Preparation and application of alcohol-resistant amine oxidase LYYOBN2 capable of degrading biogenic amine

Abstract

The invention discloses preparation and application of alcohol-resistant amine oxidase LYYOBN2 capable of degrading biogenic amine, and belongs to the technical field of molecular biology. The invention provides the amine oxidase from bacillus, and the expression of the amine oxidase in escherichia coli is realized. According to the application of the prepared amine oxidase to degradation of the biogenic amine, the enzyme is added into fermented food, the biogenic amine can be effectively degraded in an environment containing ethanol or NaCl, an enzyme library for degrading the biogenic amine 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 an alcohol-tolerant amine oxidase LYYOBN2 for degrading biodegradable biogenic amines, belonging to the technical field of molecular biology. Background Art

[0002] Biogenic amines are a class of nitrogen-containing organic compounds with biological activity, containing one or more amino groups (NH2), and can be regarded as substances in which one to three hydrogen atoms in an ammonia molecule are replaced by alkyl or aromatic groups. Amino acid decarboxylases involved in the formation of biogenic amines are widely distributed in foods and are abundant in common microorganisms in fermented foods such as yeasts, lactic acid bacteria, acetic acid bacteria, and molds. Therefore, biogenic amines are commonly present in foods. Biogenic amines are an essential endogenous physiological component in biologically active cells. There are trace amounts of endogenous biogenic amines in organisms. For example, human plasma contains 0.79 ng / mL histamine and 0.686 ng / mL tyramine. An appropriate amount of biogenic amines synthesized by the human body is helpful for maintaining the normal physiological functions of the body. However, when the body ingests high concentrations of biogenic amines or specific enzymes malfunction, excessive exogenous biogenic amines will cause a series of adverse reactions. The toxicity between different biogenic amines will also show synergistic and additive effects. Therefore, it is crucial to control the content of biogenic amines. The limit standards for biogenic amines in fermented alcoholic beverages are more stringent because about 10% of ethanol can significantly inhibit the activity of biogenic amine oxidase in the human body, indirectly causing excessive accumulation of biogenic amines and enhancing the toxic effects of biogenic amines.

[0003] Amine oxidase has a high reactivity towards biogenic amines. When applied to fermented foods, it not only retains the original flavor and quality but also ensures the safety of the food. This efficient and safe characteristic gives it better technical advantages and a broader application prospect in controlling the content of biogenic amines in foods, and thus has great potential.

[0004] For the catalytic reaction of an enzyme, ethanol, as an organic solvent, can affect the secondary and tertiary structures of proteins, resulting in conformational changes of the enzyme. Such conformational changes may interfere with the active site of the enzyme, thereby reducing its catalytic function.

[0005] Therefore, obtaining an ethanol-tolerant amine oxidase capable of degrading key biogenic amines is crucial for degrading the content of biogenic amines in fermented foods, especially fermented alcoholic beverages. However, there is currently a lack of ethanol-tolerant amine oxidase, and thus its application is limited. Summary of the Invention

[0006] The object of the present invention is to solve the problem that the content of biogenic amines in existing traditional fermented foods is generally high, but ethanol will inhibit the catalytic effect of amine oxidase. To provide an ethanol-tolerant amine oxidase LYYOBN2 derived from Bacillus cereus, and use the recombinant enzyme to degrade biogenic amines to reduce the content of biogenic amines in fermented foods and improve the quality of traditional fermented foods.

[0007] The first object of the present invention is to provide an amine oxidase, which is (a) or (b):

[0008] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.1;

[0009] (b) A protein derived from (a) in which one or several amino acids are substituted, deleted or added in the amino acid sequence and having amine oxidase activity.

[0010] The present invention also provides a gene encoding the amine oxidase.

[0011] In one embodiment, the gene contains the nucleotide sequence shown in SEQ ID NO.3.

[0012] The present invention also provides a recombinant expression plasmid carrying the gene.

[0013] 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.

[0014] 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.

[0015] In one embodiment, the recombinant expression plasmid is pET28a(+).

[0016] The present invention also provides a recombinant microbial cell expressing the amine oxidase.

[0017] In one embodiment, the recombinant microbial cell includes, but is not limited to, Escherichia coli, Bacillus or yeast.

[0018] 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.2.

[0019] In one embodiment, the genetically engineered bacterium uses Escherichia coli BL21(DE3) as a host.

[0020] In one embodiment, the genetically engineered bacterium uses a pET series plasmid as an expression vector.

[0021] In one embodiment, the genetically engineered bacterium uses pET28a(+) as an expression vector and expresses the amine oxidase shown in SEQ ID NO.2.

[0022] 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.

[0023] In one embodiment, the vector is pET28a(+).

[0024] In one embodiment, the gene sequence is ligated between the NheⅠ and HindⅢ sites of pET28a(+).

[0025] The present invention also provides a method for producing amine oxidase, which is to culture the genetically engineered bacterium in a medium and collect the amine oxidase.

[0026] In one embodiment, the method is to collect the cell bodies from the cell culture solution and break the cells to obtain a crude enzyme solution containing amine oxidase.

[0027] In one embodiment, the method includes purifying the crude enzyme solution.

[0028] 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.

[0029] In one embodiment, the affinity chromatography includes metal chelate affinity chromatography (such as purifying proteins using His tags), immunoaffinity chromatography, etc.

[0030] In one embodiment, nickel column affinity chromatography is used for the purification.

[0031] In one embodiment, the culturing is to inoculate the genetically engineered bacterium into LB medium and, when the OD600 reaches 0.6 - 0.8, induce enzyme production with IPTG.

[0032] In one embodiment, the induction is carried out at 16 - 30 °C.

[0033] In one embodiment, the final concentration of IPTG is 0.4 - 0.6 mmol·L -1 .

[0034] In one embodiment, the induction is carried out at 20 °C with an induction speed of 200 rpm for 14 h.

[0035] The present invention also provides the application of the amine oxidase in reducing biogenic amines.

[0036] In one embodiment, the application is to bring the amine oxidase into contact with biogenic amines in the environment.

[0037] In one embodiment, the environment includes a liquid environment, a semi-solid environment or a solid environment.

[0038] In one embodiment, the application includes reducing the content of biogenic amines in fermented foods.

[0039] In one embodiment, the fermented foods include fermented dairy products, fermented bean products, fermented meat products, fermented cereal products, fermented vegetable products, fermented condiments or fermented alcoholic beverages.

[0040] 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.

[0041] 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.

[0042] In one embodiment, the fermented vegetable products include, but are not limited to, pickles, sauerkraut, kimchi, pickled vegetables, etc.

[0043] In one embodiment, the fermented soybean products include, but are not limited to, fermented bean curd or natto.

[0044] In one embodiment, the fermented condiments include, but are not limited to, soy sauce, miso, vinegar, broad bean paste.

[0045] In one embodiment, the fermented alcoholic beverages include, but are not limited to, beer, wine, rice wine.

[0046] In one embodiment, the application is to add the amine oxidase to yellow rice wine to reduce the content of biogenic amines therein.

[0047] In one embodiment, the application is to add the amine oxidase to yellow rice wine and react at 25 - 28 °C.

[0048] In one embodiment, the application is to add the amine oxidase to cooking wine to reduce the content of biogenic amines therein.

[0049] 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.

[0050] In one embodiment, the application is to add the amine oxidase to soy sauce to reduce the content of biogenic amines therein.

[0051] 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.

[0052] In one embodiment, the application is to add the amine oxidase to fish sauce to reduce the content of biogenic amines therein.

[0053] 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.

[0054] In one embodiment, the application is to add the amine oxidase to red wine to reduce the content of biogenic amines therein.

[0055] 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.

[0056] 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.

[0057] The present invention provides an enzyme preparation for degrading biogenic amines, and the enzyme preparation contains an amine oxidase having an amino acid sequence as shown in SEQ ID NO.1.

[0058] 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.

[0059] In one embodiment, the stabilizer includes, but is not limited to, saccharides, polyols, proteins, polymers, metal ions, etc.

[0060] 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-biological in vivo environment.

[0061] In one embodiment, the environmental system includes, but is not limited to, dairy products, fish products, meat products, and fermented foods.

[0062] 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.

[0063] In one embodiment, the genetically engineered bacterium uses bacteria or fungi as host cells.

[0064] 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.

[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 products for degrading biogenic amines in the environmental system.

[0066] 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.

[0067] In one embodiment, the fermented foods include, but are not limited to, fermented vegetables and alcoholic beverages.

[0068] 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.

[0069] In one embodiment, the genetically engineered bacterium uses bacteria or fungi as host cells.

[0070] 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.

[0071] Beneficial effects:

[0072] (1) The recombinant amine oxidase LYYOBN2 from Bacillus provided by the present invention has strong performance in degrading biogenic amines. This enzyme also has certain low pH stability (able to retain more than 50% of the enzyme activity at pH 4.0), temperature stability (after incubation at 55 °C, the remaining enzyme activities of LYYOBN2 towards substrates HIS, TYR, PUT, and CAD all exceed 40%), and can tolerate a certain concentration of ethanol (when the ethanol concentration is 25% vol, LYYOBN2 retains 63.78 ± 3.14% of the enzyme activity towards TYR), which helps to achieve the degradation of biogenic amines in fermented foods, especially fermented alcoholic beverages.

[0073] (2) The present invention also provides the application of the amine oxidase LYYOBN2 in degrading biogenic amines in commercially available soy sauce. This enzyme can degrade phenethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), and tyramine (TYR) within 7 days, with the degradation rates reaching 19.5%, 15.96%, 100%, 43.22%, and 12.19% respectively, and the total degradation rate being 30%.

[0074] (3) The present invention also provides the application of the amine oxidase LYYOBN2 in degrading biogenic amines in Chinese rice wine. This enzyme can degrade putrescine (PUT), cadaverine (CAD), histamine (HIS), and tyramine (TYR) within 7 days, with the degradation rates reaching 10.97%, 24.06%, 30.18%, and 49.73% respectively. Meanwhile, phenethylamine (PHE) is completely degraded (degradation rate is 100%). The total degradation rate of this enzyme in Chinese rice wine is 21.22%.

[0075] (4) The present invention also provides the application of the amine oxidase LYYOBN2 in degrading biogenic amines in cooking wine. The degradation rates of this enzyme for tryptamine (TRY), phenethylamine (PHE), putrescine (PUT), cadaverine (CAD), and histamine (HIS) are 50.2%, 3.54%, 29.46%, 37%, and 16.04% respectively. The degradation rate of tyramine (TYR) is 13.26%. The total degradation rate of this enzyme in cooking wine is 30.13%.

[0076] (5) The present invention also provides the application of the amine oxidase LYYOBN2 in degrading biogenic amines in fish sauce. The degradation rates of this enzyme for tryptamine (TRY), phenethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), tyramine (TYR), and spermidine (SPD) are 13.37%, 7.62%, 17.41%, 84.36%, 8.88%, 38.33%, and 43.65% respectively. The total degradation rate of this enzyme in fish sauce is approximately 30.32%.

[0077] (6) The present invention also provides the application of the amine oxidase LYYOBN2 in degrading biogenic amines in red wine. This enzyme exhibits extremely strong degradation ability and can significantly degrade phenethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), tyramine (TYR), and spermidine (SPD) within 7 days, with the degradation rates reaching 99.49%, 8.72%, 81.38%, 47.68%, 51.26%, and 4.25% respectively. The degradation rate of tryptamine (TRY) is 23.78%. The total degradation rate of this enzyme in red wine is approximately 35.14%.

[0078] (7) The present invention also provides the application of amine oxidase LYYOBN2 in degrading biogenic amines in low-salt soy sauce. In low-salt soy sauce, the degradation rates of this enzyme for phenethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), tyramine (TYR) and spermidine (SPD) are 23.11%, 4.95%, 36.27%, 11.7%, 18.36% and 1.08% respectively. The total degradation rate of this enzyme in low-salt soy sauce is about 9.05%.

[0079] (8) The present invention also constructs a recombinant Escherichia coli expressing amine oxidase LYYOBN2. After inducing culture at 20 °C and 200 r·min -1 for 14 h, ultrasonic disruption is carried out, and 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 1200 μg of LYYOBN2 enzyme protein. Description of the Drawings

[0080] Figure 1 Agarose gel electrophoresis verification diagram for the amine oxidase gene: M: DNA Maker; 1: PCR amplification product (1461 bp).

[0081] Figure 2 Plasmid map of the amine oxidase pET28a-LYYOBN2 expression vector.

[0082] Figure 3 Protein electrophoresis diagram and enzyme activity of the recombinant enzyme LYYOBN2 under different induction conditions. Among them, (A): M: PageRuler TM Prestained protein molecular weight standard, 10 to 180 kDa; 1: pET28a empty vector; 2-3: supernatant and precipitate of the crude enzyme solution under the induction conditions of 0.2 mM IPTG, 16 °C, 10 h; 4-5: supernatant and precipitate of the crude enzyme solution under the induction conditions of 0.5 mM IPTG, 20 °C, 14 h; 6-7: supernatant and precipitate of the crude enzyme solution under the induction conditions of 0.4 mM IPTG, 25 °C, 10 h; 8-9: supernatant and precipitate of the crude enzyme solution under the induction conditions of 0.2 mM IPTG, 30 °C, 10 h; (B): Enzyme activities of different biogenic amines under different induction conditions.

[0083] Figure 4 Electrophoresis diagram of the protein separation and purification process of the recombinant enzyme LYYOBN2.

[0084] Figure 5 Relative enzyme activities of amine oxidase LYYOBN2 at different reaction temperatures; putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenethylamine (PHE), tryptamine (TRY), spermine (SPE) and spermidine (SPD).

[0085] Figure 6 is the temperature stability of amine oxidase LYYOBN2; putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenethylamine (PHE), tryptamine (TRY), spermine (SPE) and spermidine (SPD).

[0086] Figure 7 is the relative enzyme activity of amine oxidase LYYOBN2 at different pH values; putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenethylamine (PHE), tryptamine (TRY), spermine (SPE) and spermidine (SPD).

[0087] Figure 8 is the pH stability of amine oxidase LYYOBN2. Putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenethylamine (PHE), tryptamine (TRY), spermine (SPE) and spermidine (SPD).

[0088] Figure 9 is the relative residual enzyme activity of amine oxidase LYYOBN2 under different ethanol conditions; putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenethylamine (PHE), tryptamine (TRY), spermine (SPE) and spermidine (SPD).

[0089] Figure 10 is the relative residual enzyme activity of amine oxidase LYYOBN2 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

[0090] (I) Technical terms:

[0091] 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.

[0092] 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.

[0093] 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.

[0094] The "fermented foods" involved in the present invention are foods made through the fermentation of microorganisms (such as bacteria, yeast or fungi), and the types of the fermented foods 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 soya beans, yellow rice wine, beer, grape wine, etc.

[0095] The "biogenic amines" involved in the present invention are bioactive low-molecular-weight organic compounds containing amino groups, including but not limited to tryptamine (TRY), phenethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), tyramine (TYR), spermidine (SPD), spermine (SPE).

[0096] (II) Reagents

[0097] The yellow rice wine, soy sauce, low-salt soy sauce, cooking wine, fruit wine and fish sauce involved in the following examples are purchased from a certain supermarket in Wuxi, Jiangsu Province.

[0098] (III) Culture medium

[0099] LB medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, pH adjusted to 7.0 with NaOH, autoclaved at 121 °C for 20 min.

[0100] TB medium: Purchased from Qingdao Haibo Company. Autoclaved at 121 °C for 20 min.

[0101] (IV) Detection method

[0102] The content of biogenic amines was detected by high performance liquid chromatography (HPLC).

[0103] Determination of enzyme activity: The activity of biogenic amine oxidase was determined by an indirect 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 absorption value at 510 nm. The activity of amine oxidase is linearly related to the color depth of the product within a certain range. Therefore, the activity of amine oxidase can be determined by measuring the change in A510.

[0104] 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 prepared solution (including potassium phosphate buffer with a concentration of 200 mmol·L -1 and 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 with a concentration of 10 mmol·L -1 and 70 μL of peroxidase with a concentration of 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).

[0105] Definition of specific activity of amine oxidase (U / mg): The enzyme activity contained in each milligram of protein.

[0106] Degradation rate = (content of biogenic amines before reaction - content of biogenic amines after reaction) ÷ content of biogenic amines before reaction.

[0107] Example 1: PCR amplification of amine oxidase gene LYYOBN2

[0108] (1) Primers were designed based on the amine oxidase gene (QCX93949.1) in Bacillus cereus from the NCBI database. Using the DNA of Bacillus cereus LH5 preserved by the inventor's team as a template, the amine oxidase gene LYYOBN2 was amplified. The primers required for amplification are as follows:

[0109] F: 5'-CTAgctagcGTGCAGAAAGATATAATGTATAACACTGAAATGGATGAAGCGTT-3';

[0110] R: 5'-CCCaagcttCTCATTCACTTCCdyCGCTACAmrAATTCCAGATTCAATCGCACCTT-3'.

[0111] According to the requirements of the reaction system of TaKaRa high-fidelity enzyme, the PCR reaction solution was prepared. The PCR amplification system is as follows: pre-denaturation at 98°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C (1 min·kb -1 ) for 35 cycles.

[0112] (2) The PCR product was verified for the amplification result by 1.2% agarose gel electrophoresis. As Figure 1 shown, the size of the amplified sequence is the same as that of the target gene sequence, approximately 1461 bp, indicating successful amplification. After purifying the PCR product, it was sent to the company for sequencing, and the sequencing result is shown as SEQ ID NO.2.

[0113] Example 2: Construction of a genetically engineered bacterium producing amine oxidase gene LYYOBN2

[0114] The amine oxidase gene LYYOBN2 shown in SEQ ID NO.2 amplified in Example 1 was ligated to a plasmid and transformed into microbial cells to construct a genetically engineered bacterium producing amine oxidase gene LYYOBN2.

[0115] 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.

[0116] Optionally, the host is a bacterial cell or a fungal cell, including but not limited to Escherichia coli, Bacillus or yeast.

[0117] Taking recombinant Escherichia coli as an example, the construction process of the genetically engineered bacterium pET28a-LYYOBN2 is described as follows:

[0118] (1) Construction of recombinant plasmid: The plasmid pET-28a(+) and the amine oxidase gene fragment LYYOBN2 shown in SEQ ID NO.2 obtained in Example 1 were double-digested with restriction enzymes respectively. The digestion system is as follows: 50 μL of plasmid, 2.5 μL each of restriction enzymes Nhe I and HindIII, 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 double-digested gene fragment and plasmid 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(+)-LYYOBN2.

[0119] (2) Transformation.

[0120] After placing the E.coli BL21(DE3) competent cells stored at -80°C on ice for 10 min, 10 μL of the ligation product to be transformed obtained in step (2) 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-bathing was completed, 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 out, PCR verification was carried out to screen positive transformants.

[0121] (3) Enzyme digestion verification.

[0122] The plasmid of the recombinant bacterium was extracted and double-digested with Nhe I and EcoR I to obtain the pET-28a(+) fragment and the target fragment LYYOBN2 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 bacterium E.coli BL21 / pET-28a(+)-LYYOBN2 was successfully constructed. The recombinant enzyme expressed by this strain was named LYYOBN2. The construction process is as Figure 2 shown.

[0123] Example 3: Induced Expression and Purification of Recombinase LYYOBN2

[0124] (1) Inoculate the recombinant bacterium E. coli BL21 / pET-28a(+)-LYYOBN2 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 OD600 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 bacteria, resuspend the bacteria in 0.1 mol·L -1 sodium phosphate buffer (pH 7.4), and then centrifuge to collect the bacteria. Repeat the above steps twice. Use an ultrasonic cell disruptor to disrupt the bacteria. 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. Measure the protein content of the collected crude enzyme solution. The results show that 100 mL of bacterial solution with an OD of 1 contains 1200 μg of LYYOBN2 enzyme protein.

[0127] (4) Optimal Induction Conditions and Enzyme Activity of Recombinase LYYOBN2

[0128] Cultivate according to the method in step (2), except that different induction conditions are set as follows:

[0129] 1) 0.2 mM IPTG, induction temperature 16 °C, induction time 10 hours; 2) 0.5 mM IPTG, induction temperature 20 °C, induction time 14 hours; 3) 0.4 mM IPTG, induction temperature 25 °C, induction time 10 hours; 4) 0.2 mM IPTG, induction temperature 30 °C, induction time 10 hours.

[0130] Collect the fermented broth after cultivation, prepare the crude enzyme solution according to the method in step (2), and measure the catalytic activity against different biogenic amines. The results show that after optimizing different induction conditions, it is found that the crude enzyme solution of the recombinant enzyme LYYOBN2 has the highest relative enzyme activity under the conditions of an IPTG concentration of 0.5 mM, an induction temperature of 20 °C, and a time of 14 h, which is used as its optimal induction condition. LYYOBN2 has relatively high enzyme activities against HIS, TYR, PUT, and CAD( Figure 3 ).

[0131] (5) Purification of the recombinant enzyme LYYOBN2

[0132] Perform affinity chromatography on the supernatant obtained in step (4) using a His Trap TM HP (GE Healthcare) to purify the protein, and use an AKTAavant 25 instrument to separate and purify the target protein.

[0133] Perform gel electrophoresis analysis on the supernatant of the crude enzyme solution before purification of LYYOBN2 and the protein after purification of LYYOBN2 respectively. The results are as Figure 4 shown.

[0134] The results show that the molecular weight of the recombinant amine oxidase LYYOBN2 is approximately 59.09 kDa. The successful expression of the recombinant engineering bacteria was confirmed by SDS-PAGE gel electrophoresis analysis of the crude enzyme solution. Lane 2 proves that the target protein was successfully separated and purified by an affinity chromatography nickel column to obtain pure enzyme. The biogenic amine catalytic ability of the prepared enzyme protein was detected, and the results are shown in Table 1.

[0135] Table 1 Specific enzyme activity of pure amine oxidase LYYOBN2 (unit U / mg)

[0136]

[0137] Example 4: Preparation of the amine oxidase LYYOBN2 enzyme preparation

[0138] Mix the purified amine oxidase LYYOBN2 in Example 3 with an enzyme stabilizer to prepare an enzyme preparation containing amine oxidase LYYOBN2. Among them, the stabilizer refers to a substance that can protect the stability of the enzyme during production, storage, and use, preventing enzyme inactivation or degradation, including but not limited to sugars, polyols, proteins, polymers, metal ions, etc.

[0139] Example 5: Degradation of biogenic amines by the recombinant enzyme LYYOBN2 at different reaction temperatures and stability at different temperatures

[0140] The reaction system follows the standard, that is, the reaction is carried out in a 96-well plate. The reaction system includes 10 μL of a concentration of 200 mg·L -1Enzyme solution, 100 μL of the preparation solution (including 200 mmol·L -1 , potassium phosphate buffer solution 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, add 20 μL of the biogenic amine solution (10 mmol·L -1 ) and 70 μL of peroxidase (1.4 mg·mL -1 ). Measure the absorbance at 510 nm. The difference is that the reaction system temperature is changed to 20 °C, 28 °C, 37 °C, 45 °C, 50 °C, 55 °C, and 60 °C respectively, and the enzyme activity of the recombinant enzyme LYYOBN2 is measured under different temperature reaction conditions. Taking the highest enzyme activity measured at the above temperatures as 100%, calculate the relative enzyme activity at each temperature with a single biogenic amine as the substrate. The results are as Figure 5 shown. As the temperature increases, the enzyme activity of LYYOBN2 increases. However, when amine oxidase is applied to fermented foods, it is generally at room temperature.

[0141] After the purified enzyme solution in Example 3 is incubated in a water bath at 20 °C, 28 °C, 37 °C, 45 °C, 50 °C, 55 °C, and 60 °C respectively, according to the standard enzyme reaction system, measure the enzyme activity to determine its stable temperature range. The results are shown in Figure 6 . As the temperature increases, the thermal stability of the enzyme decreases. After incubation at 55 °C, the remaining enzyme activities of LYYOBN2 for substrates HIS (52.81 ± 4.52%), TYR (41.65 ± 2.22%), PUT (45.89 ± 4.69%), and CAD (41.20 ± 3.74%) all exceed 40%.

[0142] Example 6: Degradation of biogenic amines by recombinant enzyme LYYOBN2 at different reaction pH values and enzyme stability under different pH conditions

[0143] The reaction system is carried out in a 96-well plate according to the standard. The reaction system includes 10 μL of the enzyme solution with a concentration of 200 mg·L -1 , 100 μL of the preparation solution (including 200 mmol·L -1 , potassium phosphate buffer solution 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, add 20 μL of the 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 the pH of the reaction system was adjusted to 4.0 - 9.0, and the enzyme activity of recombinant enzyme LYYOBN2 under different pH reaction conditions was measured. Taking the highest enzyme activity as 100%, the relative enzyme activities at each temperature were calculated using a single biogenic amine as the substrate. The results are as Figure 7 shown, and the optimal reaction pH of LYYOBN2 is 7.0.

[0144] The purified recombinant enzyme solution from Example 3 was placed in different pH environmental conditions, and the enzyme was incubated for 30 min. The enzyme activity was measured, and the results are shown in Figure 8 . The stability was the strongest at pH 6.0. At pH 4.0, LYYOBN2 could retain more than about 50% of the enzyme activity for the substrates HIS (50.45 ± 4.06%), TYR (66.38 ± 5.10%), PUT (59.07 ± 3.18%), and CAD (60.91 ± 2.17%). The relatively wide pH application and stability range enable amine oxidase LYYOBN2 to have the potential for application in fermented foods.

[0145] Example 7: Effects of Different Concentrations of Ethanol on Amine Oxidase LYYOBN2

[0146] The reaction system was the same as in Example 3, except that different final concentrations of ethanol (≤ 25 vol%) were added, and the reaction system without ethanol was used as a control. The results showed that the enzyme activities of recombinant amine oxidase LYYOBN2 in the reaction system towards each biogenic amine were inhibited to varying degrees. When the ethanol concentration was 10% vol, LYYOBN2 could retain more than 75% of the enzyme activity for TYR, PUT, and CAD, except for HIS. When the ethanol concentration was 25% vol, LYYOBN2 still retained more than 20% of the enzyme activity. LYYOBN2 had the highest enzyme activities towards PUT, CAD, PHE, TRY, SPE, and SPD, which were 33.11 ± 5.16 U / mg, 36.22 ± 2.68 U / mg, 29.56 ± 5.10 U / mg, 30.00 ± 4.28 U / mg, 40.44 ± 3.17 U / mg, and 44.89 ± 4.66 U / mg, respectively.

[0147] LYYOBN2 has good tolerance to low - concentration ethanol solutions and can maintain good catalytic ability towards biogenic amines. This characteristic provides a good basis for its application in the degradation of biogenic amines in alcoholic beverages containing low - concentration ethanol.

[0148] Table 2 Enzyme Activity Values of Amine Oxidase LYYOBN2 under High - Concentration Ethanol (25% vol) Conditions (Unit: U / mg)

[0149]

[0150] Example 8: Effects of Different Concentrations of NaCl on Amine Oxidase LYYOBN2

[0151] The reaction system was the same as that in Example 3, except that different concentrations of NaCl (≤200 g / L) were added respectively. The results showed that as the NaCl concentration increased (4 - 16%), the relative enzyme activity of LYYOBN2 towards biogenic amines remained above about 80%. When the NaCl concentration exceeded 160 g / L, the enzyme activity decreased. Under the condition of high-concentration NaCl (200 g / L), the enzyme activity of amine oxidase LYYOBN2 towards tyramine and putrescine was the highest, and the relative enzyme activities remained above 60%, indicating that amine oxidase LYYOBN2 had good tolerance to NaCl within 200 g / L.

[0152] Table 3 Enzyme Activity Values (Unit: U / mg) of Amine Oxidase LYYOBN2 under the Condition of High-Concentration NaCl (20%, w / v)

[0153]

[0154] This amine oxidase LYYOBN2 had the catalytic ability towards biogenic amines in the system containing NaCl, and this characteristic provided a good basis for its application in the degradation of biogenic amines in fermented foods containing NaCl.

[0155] Example 9: Application of Amine Oxidase LYYOBN2 in Commercial Soy Sauce

[0156] Soy sauce is mainly brewed from soybeans or black beans, wheat or bran, and table salt through processes such as oil production and fermentation. The composition of soy sauce is relatively complex, and the range of the total biogenic amine content in the sample is 10 - 1898.17 mg / L. 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.

[0157] The recombinant amine oxidase LYYOBN2 prepared in Example 3 was ultrafiltered and concentrated, and the protein concentration was adjusted to 150 μg / ml, and then added to commercial soy sauce to make the enzyme concentration in soy sauce 75 μg / mL. It was left standing at room temperature (25°C) for 7 days. The results showed that amine oxidase LYYOBN2 could effectively reduce the biogenic amine content in low-salt soy sauce.

[0158] For commercial soy sauce, this enzyme could degrade phenethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), and tyramine (TYR) within 7 days, and the degradation rates reached 19.5%, 15.96%, 100%, 43.22%, and 12.19% respectively. The total degradation rate of this enzyme in soy sauce was about 30%.

[0159] Table 4 Contents (mg / L) of soy sauce before and after adding LYYOBN2 and degradation rates (%) of corresponding biogenic amines

[0160]

[0161] Example 10: Application of amine oxidase LYYOBN2 in Chinese rice wine

[0162] Chinese rice wine is a fermented wine brewed mainly from rice, millet, corn, wheat, water, etc. by adding 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, ranging from 8.1 to 305 mg / L. 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.

[0163] Adjust the protein concentration of the recombinant amine oxidase LYYOBN2 to 150 μg / ml and add it to Chinese rice wine at a final concentration of 75 μg / mL. React at room temperature (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 5.

[0164] For Chinese rice wine, this enzyme also shows significant degradation effect and can degrade putrescine (PUT), cadaverine (CAD), histamine (HIS) and tyramine (TYR) within 7 days, and the degradation rates reach 10.97%, 24.06%, 30.18% and 49.73% respectively. At the same time, phenethylamine (PHE) is completely degraded (degradation rate is 100%). The total degradation rate of this enzyme in Chinese rice wine is about 21.21%.

[0165] Table 5 Contents (mg / L) of Chinese rice wine before and after adding LYYOBN2 and degradation rates (%) of corresponding biogenic amines

[0166]

[0167] Example 11: Application of amine oxidase LYYOBN2 in cooking wine

[0168] "Cooking wine" is the name for wine used in cooking, which is brewed by adding Chinese rice wine and has a low alcohol concentration, less than 15% (v / v). Cooking wine contains both ethanol and NaCl, which is very unfavorable for the enzymatic catalytic reaction.

[0169] Adjust the protein concentration of the recombinant amine oxidase LYYOBN2 to 150 μg / ml and add it to cooking wine at a final concentration of 75 μg / mL. React at room temperature (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 6.

[0170] This enzyme can degrade tryptamine (TRY), phenethylamine (PHE), putrescine (PUT), cadaverine (CAD), and histamine (HIS) in cooking wine at degradation rates of 50.2%, 3.54%, 29.46%, 37%, and 16.04% respectively within 7 days. The degradation rate of tyramine (TYR) is 13.26%. The total degradation rate of this enzyme in cooking wine is approximately 30.13%.

[0171] Table 6 Contents (mg / L) of cooking wine before and after adding LYYOBN2 and degradation rates (%) of corresponding biogenic amines

[0172]

[0173] Example 12: Application of amine oxidase LYYOBN2 in fish sauce

[0174] Fish sauce is a fish soy sauce product made from fish by-products or fish viscera as raw materials, adding 30% - 40% (mass fraction) of salt and fermenting under the sun for 1 - 2 years. It is highly nutritious and delicious. 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 contents in current fish sauce products on the market vary widely, and the total biogenic amine content in some fish sauce samples can exceed 1000 mg / kg. The specific steps are as follows:

[0175] Adjust the protein concentration of the recombinant amine oxidase LYYOBN2 to 150 μg / ml and add it to fish sauce at a final concentration of 75 μg / mL, and react at room temperature of about 28°C for 7 days. The control group is a commercially available fish sauce with the enzyme replaced by PBS buffer. The results are shown in Table 7.

[0176] In fish sauce, the degradation rates of this enzyme for tryptamine (TRY), phenethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), tyramine (TYR), and spermidine (SPD) are 13.37%, 7.62%, 17.41%, 84.36%, 8.88%, 38.33%, and 43.65% respectively. The total degradation rate of this enzyme in fish sauce is approximately 30.32%.

[0177] Table 7 Contents (mg / L) of fish sauce before and after adding LYYOBN2 and degradation rates (%) of corresponding biogenic amines

[0178]

[0179] Example 13: Application of amine oxidase LYYOBN2 in red wine

[0180] 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 concentration range of the total amount of biogenic amines in red wine varies due to differences in detection methods and samples, but it is usually around 1.5 μg / L to 100 mg / L. Adjust the protein concentration of the recombinant amine oxidase LYYOBN2 to 150 μg / ml and add it to red wine at a final concentration of 75 μg / mL, and 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 8.

[0181] This enzyme exhibits extremely strong degradation ability and can significantly degrade phenethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), tyramine (TYR), and spermidine (SPD) within 7 days, with degradation rates reaching 99.49%, 8.72%, 81.38%, 47.68%, 51.26%, and 4.25% respectively. The degradation rate of tryptamine (TRY) is 23.78%. The total degradation rate of this enzyme in red wine is approximately 35.14%.

[0182] Table 8 Content (mg / L) of red wine before and after adding LYYOBN2 and the degradation rate (%) of corresponding biogenic amines

[0183]

[0184] Example 14: Application of amine oxidase LYYOBN2 in commercially available soy sauce 2

[0185] 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 range of the total biogenic amine content in the sample is 10 - 1898.17 mg / L. The salt content of low-salt soy sauce is approximately 8 g NaCl / 100 mL. The pH is about 4.4 - 4.6. NaCl and acidity limit the enzymatic catalytic reaction.

[0186] Ultrafilter and concentrate the recombinant amine oxidase LYYOBN2 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, and let it stand at room temperature (25°C) for 7 days. The results show that amine oxidase LYYOBN2 can effectively reduce the biogenic amine content in low-salt soy sauce.

[0187] In low-salt soy sauce, the degradation rates of this enzyme for phenethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), tyramine (TYR), and spermidine (SPD) are 23.11%, 4.95%, 36.27%, 11.7%, 18.36%, and 1.08% respectively. The total degradation rate of this enzyme in low-salt soy sauce is approximately 9.05%.

[0188] Table 9 Contents (mg / L) of low-salt soy sauce before and after adding LYYOBN2 and degradation rates (%) of corresponding biogenic amines

[0189]

[0190] Example 15: Application of amine oxidase LYYOBN2 in fermented sausage

[0191] 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 meat pieces of 4 - 5 cm. 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 LYYOBN2 prepared in Example 3 to adjust the protein concentration to 150 μg / 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 sausage stuffed into the casings is marinated at 4°C for 12 h, raise the temperature to 30°C, ferment until the pH drops to about 5.1, and mature at 14 - 16°C for 1 - 10 days. Compared with the fermented sausage without adding amine oxidase LYYOBN2, the results show that amine oxidase LYYOBN2 can effectively reduce the biogenic amine content in fermented sausage.

[0192] Currently, the research on the degradation of biogenic amines in fermented food systems by amine oxidase is almost blank. This application lays a technical foundation for the catalytic degradation of biogenic amines by this amine oxidase 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 ethanol conditions.

[0193] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and decorations 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. Amine oxidase LYYOBN2 for degrading 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 LYYOBN2 as claimed in claim 1.

3. A recombinant expression plasmid carrying the gene as claimed in claim 2.

4. A recombinant microbial cell expressing the amine oxidase as claimed in claim 1.

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

2.

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, expressing 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 shown in SEQ ID NO.2 with a vector and transform it into Escherichia coli cells.

8. A method for preparing the amine oxidase LYYOBN2 according to claim 1, characterized in that, Cultivate the recombinant microbial cell as claimed in claim 4 or the genetically engineered bacterium as claimed in any one of claims 5 to 6 in a medium for a period of time, and collect the amine oxidase LYYOBN2.

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

10. Use of the amine oxidase LYYOBN2 as claimed in claim 1 in reducing biogenic amines.

11. The application according to claim 10, wherein, The use is to reduce the content of biogenic amines in fermented foods.

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, spermine.

13. An enzyme preparation for degrading biogenic amines, characterized in that, The enzyme preparation contains the amine oxidase LYYOBN2 as claimed in claim 1.

14. A method for degrading biogenic amines in an environmental system, characterized in that, Contact the amine oxidase LYYOBN2 as claimed in claim 1, or the recombinant microbial cell as claimed in claim 4, or the genetically engineered bacterium as claimed in any one of claims 5 to 6 with biogenic amines in the environment to degrade the biogenic amines.

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