IPTG, 30 °C, 10 h; (B): Enzyme activity of different biogenic amines under different induction conditions.

[0082] Figure 4 Electrophoresis diagram of the protein separation and purification process of recombinant amine oxidase KCYOBN; M: Unstained Protein Ladder; 1: Purified protein amine oxidase KCYOBN (about 58.71 kDa).

[0083] Figure 5 Relative enzyme activity of amine oxidase KCYOBN at different temperatures. Putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenethylamine (PHE), tryptamine (TRY), spermine (SPE) and spermidine (SPD).

[0084] Figure 6 Temperature stability of amine oxidase KCYOBN. Putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenethylamine (PHE), tryptamine (TRY), spermine (SPE) and spermidine (SPD).

[0085] Figure 7 Relative enzyme activity of amine oxidase KCYOBN at different pH values. Putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenethylamine (PHE), tryptamine (TRY), spermine (SPE) and spermidine (SPD).

[0086] Figure 8 pH stability of amine oxidase KCYOBN. Putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenethylamine (PHE), tryptamine (TRY), spermine (SPE) and spermidine (SPD).

[0087] Figure 9 The relative residual enzyme activity of amine oxidase KCYOBN under different ethanol conditions; putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenethylamine (PHE), tryptamine (TRY), spermine (SPE) and spermidine (SPD).

[0088] Figure 10 The relative residual enzyme activity of amine oxidase KCYOBN under different concentrations of NaCl; putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenethylamine (PHE), tryptamine (TRY), spermine (SPE) and spermidine (SPD). Detailed implementation manners

[0089] (I) Technical terms:

[0090] The "dairy products" involved in the present invention refer to foods made from animal milk through different processing methods, and their types include but are not limited to pasteurized milk, sterilized milk, formulated milk, fermented milk, whole milk powder, skim milk powder, whole milk with added sugar powder, flavored milk powder, infant milk powder and other formula milk powders, condensed milk, milk fat, cheese, ice cream, casein, milk tablets, lactose, etc.

[0091] The "fish products" involved in the present invention refer to products made mainly from fish meat or certain organs of fish through various processing methods; the processing methods include pickling, smoking, drying, freezing, canning, fermentation, etc.

[0092] The "meat products" involved in the present invention refer to products made from animal muscle tissues or edible internal organs as the main raw materials through various processing methods; according to the national standard of "Meat and Meat Products Terms" (GB / T 19480-2009), meat products are divided into two major categories: Chinese-style meat products and Western-style meat products; the Chinese-style meat products include: Cured meat, Corned meat, Chinese ham, Dried meat floss, Dried meat dice, Dried meat slice, Stewed meat in seasoning, Meat flavored with fermented rice, Smoked meat products, Chinese sausage, Sausage, Air-dried sausage, Fresh sausage product, Smoked and fresh sausage, Semi-dry sausage, Dry sausage, Prepare meat products, Meat cake, Salted meat; the Western-style meat products include cooked and smoked ham, cooked and smoked sausage, sausage products, blood sausage, fermented sausage, bacon, ham or meat enema.

[0093] The "fermented food" involved in the present invention is a food made through the fermentation of microorganisms (such as bacteria, yeast or fungi), and the types of the fermented food include but are not limited to alcoholic beverages, fish and shrimp sauce, fruit wine, soy sauce, yogurt, cheese, fermented glutinous rice, pickled vegetables, soy sauce, vinegar, fermented soybeans, yellow rice wine, beer, wine, etc.

[0094] The "biogenic amines" involved in the present invention are low-molecular-weight organic compounds with biological activity and containing amino groups, including but not limited to tryptamine (TRY), phenethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), tyramine (TYR), spermidine (SPD), spermine (SPE).

[0095] (II) Reagents

[0096] The yellow rice wine, soy sauce, low-salt soy sauce, cooking wine, fruit wine, fish sauce, etc. involved in the following examples are purchased from a certain supermarket in Wuxi, Jiangsu Province.

[0097] (III) Culture Medium

[0098] LB medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, adjust the pH to 7.0 with NaOH, autoclave at 121 °C for 20 min.

[0099] TB medium: Purchased from Qingdao Haibo Company. Autoclave at 121 °C for 20 min.

[0100] (IV) Detection method

[0101] The content of biogenic amines was detected by high performance liquid chromatography (HPLC).

[0102] Determination of enzyme activity: The activity of biogenic amine oxidase was determined by an indirect assay method using catalase. Amine oxidase acts on biogenic amines, degrading them into corresponding aldehydes and hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide reacts with 4-aminoantipyrine and 2,4,6-tribromo-3-hydroxybenzoic acid to form a quinone dye. This product has a maximum absorbance at 510 nm. The activity of amine oxidase is linearly related to the color intensity of the product within a certain range. Therefore, the activity of amine oxidase can be determined by measuring the change in A510.

[0103] The reaction was carried out in a 96-well plate. The reaction system included 10 μL of enzyme solution (200 mg·L -1 ), 100 μL of prepared solution (including 200 mmol·L -1 , potassium phosphate buffer with pH = 7.6, 1.5 mmol·L -1 4-aminoantipyrine, 1 mmol·L -1 2,4,6-tribromo-3-hydroxybenzoic acid). To initiate the reaction, 20 μL of biogenic amine solution (10 mmol·L -1 ) and 70 μL of peroxidase (1.4 mg·mL -1 ) were added. The absorbance was measured at 510 nm. The reaction temperature was 37 °C and the reaction time was 30 min. A change in absorbance of 0.01 per minute was defined as one enzyme activity unit (U).

[0104] Definition of specific activity of amine oxidase (U / mg): The enzyme activity contained in each milligram of protein.

[0105] Example 1: PCR amplification of amine oxidase gene KCYOBN

[0106] (1) Primers were designed based on the amine oxidase gene (WFA13853.1) in Bacillus subtilis in the NCBI database. Using the DNA of Bacillus subtilis LH100-20 stored in our laboratory as a template, the amine oxidase gene KCYOBN was amplified. The primers required for amplification were as follows:

[0107] F: 5'-CGCggatccTTGAATTCTCTAATGAATGATGACATGGT-3';

[0108] R: 5'-CCCaagcttAGGTAAGCGATTGACCTCATAAGCAA-3'.

[0109] Prepare the PCR reaction solution according to the requirements of the reaction system of TaKaRa high-fidelity enzyme. The PCR amplification system is as follows: pre-denature at 98°C for 10 s, anneal at 60°C for 30 s, extend at 72°C (1 min·kb -1 ) for 35 cycles.

[0110] (2) Verify the amplification result by 1.2% agarose gel electrophoresis of the PCR product. As Figure 1 shown, the size of the amplified sequence is the same as that of the target gene sequence, about 1434 bp, indicating successful amplification. Purify the PCR product and send it to the company for sequencing. The sequencing result is shown in SEQ ID NO.2.

[0111] Example 2: Construction of a genetically engineered bacterium with the amine oxidase gene KCYOBN

[0112] Ligate the amine oxidase gene KCYOBN shown in SEQ ID NO.2 amplified in Example 1 with a plasmid and transform it into microbial cells to construct a genetically engineered bacterium with the amine oxidase gene KCYOBN.

[0113] Optionally, the plasmid includes but is not limited to pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K, pPIC9K or pTrc series vectors; the pET series vectors include pET24a(+), pET28a(+), pET29a(+), pET30a(+); the Duet series vectors include pRSFDuet-1, pCDFDuet-1; the pTrc series vectors include pTrc99a.

[0114] Optionally, the host is a bacterial cell or a fungal cell, including but not limited to Escherichia coli, Bacillus or Saccharomyces cerevisiae.

[0115] Taking recombinant Escherichia coli as an example, the construction process of the genetically engineered bacterium pET28a-KCYOBN is described as follows:

[0116] (1) Digestion and ligation.

[0117] The plasmid pET-28a(+) and the gene fragment shown in SEQ ID NO.2 obtained in Example 1 were respectively double digested with restriction enzymes. The digestion system was as follows: 50 μL of plasmid, 2.5 μL each of restriction enzymes Hind III and BamHI, and 5 μL of Green Buffer. The components in the digestion system were thoroughly mixed and then reacted in a 37°C metal bath for 60 min. After the gene fragment and plasmid after double digestion were recovered and purified, they were mixed at a molar ratio of 3 - 10:1, and an equal volume of Solution I ligase was added. After thorough mixing, it was incubated in a 16°C metal bath overnight to prepare the recombinant vector pET-28a(+)-KCYOBN.

[0118] (2) Transformation.

[0119] The E.coli BL21(DE3) competent cells stored at -80°C were placed on ice for 10 min. Then, 10 μL of the ligation product to be transformed obtained in step (1) was aspirated with a pipette and added to the competent cells, and gently pipetted and mixed evenly. After uniform mixing, it was ice-bathed for 30 min. After the ice-bath ended, it was heat-shocked at 42°C for 45 s, and immediately taken out and placed in ice for 2 min after completion. Then, 700 μL of LB liquid medium was added, and it was cultured at 37°C with shaking at 200 r·min -1 for 60 min. Centrifuged at 8000 r·min -1 for 1 min, most of the supernatant was discarded, and about 200 μL of the supernatant was left to resuspend the cells. The cell suspension was evenly spread on an LB solid medium plate containing 50 mg·L -1 kanamycin, and inverted and cultured in a 37°C incubator overnight. After culturing until single colonies grew, PCR verification was carried out to screen positive transformants.

[0120] (3) Enzyme digestion verification.

[0121] The plasmid of the recombinant bacteria was extracted and double digested with Hind III and BamHI to obtain the pET-28a(+) fragment and the target fragment KCYOBN respectively. The target fragment was sent to the company for sequencing, and the sequencing result was consistent with the target gene sequence, verifying that the recombinant bacteria E.coli BL21 / pET-28a(+)-KCYOBN was successfully constructed. The recombinant enzyme expressed by this strain was named KCYOBN. The construction process was as Figure 2 shown.

[0122] Example 3: Induced expression and purification of recombinant enzyme KCYOBN

[0123] The specific steps are as follows:

[0124] (1) Inoculate the recombinant bacterium E. coli BL21 / pET-28a(+)-KCYOBN constructed in Example 2 into LB medium containing 50 mg·L -1 kanamycin, and culture it at 37 °C and 150 r·min -1 for 14 h to prepare a seed solution.

[0125] (2) Transfer the obtained seed solution to TB fermentation medium containing 50 mg·L -1 kanamycin at an inoculation amount of 5% (v / v), and culture it at 37 °C and 160 r·min -1 until the OD 600 reaches 0.4 - 0.6, and then culture it under different induction conditions to obtain a bacterial solution.

[0126] (3) Centrifuge the bacterial solution at 4 °C and 12000 r·min -1 for 10 min, collect the lower-layer bacterial cells, resuspend the bacterial cells with 0.1 mol·L -1 sodium phosphate buffer (pH 7.4), and then centrifuge to collect the bacterial cells. Repeat the above steps twice. Use an ultrasonic cell disruptor to disrupt the bacterial cells. The ultrasonic conditions are: 400 W, working for 2 s, with an interval of 3 s, and the disruption time is 30 min. After disruption, centrifuge at 4 °C and 12000 r·min -1 to collect the upper-layer supernatant, filter it through a 0.22 μM filter membrane, and store it at low temperature for later use.

[0127] (4) Optimal induction conditions and enzyme activity of recombinant enzyme KCYOBN

[0128] Culture according to the method in step (2), except that different induction conditions are set as follows: 1) 0.2 mM IPTG, induction temperature 16 °C, induction time 10 h; 2) 0.5 mM IPTG, induction temperature 20 °C, induction time 14 h; 3) 0.4 mM IPTG, induction temperature 25 °C, induction time 10 h; 4) 0.2 mM IPTG, induction temperature 30 °C, induction time 10 h.

[0129] The fermented broth after cultivation was collected, and crude enzyme solution was prepared according to the method of Example 3. The protein content of the collected crude enzyme solution was measured. The results showed that 100 mL of bacterial solution (OD was 1) under the optimal induction conditions contained 2000 μg of KCYOBN enzyme protein. The catalytic activities towards different biogenic amines were also measured. The results showed that after optimization of different induction conditions, it was found that the enzyme activity of recombinant enzyme KCYOBN was relatively the highest under the conditions of IPTG concentration of 0.4 mM, induction temperature of 25 °C, and time of 10 h, which was taken as its optimal induction conditions. The enzyme activities of KCYOBN towards HIS, TYR, PUT, CAD, and PHE were relatively high, being 14.72 U, 13.88 U, 15.14 U, 14.78 U, and 15.75 U( Figure 3 ).

[0130] (5) Purification of recombinant enzyme KCYOBN

[0131] The supernatant obtained in step (4) was subjected to affinity chromatography column His Trap TM HP (GE Healthcare) to purify the protein, and an AKTA avant 25 instrument was used to separate and purify the target protein.

[0132] The supernatant of the crude enzyme solution before purification of KCYOBN and the protein after purification of KCYOBN were respectively subjected to gel electrophoresis analysis, and the results were as Figure 4 shown.

[0133] The results showed that: the molecular weight of recombinant amine oxidase KCYOBN was about 58.71 kDa. The SDS-PAGE gel electrophoresis analysis of the crude enzyme solution confirmed the successful expression of the recombinant engineering bacteria. Lane 1 proved that the target protein was successfully separated and purified by the affinity chromatography nickel column to obtain pure enzyme. The specific enzyme activities of the purified KCYOBN towards substrates HIS, TYR, PUT, CAD, PHE, TRY, SPE, and SPD were measured to be 32.67 U / mg, 76.67 U / mg, 64.45 U / mg, 32.67 U / mg, 28.22 U / mg, 26.89 U / mg, 28.44 U / mg, and 28.44 U / mg respectively.

[0134] Example 4: Preparation of amine oxidase KCYOBN enzyme preparation

[0135] The purified amine oxidase KCYOBN in Example 3 was mixed with an enzyme stabilizer to prepare an enzyme preparation containing amine oxidase KCYOBN. Among them, the stabilizer refers to a substance that can protect the stability of the enzyme during production, storage, and use, preventing the enzyme from inactivating or degrading, including but not limited to sugars, polyols, proteins, polymers, metal ions, etc.

[0136] Example 5: Degradation of biogenic amines by recombinant enzyme KCYOBN at different reaction temperatures and its stability at different temperatures

[0137] The reaction system was carried out according to the standard, that is, the reaction was carried out in a 96-well plate. The reaction system included 10 μL of enzyme solution with a concentration of 200 mg·L -1 , 100 μL of preparation solution (including 200 mmol·L -1 , potassium phosphate buffer with pH = 7.6, 1.5 mmol·L -1 4-aminoantipyrine, 1 mmol·L -1 2,4,6-tribromo-3-hydroxybenzoic acid). To start the reaction, 20 μL of biogenic amine solution (10 mmol·L -1 ) and 70 μL of peroxidase (1.4 mg·mL -1 ) were added. The absorbance was measured at 510 nm. The difference was that the temperature of the reaction system was changed to 20 °C, 28 °C, 37 °C, 45 °C, 50 °C, 55 °C, and 60 °C respectively. The enzyme activity of recombinant enzyme KCYOBN under different temperature reaction conditions was measured. The highest enzyme activity measured at the above temperatures was defined as 100%. The relative enzyme activities at each temperature were calculated using each biogenic amine as a substrate. The results are as Figure 5 shown. The optimal temperature of KCYOBN was 37 °C. Its activity increased with the increase of temperature between 20 and 37 °C, but when the temperature exceeded 45 °C, the enzyme activity decreased rapidly. When the temperature was 55 °C, the remaining enzyme activities for HIS (24.95 ± 5.25%), TYR (8.44 ± 3.79%), PUT (21.61 ± 3.09%), and CAD (20.26 ± 5.04%) were no more than 25%.

[0138] The purified enzyme solution in Example 3 was incubated in a water bath at 20 °C, 28 °C, 37 °C, 45 °C, 50 °C, 55 °C, and 60 °C for 1 h respectively, and then the enzyme activity was measured according to the standard enzyme reaction system to determine its stable temperature range. The results are shown in Figure 6 . As the incubation temperature increased, the stability of the enzyme decreased.

[0139] Example 6: Degradation of biogenic amines by recombinant enzyme KCYOBN at different reaction pH values and its enzyme stability under different pH conditions

[0140] The reaction system was carried out according to the standard in a 96-well plate. The reaction system included 10 μL of enzyme solution with a concentration of 200 mg·L -1 , 100 μL of preparation solution (including 200 mmol·L -1 , potassium phosphate buffer with different pH values, 1.5 mmol·L -1 4-aminoantipyrine, 1 mmol·L -12,4,6 - tribromo - 3 - hydroxybenzoic acid), to initiate the reaction, 20 μL of biogenic amine solution (10 mmol·L -1 ) and 70 μL of peroxidase (1.4 mg·mL -1 ) were added, the absorbance was measured at 510 nm, and the reaction temperature was 37 °C. The difference is that the pH of the reaction system was adjusted to 4.0 - 9.0 respectively, the enzyme activity of recombinant enzyme KCYOBN under different pH reaction conditions was measured, with the highest enzyme activity taken as 100%, and the relative enzyme activities at each temperature were calculated using each individual biogenic amine as the substrate. The results are as Figure 7 shown, the optimal reaction pH of KCYYOBN is in the range of 7.0 - 8.0.

[0141] After diluting the purified recombinant enzyme solution in Example 3 by an appropriate multiple, the enzyme was incubated for 30 min under different pH environmental conditions, and the enzyme activity was measured. The results are shown in Figure 8 . Generally speaking, when the enzyme is around pH 6.0, the enzyme is the most stable. KCYOBN can retain about 60% or more of its enzyme activity between pH 4.0 - 8.0. The relatively wide pH applicability and stability range enable amine oxidase KCYOBN to have the potential to be applied to fermented foods.

[0142] Example 7: Effects of different concentrations of ethanol on amine oxidase KCYOBN

[0143] The reaction system was carried out in a 96 - well plate according to the standard. The reaction system included 10 μL of enzyme solution with a concentration of 200 mg·L -1 , 100 μL of formulated solution (including 200 mmol·L -1 , potassium phosphate buffer with pH = 7.6, 1.5 mmol·L -1 4 - aminoantipyrine, 1 mmol·L -1 2,4,6 - tribromo - 3 - hydroxybenzoic acid), to initiate the reaction, 20 μL of biogenic amine solution (10 mmol·L -1 ) and 70 μL of peroxidase (1.4 mg·mL -1) The absorbance was measured at 510 nm and the reaction temperature was 37 °C. The difference was that ethanol with different final concentrations (5% vol - 25% vol) was added respectively, and the reaction system without ethanol (0% vol) was used as a control. The results showed that the enzyme activities of recombinant amine oxidase KCYOBN against various biogenic amines in the reaction system were inhibited to varying degrees. Under the condition of 5% vol ethanol concentration, KCYOBN could retain more than 80% of the enzyme activity for HIS, TYR, PUT and CAD. When the ethanol concentration was 10% vol, the enzyme activity of KCYOBN could be maintained above 60%. When the ethanol concentration exceeded 20% vol, KCYOBN remained more than 50% of the enzyme activity. When the ethanol concentration was 25% vol, KCYOBN remained about 20% or more of the enzyme activity (Table 1).

[0144] Table 1 Enzyme activity values (unit: U / mg) of amine oxidase KCYOBN under the condition of high - concentration ethanol (25% vol)

[0145]

[0146] Thus, it can be seen that this amine oxidase KCYOBN has the catalytic ability for biogenic amines in a system containing a low - concentration ethanol solution, and this characteristic provides a good basis for its application in the degradation of biogenic amines in alcoholic beverages containing low - concentration ethanol.

[0147] Example 8: Effects of different concentrations of NaCl on amine oxidase KCYOBN

[0148] The reaction system was carried out in a 96 - well plate according to the standard. The reaction system included 10 μL of enzyme solution with a concentration of 200 mg·L -1 , 100 μL of prepared solution (including potassium phosphate buffer with a concentration of 200 mmol·L -1 and pH = 7.6, 1.5 mmol·L -1 4 - aminophenazone, 1 mmol·L -1 2,4,6 - tribromo - 3 - hydroxybenzoic acid). To start the reaction, 20 μL of biogenic amine solution (10 mmol·L -1 ) and 70 μL of peroxidase (1.4 mg·mL -1) The absorbance was measured at 510 nm and the reaction temperature was 37 °C. The difference was that different final concentrations of NaCl (4% - 20%, w / v) were added respectively, and the reaction system without NaCl (0%, w / v) was used as a control. The results showed that as the NaCl concentration increased, the relative enzyme activity of KCYOBN first increased and then decreased. Especially when the NaCl concentration reached 16%, the relative enzyme activities of KCYOBN against HIS, TYR, PUT, and CAD reached 176.87 ± 3.01%, 197.01 ± 4.73%, 112.76 ± 3.75%, and 159.86 ± 5.19%, respectively. When the NaCl concentration exceeded 16%, the enzyme activity decreased (Table 2). When the NaCl concentration was 20%, KCYOBN maintained a relative enzyme activity of more than 84% against biogenic amines.

[0149] Table 2 Enzyme activity values (unit: U / mg) of amine oxidase KCYOBN under the condition of high - concentration NaCl (20%, w / v)

[0150]

[0151] It can be seen from this that the amine oxidase KCYOBN has good catalytic ability for biogenic amines in the system containing NaCl, and this characteristic provides a good basis for its application in the degradation of biogenic amines in fermented foods containing NaCl.

[0152] Example 9: Application of amine oxidase KCYOBN in commercially available soy sauce

[0153] Soy sauce is mainly brewed from soybeans or black beans, wheat or bran, and salt through processes such as oil production and fermentation. The composition of soy sauce is relatively complex. The range of the total biogenic amine content in the sample is 10 - 1898.17 mg / L, and the salt content of ordinary soy sauce is about 12 g NaCl / 100 mL. The pH is about 4.4 - 4.6. NaCl and acidity limit the progress of the enzymatic reaction.

[0154] The recombinant amine oxidase KCYOBN prepared in Example 3 was ultra - filtered and concentrated, and the protein concentration was adjusted to 150 μg / ml, and then added to commercially available soy sauce so that the enzyme concentration in the soy sauce was 75 μg / mL. It was left standing at room temperature (25 °C) for 7 days. The results showed that the amine oxidase KCYOBN could effectively reduce the biogenic amine content in low - salt soy sauce.

[0155] As shown in Table 3, the degradation rates of this enzyme for phenethylamine, putrescine, cadaverine, histamine, tyramine, and spermidine in commercially available soy sauce 1 reached 17.70%, 33.10%, 50.14%, 56.11%, 44.63%, and 15.73% respectively within 7 days. The total degradation rate reached 29.02%.

[0156] Table 3 Biogenic amine content (mg / L) in soy sauce before and after adding KCYOBN and the degradation rate (%) of the corresponding biogenic amines

[0157]

[0158] Example 10: Application of amine oxidase KCYOBN in Chinese rice wine

[0159] Chinese rice wine is a fermented wine brewed mainly from rice, millet, corn, wheat, water, etc. with the addition of koji and / or some enzyme preparations, yeast and other saccharification and fermentation agents. The alcohol content is about 14%vol - 20%vol. Some organic acids are produced during the brewing process, and its pH value is about 3.5 - 4.5. The total biogenic amine content varies. The relatively high concentration of ethanol and high acidity in Chinese rice wine are very unfavorable for the enzymatic catalytic reaction. Therefore, controlling the biogenic amine content in Chinese rice wine by enzymatic method requires more stringent conditions. The specific steps are as follows:

[0160] Adjust the protein concentration of the recombinant amine oxidase KCYOBN to 150 μg / ml and add it to Chinese rice wine at a final concentration of 75 μg / mL. React at room temperature of about 28°C for 7 days. The control group is a commercially available Chinese rice wine (18%vol) with the enzyme replaced by PBS buffer. The results are shown in Table 4.

[0161] As shown in Table 4, this enzyme can degrade phenethylamine, putrescine, cadaverine, histamine, tyramine and spermidine in 18%vol commercially available Chinese rice wine within 7 days, and the degradation rates are 3.16%, 16.97%, 2.74%, 22.30%, 3.65% and 38.74% respectively. The total degradation rate reaches 15.24%. Table 4 Biogenic amine content (mg / L) in Chinese rice wine before and after adding KCYOBN and the degradation rate (%) of the corresponding biogenic amines

[0162]

[0163] Example 11: Application of amine oxidase KCYOBN in cooking wine

[0164] "Cooking wine" is the name of cooking wine, which is brewed by adding Chinese rice wine and has a low alcohol concentration, less than 15%. Cooking wine contains both ethanol and NaCl, which is very unfavorable for the enzymatic catalytic reaction. The specific steps are as follows:

[0165] Adjust the protein concentration of the recombinant amine oxidase KCYOBN to 150 μg / ml and add it to cooking wine at a final concentration of 75 μg / mL. React at room temperature of about 28°C for 7 days. The control group is a commercially available cooking wine with the enzyme replaced by PBS buffer. The results are shown in Table 5.

[0166] As shown in Table 5, the enzyme can degrade tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine and spermidine in commercial cooking wine within 7 days with degradation rates of 72.70%, 26.01%, 38.76%, 52.11%, 40.05%, 16.46% and 0.6%, respectively. The total degradation rate reaches 45.97%.

[0167] Table 5 Biogenic amine content (mg / L) of cooking wine before and after adding KCYOBN and the degradation rate of corresponding biogenic amines (%)

[0168]

[0169]

[0170] Example 12: Application of amine oxidase KCYOBN in fish sauce

[0171] Fish sauce is made from fish byproducts or fish viscera, with 30% to 40% (mass fraction) salt added, and fermented in the sun for 1 to 2 years. It has high nutritional value and delicious taste. Natural fermentation is a key step in producing high-quality fish sauce, but this process also produces a large amount of biogenic amines. The biogenic amine content of fish sauce products on the market varies. The specific steps are as follows:

[0172] The recombinant amine oxidase KCYOBN was adjusted to a protein concentration of 150 ug / ml, added to the fish sauce at a final concentration of 75 μg / mL, and reacted at room temperature of about 28° C. for 7 days. The control group was a commercial fish sauce in which the enzyme was replaced with PBS buffer. The results are shown in Table 6.

[0173] As shown in Table 6, the enzyme was able to degrade tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine and spermidine in commercial fish sauce within 7 days with degradation rates of 17.60%, 100%, 24.36%, 8.52%, 66.94%, 12.49% and 47.21%, respectively, and the total degradation rate reached 38.33%.

[0174] Table 6 Biogenic amine content (mg / L) of fish sauce before and after adding KCYOBN and the degradation rate of corresponding biogenic amines (%)

[0175]

[0176] Example 13: Application of amine oxidase KCYOBN in red wine

[0177] The volume fraction of ethanol in wine is generally above 13%, which not only increases the difficulty of BA degradation in wine but also significantly enhances the toxicity of BA. The specific steps are as follows: Adjust the protein concentration of the recombinant amine oxidase KCYOBN to 150 μg / ml and add it to red wine at a final concentration of 75 μg / mL. React at room temperature (about 28°C) for 7 days. The control group is red wine with the enzyme replaced by PBS buffer. The results are shown in Table 7.

[0178] As shown in Table 7, the enzyme can degrade tryptamine, phenethylamine, putrescine, cadaverine, histamine, tyramine, and spermidine in commercially available red wine within 7 days, and the degradation rates reach 99.81%, 1.99%, 2.67%, 35.55%, 6.56%, 32.02%, and 4.82% respectively. The total degradation rate reaches 27.80%.

[0179] Table 7 Biogenic amine content (mg / L) in red wine before and after adding KCYOBN and the degradation rate (%) of the corresponding biogenic amines

[0180]

[0181] Example 14: Application of amine oxidase KCYOBN in commercially available soy sauce 2

[0182] Soy sauce is mainly brewed from soybeans or black beans, wheat or bran, and salt through processes such as oil production and fermentation. The composition of soy sauce is relatively complex, and the total biogenic amine content of the sample ranges from 10 - 1898.17 mg / L. The salt content of low-salt soy sauce is about 8 g NaCl / 100 mL. The pH is about 4.4 - 4.6. NaCl and acidity limit the enzyme-catalyzed reaction.

[0183] Ultrafilter and concentrate the recombinant amine oxidase KCYOBN prepared in Example 3 to adjust the protein concentration to 150 μg / ml, and add it to commercially available low-salt soy sauce at a final concentration of 75 μg / mL. Let it stand at room temperature (25°C) for 7 days. The results show that amine oxidase KCYOBN can effectively reduce the biogenic amine content in low-salt soy sauce.

[0184] As shown in Table 8, the enzyme can degrade tryptamine, phenethylamine, putrescine, cadaverine, histamine, tyramine, and spermidine in commercially available soy sauce 2 within 7 days, and the degradation rates reach 4.07%, 16.89%, 12.00%, 24.87%, 7.85%, 8.86%, and 22.56% respectively. The total degradation rate reaches 10.76%.

[0185] Table 8 Biogenic amine content (mg / L) in low-salt soy sauce before and after adding KCYOBN and the degradation rate (%) of the corresponding biogenic amines

[0186]

[0187] Example 15: Application of Amine Oxidase KCYOBN in Fermented Sausages

[0188] By mass, take 65 - 80% lean meat and 20 - 35% fat. Wash and remove bones, tendons, muscle membranes, lymph, blood vessels, diseased and damaged parts. Separate the fat and lean, and cut into 4 - 5 cm meat pieces. Put the lean meat and about 5 - 8% ice chips into a chopper and chop for 1 - 3 min. Based on the mass of pork, add 0.01 - 0.15% sodium nitrite, 2 - 3% salt, 0.2 - 0.3% compound phosphate, and 0.05 - 0.06% sodium ascorbate. Spices, pepper, garlic, chili, and nutmeg are 0.2% - 0.3% of the raw meat. Ultrafilter and concentrate the recombinant amine oxidase KCYOBN prepared in Example 3 to adjust the protein concentration to 150 ug / ml, add it to the sausage raw materials, chop for 1 - 2 min, then add the fat and about 5 - 8% ice chips, and chop for 4 - 6 min. After marinating, stuff into sausage casings. After the stuffed sausage is marinated at 4°C for 12 h, raise the temperature to 30°C, ferment until the pH drops to about 5.1, and ripen at 14 - 16°C for 1 - 10 days. Compared with the fermented sausage without adding amine oxidase KCYOBN, the results show that amine oxidase KCYOBN can effectively reduce the biogenic amine content in fermented sausages.

[0189] Currently, the research on the degradation of biogenic amines in fermented food systems by amine oxidase is almost blank. This application lays the technical foundation for the catalytic degradation of biogenic amines in fermented food systems such as yellow rice wine, soy sauce, low-salt soy sauce, cooking wine, fruit wine, and fish sauce, especially under high-salt conditions.

[0190] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Salt-tolerant amine oxidase KCYOBN for biodegradable biogenic amines, characterized in that, It is (a) or (b): (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.1; (b) A protein derived from (a) in which one or several amino acids in the amino acid sequence are substituted, deleted or added and which has amine oxidase activity.

2. A gene encoding the amine oxidase according to claim 1.

3. A recombinant plasmid carrying the gene according to claim 2.

4. A recombinant microbial cell expressing the amine oxidase according to claim 1.

5. A genetically engineered bacterium, characterized in that, Using Escherichia coli as the host to express the amine oxidase shown in SEQ ID NO.

1.

6. The genetically engineered bacterium according to claim 5, characterized in that, Using Escherichia coli BL21(DE3) as the host and pET28a(+) as the expression vector to express the amine oxidase gene shown in SEQ ID NO.

2.

7. The method for constructing the genetically engineered bacterium according to claim 6, characterized in that, Connect the gene sequence shown in SEQ ID NO.2 with the vector and transform it into Escherichia coli cells.

8. A method for preparing the amine oxidase KCYOBN as claimed in claim 1, characterized in that, Cultivate the recombinant microbial cell according to claim 4 or the genetically engineered bacterium according to claim 5 or 6 in a medium for a period of time, and collect the amine oxidase KCYOBN.

9. The method according to claim 8, wherein The method is to collect the bacterial cells from the cell culture solution and break the cells to obtain a crude enzyme solution containing amine oxidase; Optionally, the method further purifies the crude enzyme solution.

10. Use of the amine oxidase KCYOBN according to claim 1 in reducing biogenic amines.

11. The application according to claim 10, characterized in that, The use is to reduce the content of biogenic amines in fermented foods; Optionally, the fermented foods include but are not limited to fermented vegetables and alcoholic beverages.

12. The application according to claim 10 or 11, characterized in that, The biogenic amines include but are not limited to one or more of tryptamine, phenethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine.

13. An enzyme preparation containing the amine oxidase KCYOBN according to claim 1.

14. A method for degrading biogenic amines in an environmental system, characterized in that, Contact the biogenic amines in the environment with the amine oxidase according to claim 1, the recombinant microbial cell according to claim 4, or the genetically engineered bacterium according to claim 5 or 6 to degrade the biogenic amines.

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