Preparation and application of alcohol-resistant amine oxidase LYYOBN1 capable of degrading biogenic amine

Abstract

The invention discloses preparation and application of alcohol-resistant amine oxidase LYYOBN1 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. The prepared amine oxidase is used for degrading 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 LYYOBN1 for biodegradable biogenic amines, belonging to the technical field of molecular biology. Background Art

[0002] Biogenic amines are a class of bioactive substances with potential toxicity widely present in foods, especially fermented foods. Biogenic amines in fermented foods are mainly formed by the decarboxylation of amino acids under the action of microorganisms. The content of these biogenic amines is often relatively high, and the main types include putrescine, tyramine, histamine, cadaverine, etc. Intake of excessive exogenous biogenic amines can lead to health risks, ranging 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] Currently, the content of biogenic amines in fermented foods is often reduced by controlling the levels of precursors, the growth of biogenic amine-producing strains, the activity of amino acid decarboxylases, and enhancing the activity of amine oxidases. Since the method of controlling the growth of spoilage microorganisms to reduce the content of biogenic amines in foods has certain limitations and cannot effectively reduce the high content of biogenic amines that have already been generated, using amine oxidases is an effective control measure to address unavoidable biogenic amines. Amine oxidases do not have strict specificity for the degradation of biogenic amines. One amine oxidase can degrade at least one biogenic amine, and there are correlative differences in its substrate specificity and action sites.

[0004] In the alcoholic beverage industry, the occurrence of biogenic amines has attracted increasing attention due to their potential toxicity threats related to ethanol content. Therefore, considering the potential synergistic effect between biogenic amines and ethanol, controlling the level of biogenic amines in fermented alcoholic beverages, that is, degrading multiple biogenic amines simultaneously, is crucial for reducing the risk of acute allergic reactions and ensuring food safety.

[0005] However, ethanol can inhibit the activity of amine oxidases. As an organic solvent, ethanol may affect the structure and function of the enzyme, posing a challenge for its use in controlling the content of biogenic amines in fermented alcoholic beverages. Currently, there is a lack of research on amine oxidases suitable for tolerating ethanol and their enzymatic properties. Therefore, obtaining ethanol-tolerant amine oxidases is crucial for improving the quality of fermented foods. Summary of the Invention

[0006] 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 ethanol can inhibit the catalytic action of amine oxidases. The present invention provides an ethanol-tolerant amine oxidase LYYOBN1 derived from Bacillus, and uses 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.

[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 in the amino acid sequence are substituted, deleted or added and which has amine oxidase activity.

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

[0011] In one embodiment, the gene contains the nucleotide sequence shown in SEQ ID NO.2.

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

[0013] In one embodiment, the plasmid includes, but is not limited to, vectors of the pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K, pPIC9K or pTrc series.

[0014] In one embodiment, the vectors of the pET series include pET24a(+), pET28a(+), pET29a(+), pET30a(+); the vectors of the Duet series include pRSFDuet-1, pCDFDuet-1; the vectors of the pTrc series include pTrc99a.

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

[0016] The present invention also provides a recombinant microbial cell expressing the said 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 which 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 plasmid of the pET series 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.1.

[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 bacterial cells from the cell culture broth, break the cells to obtain a crude enzyme solution containing amine oxidase.

[0027] In one embodiment, the method further 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 culture is to inoculate the genetically engineered bacterium into LB medium, and when the OD600 reaches 0.6 - 0.8, IPTG is used to induce enzyme production.

[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 25 °C with 200 rpm for 10 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 contact the amine oxidase 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 grain 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 the main 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 reaction lasts for at least 7 days.

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

[0050] 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.

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

[0052] 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.

[0053] In one embodiment, the application is to add the amine oxidase to fish sauce to reduce the biogenic amine content therein.

[0054] 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.

[0055] In one embodiment, the application is to add the amine oxidase to red wine to reduce the biogenic amine content therein.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] In one embodiment, the stabilizers include, but are not limited to, sugars, polyols, proteins, polymers, metal ions, etc.

[0061] 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.

[0062] In one embodiment, the environmental system includes, but is not limited to, dairy products, fish products, meat products, and fermented foods.

[0063] 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.

[0064] In one embodiment, the genetically engineered bacterium uses a bacterium or a fungus as a host cell.

[0065] 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.

[0066] The present invention also provides the use of the above enzyme preparation, or the amine oxidase with the amino acid sequence shown in SEQ ID NO.1, or the genetically engineered bacterium expressing the amine oxidase with the amino acid sequence shown in SEQ ID NO.1 in the preparation of products for degrading biogenic amines in the environmental system.

[0067] The present invention also provides the use of the above enzyme preparation, or the amine oxidase with the amino acid sequence shown in SEQ ID NO.1, or the genetically engineered bacterium expressing the amine oxidase with the amino acid sequence shown in SEQ ID NO.1 in the preparation of fermented foods.

[0068] In one embodiment, the fermented foods include, but are not limited to, fermented vegetables and alcoholic beverages.

[0069] 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.

[0070] In one embodiment, the host cell of the genetically engineered bacterium is a bacterium or a fungus.

[0071] The present invention also provides the use of the above enzyme preparation, or the amine oxidase with the amino acid sequence shown in SEQ ID NO.1, or the genetically engineered bacterium expressing the amine oxidase with the amino acid sequence shown in SEQ ID NO.1 in the degradation of biogenic amines in the food field.

[0072] Beneficial effects:

[0073] (1) The recombinant amine oxidase LYYOBN1 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 about 60% or more of the enzyme activity at pH 4.0), temperature stability (after incubation at 55°C, the remaining enzyme activities of LYYOBN1 against HIS, TYR, PUT, and CAD exceed 60%), and can tolerate a certain concentration of ethanol (when the ethanol concentration is 25% vol, LYYOBN1 still retains more than 50% of the enzyme activity), which helps to achieve the degradation of biogenic amines in fermented foods, especially fermented alcoholic beverages.

[0074] (2) The present invention also provides the use of the amine oxidase LYYOBN1 in the degradation of biogenic amines in fermented foods. This enzyme can degrade biogenic amines in commercially available soy sauce within 7 days, and the degradation rates of phenethylamine, putrescine, cadaverine, histamine, tyramine, and spermidine are 18.97%, 25.48%, 2.29%, 93.32%, 0.24%, and 32.71% respectively, and the total degradation rate is 29.30%.

[0075] (3) The present invention also provides the application of the amine oxidase LYYOBN1 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 21.38%, 7.15%, 8.17%, 24.70%, 36.88%, 6.63%, and 17.28% respectively, and the total degradation rate is 19.5%.

[0076] (4) The present invention also provides the application of the amine oxidase LYYOBN1 in degrading biogenic amines in fish sauce. This enzyme can degrade tryptamine (TRY), putrescine (PUT), cadaverine (CAD), and spermidine (SPD) in fish sauce to a certain extent within 7 days, and the degradation rates are 43.56%, 2.22%, 2.92%, and 26.97% respectively. The degradation rates for phenethylamine (PHE), histamine (HIS), and tyramine (TYR) are 2.68%, 0.5%, and 0% respectively. The total degradation rate of this enzyme in fish sauce is about 11.39%.

[0077] (5) The present invention also provides the application of the amine oxidase LYYOBN1 in degrading biogenic amines in red wine. This enzyme can significantly degrade histamine (HIS) in red wine within 7 days, and the degradation rate reaches 73.77%. At the same time, the degradation rates of tryptamine (TRY), putrescine (PUT), cadaverine (CAD), and tyramine (TYR) are 8.87%, 0%, 18.46%, and 3.37% respectively. The total degradation rate of this enzyme in red wine is about 22.67%.

[0078] (6) The present invention also provides the application of the amine oxidase LYYOBN1 in degrading biogenic amines in low-salt soy sauce. This enzyme can degrade phenethylamine (PHE), putrescine (PUT), cadaverine (CAD), tyramine (TYR), and spermidine (SPD) in low-salt soy sauce within 7 days, and the degradation rates are 12.7%, 1.63%, 26.31%, 14.05%, and 18% respectively. The total degradation rate of this enzyme in low-salt soy sauce is about 2.91%.

[0079] (7) The present invention also constructs a recombinant Escherichia coli expressing the amine oxidase LYYOBN1. After inducing culture at 25°C and 200 r·min -1 for 10 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 1500 μg of LYYOBN1 enzyme protein. Description of the Drawings

[0080] Figure 1 It is an agarose gel electrophoresis verification diagram of the amine oxidase gene: M: DNA Maker; 1: PCR amplification product (1434 bp).

[0081] Figure 2 Plasmid map of the amine oxidase pET28a-LYYOBN1 expression vector.

[0082] Figure 3 Degradation ability of amine oxidase LYYOBN1 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); 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 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.

[0083] Figure 4 Electrophoresis diagram of the protein separation and purification process of recombinant amine oxidase LYYOBN1; M: Unstained Protein Ladder; 1: Supernatant of crude enzyme solution before purification; 2: Purified protein amine oxidase KCYOBN (about 59.06 kDa).

[0084] Figure 5 Relative enzyme activity of amine oxidase LYYOBN1 at different temperatures; Putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenethylamine (PHE), tryptamine (TRY), spermine (SPE) and spermidine (SPD).

[0085] Figure 6 Temperature stability of amine oxidase LYYOBN1; Putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenethylamine (PHE), tryptamine (TRY), spermine (SPE) and spermidine (SPD).

[0086] Figure 7 Relative enzyme activity of amine oxidase LYYOBN1 at different pH values; Putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenethylamine (PHE), tryptamine (TRY), spermine (SPE) and spermidine (SPD).

[0087] Figure 8For the pH stability of amine oxidase LYYOBN1. Putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenethylamine (PHE), tryptamine (TRY), spermine (SPE) and spermidine (SPD).

[0088] Figure 9 For the relative residual enzyme activity of amine oxidase LYYOBN1 under different ethanol conditions; putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenethylamine (PHE), tryptamine (TRY), spermine (SPE) and spermidine (SPD).

[0089] Figure 10 For the relative residual enzyme activity of amine oxidase LYYOBN1 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 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).

[0096] (II) Reagents

[0097] 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.

[0098] (III) Culture medium

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

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

[0101] (IV) Detection methods

[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 - aminophenazone 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 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 - aminophenazone, 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] Example 1: PCR amplification of amine oxidase gene LYYOBN1

[0107] (1) Primers were designed according to the amine oxidase gene (QCX93858.1) in Bacillus cereus in the NCBI database. Using the DNA of Bacillus cereus LH5 preserved by the inventor's team as a template, the amine oxidase gene LYYOBN1 was amplified. The primers required for amplification were as follows:

[0108] F: 5'-CTAgctagcATGGGGAATCCATTAGCAATGGAAGAAATG-3';

[0109] R: 5'-CCGgaattcCTGTTCATTTACTTCATATGCAACTCTTACTCCA-3'.

[0110] 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-denaturation at 98°C for 10 s, annealing at 60°C for 30 s, extension at 72°C (1 min·kb -1 ) for 35 cycles.

[0111] (2) Verify the amplification result of the PCR product 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, about 1434 bp, indicating successful amplification. After purifying the PCR product, send it to the company for sequencing, and the sequencing result is shown in SEQ ID NO.2.

[0112] Example 2: Construction of a genetically engineered bacterium of amine oxidase gene LYYOBN1

[0113] Ligate the amine oxidase gene LYYOBN1 shown in SEQ ID NO.2 amplified in Example 1 with a plasmid and transform it into a microbial cell to construct a genetically engineered bacterium of amine oxidase gene LYYOBN1.

[0114] 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.

[0115] Optionally, the host is a bacterial cell or a fungal cell, including but not limited to Escherichia coli, Bacillus or yeast.

[0116] Taking recombinant Escherichia coli as an example, the construction process of the genetically engineered bacterium pET28a-LYYOBN1 is described as follows:

[0117] (1) 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 Nhe I and EcoR I, and 5 μL of Green Buffer. The components in the digestion system were thoroughly mixed and then placed 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 placed in a 16°C metal bath for overnight incubation to prepare the recombinant vector pET-28a(+)-LYYOBN1.

[0118] (2) 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 (1) was aspirated with a pipette and added to the competent cells. It was gently pipetted and mixed evenly, and then incubated on ice for 30 min. After the ice bath ended, it was heat shocked at 42°C for 45 s, and immediately taken out and placed on 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. It was centrifuged at 8000 r·min -1 for 1 min, and most of the supernatant was discarded, leaving about 200 μL of supernatant to resuspend the bacteria. The bacterial solution 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.

[0119] (3) 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 LYYOBN1 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(+)-LYYOBN1 was successfully constructed. The recombinant enzyme expressed by this strain was named LYYOBN1. The construction process was as Figure 2 shown.

[0120] Example 3: Induced expression and purification of recombinant enzyme LYYOBN1

[0121] (1) The recombinant bacterium E.coli BL21 / pET-28a(+)-LYYOBN1 constructed in Example 2 was inoculated into an LB medium containing 50 mg·L -1 kanamycin and cultured at 37°C with shaking at 150 r·min -1 for 14 h to prepare a seed solution.

[0122] (2) Transfer the obtained seed liquid to a TB fermentation medium containing 50 mg·L -1 kanamycin at an inoculation amount of 5% (v / v), and culture at 37 °C and 160 r·min -1 until the OD600 reaches 0.4 - 0.6, and then culture under different induction conditions to obtain the bacterial liquid.

[0123] (3) Centrifuge the bacterial liquid at 4 °C and 12,000 r·min -1 for 10 min, collect the lower-layer bacterial cells, add 0.1 mol·L -1 sodium phosphate buffer (pH 7.4) to resuspend the bacterial cells, 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, interval of 3 s, and disruption time of 30 min. After disruption, centrifuge at 4 °C and 12,000 r·min -1 to collect the 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 liquid with an OD of 1 contains 1500 μg of LYYOBN1 enzyme protein.

[0124] (4) Optimal induction conditions and enzyme activity of recombinant enzyme LYYOBN1

[0125] Cultivate according to the method in step (2), with the difference that different induction conditions are set respectively, as follows: 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.

[0126] Collect the fermented liquid 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 recombinant enzyme LYYOBN1 has the relatively highest enzyme activity under the conditions of IPTG concentration of 0.4 mM, induction temperature of 25 °C, and time of 10 h. The enzyme activities of LYYOBN1 against HIS, TYR, PUT, and CAD are 23.83 ± 0.39, 25.34 ± 0.26, 26.13 ± 1.17, and 25.80 ± 0.22 U( Figure 3 ).

[0127] (5) Purification of recombinant enzyme LYYOBN1

[0128] Perform affinity chromatography on the supernatant obtained in step (4) using a His TrapTM The protein was purified by HP (GE Healthcare), and the target protein was separated and purified using an AKTA avant 25 instrument.

[0129] The supernatant of the crude enzyme solution before purification of LYYOBN1 and the protein after purification of LYYOBN1 were analyzed by gel electrophoresis, and the results are as Figure 4 shown. The molecular weight of the recombinant amine oxidase LYYOBN1 is approximately 59.06 kDa. The successful expression of the recombinant engineering bacteria was confirmed by SDS-PAGE gel electrophoresis analysis of the crude enzyme solution. Lane 2 proved that the target protein was successfully separated and purified through 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.

[0130] Table 1 Specific enzyme activity of pure amine oxidase LYYOBN1 (unit U / mg)

[0131]

[0132] Example 4: Preparation of amine oxidase LYYOBN1 enzyme preparation

[0133] The purified amine oxidase LYYOBN1 in Example 3 was mixed with an enzyme stabilizer to prepare an enzyme preparation containing amine oxidase LYYOBN1. 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.

[0134] Example 5: Degradation of biogenic amines by recombinant enzyme LYYOBN1 at different reaction temperatures and stability at different temperatures

[0135] 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 the prepared 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, 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. 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, and the enzyme activity of the recombinant enzyme LYYOBN1 was measured under different temperature reaction conditions. The highest enzyme activity measured at the above temperatures was defined as 100%, and the relative enzyme activities at each temperature were calculated using each single biogenic amine as the substrate. The results are as Figure 5 shown. As the temperature increased, the enzyme activity of LYYOBN1 increased.

[0136] After 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 respectively, 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 temperature increased, the thermal stability of the enzyme decreased. After incubation at 55 °C, the remaining enzyme activities of LYYOBN1 against HIS (64.66 ± 5.02%), TYR (64.78 ± 3.58%), PUT (61.46 ± 4.76%), and CAD (61.26 ± 2.79%) exceeded 60%.

[0137] Example 6: Degradation of biogenic amines by recombinant enzyme LYYOBN1 at different reaction pHs and enzyme stability under different pH conditions

[0138] 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 the prepared solution (including 200 mmol·L -1 , potassium phosphate buffer with different pHs, 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, and the absorbance was measured at 510 nm. The reaction temperature was 37 °C. The difference was that the pH of the reaction system was adjusted to 4.0 - 9.0 respectively, and the enzyme activity of the recombinant enzyme LYYOBN1 was measured under different pH reaction conditions. Using the highest enzyme activity as 100%, the relative enzyme activities at each temperature were calculated using each single biogenic amine as the substrate. The results are as Figure 7 shown. The optimal reaction pH of LYYOBN1 was around 5.0.

[0139] The purified recombinant enzyme solution in Example 3 was placed in different pH environmental conditions, and the enzyme was incubated for 30 min, and the enzyme activity was measured. The results are shown in Figure 8。The stability is the strongest at pH 6.0. LYYOBN1 can retain more than about 60% of its enzyme activity at pH 4.0. The relatively wide pH application and stability range enable the amine oxidase LYYOBN1 to have the potential for application in fermented foods.

[0140] Example 7: Effects of Ethanol at Different Concentrations on Amine Oxidase LYYOBN1

[0141] The reaction system was the same as that in Example 3, except that ethanol at different final concentrations (≤25 vol%) was added respectively, and the reaction system without ethanol was used as a control. The results showed ( Figure 9 ), the enzyme activities of the recombinant amine oxidase LYYOBN1 in the reaction system towards various biogenic amines were inhibited to varying degrees. Under the condition of 5% vol ethanol concentration, LYYOBN1 could retain more than 80% of its enzyme activity towards HIS, TYR, PUT, and CAD. When the ethanol concentration was 10% vol, the enzyme activity of LYYOBN1 could be maintained above 75%. When the ethanol concentration exceeded 20% vol, LYYOBN1 could retain more than 55% of its enzyme activity. When the ethanol concentration was 25% vol, LYYOBN1 still retained more than 50% of its enzyme activity. And its enzyme activities towards putrescine and cadaverine were the highest.

[0142] Thus, it can be seen that the amine oxidase LYYOBN1 has good catalytic ability towards 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.

[0143] Table 2 Enzyme Activity Values (Unit: U / mg) of Amine Oxidase LYYOBN1 under the Condition of High-Concentration Ethanol (25% vol)

[0144]

[0145] Example 8: Effects of NaCl at Different Concentrations on Amine Oxidase LYYOBN1

[0146] The reaction system was the same as that in Example 3, except that NaCl at different concentrations (≤200 g / L) was added respectively, and the reaction system without NaCl was used as a control. The results showed ( Figure 10 ), as the NaCl concentration increased, the relative enzyme activity of LYYOBN1 first increased and then decreased. When the NaCl concentration exceeded 160 g / L, the enzyme activity decreased. Under the condition of high-concentration NaCl (200 g / L), the amine oxidase LYYOBN1 had the highest enzyme activity towards tyramine.

[0147] Table 3 Enzyme Activity Values (Unit: U / mg) of Amine Oxidase LYYOBN1 under the Condition of High-Concentration NaCl (20%, w / v)

[0148]

[0149] The amine oxidase LYYOBN1 has the catalytic ability for biogenic amines in a system containing NaCl, and this characteristic provides a good basis for its application in the degradation of biogenic amines in fermented foods containing NaCl.

[0150] Example 9: Application of amine oxidase LYYOBN1 in commercially available soy sauce

[0151] 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 total biogenic amine content of the sample ranges from 10 - 1898.17 mg / L. The salt content of ordinary soy sauce is about 12 g NaCl / 100 mL, and the pH is about 4.4 - 4.6. NaCl and acidity limit the progress of the enzymatic catalytic reaction.

[0152] The recombinant amine oxidase LYYOBN1 prepared in Example 3 was ultrafiltered 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 LYYOBN1 could effectively reduce the biogenic amine content in low-salt soy sauce.

[0153] This enzyme can degrade phenethylamine, putrescine, cadaverine, histamine, tyramine, and spermidine in commercially available soy sauce within 7 days, and the degradation rates are 18.97%, 25.48%, 2.29%, 93.32%, 0.24%, and 32.71% respectively. The total degradation rate is 29.30% (Table 4).

[0154] Table 4 Contents (mg / L) of soy sauce and degradation rates (%) of corresponding biogenic amines before and after adding LYYOBN1

[0155]

[0156] Example 10: Application of amine oxidase LYYOBN1 in Chinese rice wine

[0157] Chinese rice wine is a fermented wine mainly made from rice, millet, broomcorn millet, corn, wheat, water, etc., brewed with 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.

[0158] The recombinant amine oxidase LYYOBN1 was ultrafiltered and concentrated and added to Chinese rice wine at a final concentration of 75 μg / mL, and reacted at room temperature (about 28°C) for 7 days. The control group was a commercially available Chinese rice wine (18% vol) with the enzyme replaced by PBS buffer. The results are shown in Table 5.

[0159] 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 0.38%, 16.68%, 26.90%, 24.03%, 85.37% and 6.96% respectively. The total degradation rate is 32.93%.

[0160] Table 5 Biogenic amine content (mg / L) in Chinese rice wine before and after adding LYYOBN1 and the degradation rate (%) of corresponding biogenic amines

[0161]

[0162] Example 11: Application of amine oxidase LYYOBN1 in cooking wine

[0163] "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% (v / v). Cooking wine contains both ethanol and NaCl, which is very unfavorable for the enzymatic catalytic reaction.

[0164] The recombinant amine oxidase LYYOBN1 was added to cooking wine at a final concentration of 75 μg / mL, and reacted at room temperature (about 28°C) for 7 days. The control group was a commercially available cooking wine with the enzyme replaced by PBS buffer. The results are shown in Table 6.

[0165] This enzyme can degrade tryptamine, phenethylamine, putrescine, cadaverine, histamine, tyramine and spermidine in commercially available cooking wine within 7 days, and the degradation rates are 21.38%, 7.15%, 8.17%, 24.70%, 36.88%, 6.63% and 17.28% respectively. The total degradation rate is 19.5%.

[0166] Table 6 Biogenic amine content (mg / L) in cooking wine before and after adding LYYOBN1 and the degradation rate (%) of corresponding biogenic amines

[0167]

[0168] Example 12: Application of amine oxidase LYYOBN1 in fish sauce

[0169] Fish sauce is a fish soy sauce product made from fish by-products or fish viscera as raw materials, added with 30% - 40% (mass fraction) of salt, and fermented under the sun for 1 - 2 years. It has high nutritional value and a delicious taste. Natural fermentation is a key step in producing high-quality fish sauce, but a large amount of biogenic amines will also be produced during this process. The biogenic amine content in fish sauce products on the market is uneven, and the total biogenic amine content in some fish sauce samples can exceed 1000 mg / kg.

[0170] Adjust the protein concentration of the recombinant amine oxidase LYYOBN1 to 150 μg / ml, add it to fish sauce at a final concentration of 75 μg / mL, and react at room temperature (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.

[0171] This enzyme can degrade tryptamine (TRY), putrescine (PUT), cadaverine (CAD), and spermidine (SPD) in fish sauce to a certain extent within 7 days, and the degradation rates are 43.56%, 2.22%, 2.92%, and 26.97% respectively. The degradation rates for phenethylamine (PHE), histamine (HIS), and tyramine (TYR) are 2.68%, 0.5%, and 0% respectively. The total degradation rate of this enzyme in fish sauce is about 11.39%.

[0172] Table 7 Biogenic amine content (mg / L) in fish sauce before and after adding LYYOBN1 and the degradation rate (%) of the corresponding biogenic amines

[0173]

[0174] Example 13: Application of amine oxidase LYYOBN1 in red wine

[0175] 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. After ultrafiltration and concentration of the recombinant amine oxidase LYYOBN1, 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.

[0176] This enzyme can significantly degrade histamine (HIS) in red wine within 7 days, and the degradation rate reaches 73.77%. At the same time, the degradation rates for tryptamine (TRY), putrescine (PUT), cadaverine (CAD), and tyramine (TYR) are 8.87%, 0%, 18.46%, and 3.37% respectively. The total degradation rate of this enzyme in red wine is about 22.67%.

[0177] Table 8 Content (mg / L) of red wine before and after adding LYYOBN1 and the degradation rate (%) of the corresponding biogenic amines

[0178]

[0179] Example 14: Application of Amine Oxidase LYYOBN1 in Commercial Soy Sauce 2

[0180] Soy sauce is mainly brewed from soybeans or black soybeans, 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 in the sample ranges from 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.

[0181] The recombinant amine oxidase LYYOBN1 prepared in Example 3 was ultrafiltered and concentrated and added to commercial low-salt soy sauce, and the protein concentration after addition was 75 μg / mL. It was left standing at room temperature (25°C) for 7 days. The results showed that amine oxidase LYYOBN1 could effectively reduce the biogenic amine content in low-salt soy sauce.

[0182] This enzyme could achieve degradation rates of 12.7%, 1.63%, 26.31%, 14.05%, and 18% for phenethylamine (PHE), putrescine (PUT), cadaverine (CAD), tyramine (TYR), and spermidine (SPD) in low-salt soy sauce within 7 days. The total degradation rate of this enzyme in low-salt soy sauce was approximately 2.91%.

[0183] Table 9 Contents (mg / L) of Low-Salt Soy Sauce and Degradation Rates (%) of Corresponding Biogenic Amines before and after Adding LYYOBN1

[0184]

[0185] Example 15: Application of Amine Oxidase LYYOBN1 in Fermented Sausages

[0186] By mass, take 65 - 80% of lean meat and 20 - 35% of fat. Wash, and remove bones, tendons, muscle membranes, lymph, blood vessels, diseased and damaged parts. Separate the fat and lean, and cut into pieces about 4 - 5 cm in size. 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 LYYOBN1 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 stuffing the sausage into casings, marinate at 4°C for 12 h, then 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 LYYOBN1, the results show that amine oxidase LYYOBN1 can effectively reduce the biogenic amine content in the fermented sausage.

[0187] 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 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 concentration conditions.

[0188] 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 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 LYYOBN1 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 the amino acid sequence in (a) is substituted, deleted or added with one or several amino acids and has amine oxidase activity.

2. A gene encoding the amine oxidase described in claim 1.

3. A recombinant plasmid carrying the gene described in claim 2.

4. A recombinant microbial cell expressing the amine oxidase described in 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, wherein 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 shown in SEQ ID NO.2 with the vector and transform it into Escherichia coli cells.

8. A method for preparing the amine oxidase LYYOBN1 as claimed in claim 1, characterized in that, Cultivate the recombinant microbial cell described in claim 4 or the genetically engineered bacterium described in any one of claims 5 to 6 in a medium, and collect the amine oxidase LYYOBN1.

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

10. The application of the amine oxidase LYYOBN1 described in claim 1 in reducing biogenic amines.

11. The application according to claim 10, characterized in that, The application 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, and spermine.

13. An enzyme preparation containing the amine oxidase LYYOBN1 described in claim 1.

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

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