A monoamine oxidase MAO7 derived from *Saccharomyces cerevisiae* that degrades biogenic amines SH and its applications

Abstract

The application discloses a single amine oxidase MAO7 of a Saccharopolyspora hirsuta-derived degradable biological amine SH and application thereof, and belongs to the technical field of molecular biology. The application provides the single amine oxidase of the Saccharopolyspora hirsuta, and realizes expression of the enzyme in Escherichia coli. The application also provides application of the single amine oxidase in degrading biological amine. The enzyme is added into commercially available yellow rice wine, and can effectively degrade tryptamine, phenylethylamine and cadaverine, with a degradation rate of more than 33.76%, which is helpful to further improve the safety of fermented food.

Description

Technical Field

[0001] This invention relates to a monoamine oxidase MAO7 derived from *Saccharomyces cerevisiae* that can degrade biogenic amines. SH Its applications belong to the field of molecular biology technology. Background Technology

[0002] Biogenic amines are a class of low-molecular-weight nitrogen-containing organic compounds with significant biological activity. They are commonly found in various fermented foods, such as dairy products, fish products, meat products, fermented vegetables, and alcoholic beverages. The content of these substances varies considerably among different foods and is generally high. Major types include putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenylethylamine (PHE), tryptamine (TRY), spermine (SPE), and spermidine (SPD). Numerous studies have shown that high doses of biogenic amines can cause various toxicological problems. Notably, alcohol intake can exacerbate the adverse physiological effects of biogenic amines, indicating that biogenic amines are a key factor contributing to hangovers and headaches caused by alcoholic beverages. Therefore, improving the quality of these fermented foods and strictly controlling their biogenic amine content is crucial.

[0003] Studies have shown that biogenic amines cannot be removed by heating and cooking; their formation can only be controlled and reduced during fermentation. However, for biogenic amines already present in food, the most promising method with the least impact on the flavor of fermented foods is catalytic degradation via microbial enzymatic hydrolysis. Amine oxidases from microorganisms play a crucial role in degrading biogenic amines in food and ensuring food safety. Based on the different cofactors they contain, amine oxidases can be divided into two types: copper-containing amine oxidases and flavin-containing amine oxidases. Among these, monoamine oxidases (MAOs), specifically those with a cofactor of FAD, belong to the flavin-containing amine oxidase class. MAOs are a class of enzymes found in many microorganisms, playing a vital role in the metabolism of biogenic amines, similar to copper-containing amine oxidases (with a cofactor of Cu). 2+ These enzymes (with cofactors) do not belong to the same category. They possess the ability to oxidize and remove the amino group from biogenic amines, thereby converting them into the corresponding aldehydes. Specifically, MAO oxidizes the amino group (-NH2) of biogenic amines to an imino group (-NH), while simultaneously reducing molecular oxygen (O2) to generate hydrogen peroxide (H2O2). The resulting imino group is further hydrolyzed to form the corresponding aldehyde compound and ammonia (NH3). This process is particularly crucial for controlling and degrading the content of biogenic amines in food.

[0004] Amine oxidases are effective in degrading biogenic amines in prepared solutions, but their activity is significantly reduced or even inactivated when used to degrade biogenic amines in fermented foods. Currently, there are no reports on monoamine oxidases used to degrade common biogenic amines such as histamine, tyramine, cadaverine, and putrescine in fermented food systems, nor are there studies on their related enzymatic properties. To address this key issue, obtaining monoamine oxidases from microorganisms derived from fermented foods to rationally regulate and reduce biogenic amine content is of great significance for improving the quality of fermented foods, especially rice wine. Summary of the Invention

[0005] The purpose of this invention is to solve the problem of generally high biogenic amine content in existing traditional fermented foods, and to provide a monoamine oxidase derived from Polysaccharidobacterium tumefaciens that can degrade biogenic amines, and to use a recombinant enzyme to degrade biogenic amines to reduce the biogenic amine content in fermented foods such as rice wine, thereby improving the quality of traditional fermented foods.

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

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

[0008] (b) A protein derived from (a) whose amino acid sequence in (a) has been substituted, deleted or added with one or more amino acids and has monoamine oxidase activity.

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

[0010] In one embodiment, the gene contains the nucleotide sequence shown in SEQ ID NO.3.

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

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

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

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

[0015] The present invention also provides recombinant microbial cells expressing the monoamine oxidase.

[0016] In one embodiment, the recombinant microbial cells include, but are not limited to, Escherichia coli, Bacillus, or yeast.

[0017] The present invention also provides a genetically engineered bacterium that uses Escherichia coli as a host to express the monoamine oxidase shown in SEQ ID NO.3.

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

[0019] In one embodiment, the genetically engineered bacteria use pET series plasmids as expression vectors.

[0020] In one embodiment, the genetically engineered bacteria use pET28a(+) as an expression vector to express the monoamine oxidase shown in SEQ ID NO.2.

[0021] The present invention also provides a method for constructing the genetically engineered bacteria, which involves linking the gene sequence shown in SEQ ID NO.2 or SEQ ID NO.3 with a vector and transforming it into Escherichia coli cells.

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

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

[0024] The present invention also provides a method for producing monoamine oxidase, wherein the genetically engineered bacteria are inoculated into a culture medium for cultivation and the monoamine oxidase is collected.

[0025] In one embodiment, the method involves collecting bacterial cells from a cell culture medium and rupturing the cells to obtain a crude enzyme solution containing monoamine oxidase.

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

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

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

[0029] In one embodiment, the purification is performed using nickel column affinity chromatography.

[0030] In one embodiment, the culture involves inoculating the genetically engineered bacteria into LB medium and culturing until the OD600 reaches 0.6-0.8, then inducing enzyme production with IPTG.

[0031] In one embodiment, the induction is performed at 16–37°C.

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

[0033] In one embodiment, the induction is performed at 20°C and 200 rpm for 16 hours.

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

[0035] In one embodiment, the application involves contacting the monoamine oxidase with biogenic amines in the environment.

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

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

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

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

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

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

[0042] In one embodiment, the fermented soy product includes, but is not limited to, fermented bean curd or natto.

[0043] In one embodiment, the fermented condiment includes, but is not limited to, soy sauce, miso, vinegar, and fermented soybean paste.

[0044] In one embodiment, the fermented alcoholic beverage includes, but is not limited to, beer, wine, and rice wine.

[0045] In one embodiment, the application involves adding the monoamine oxidase to rice wine to reduce the content of the main biogenic amines.

[0046] In one embodiment, the application involves adding the monoamine oxidase to rice wine and reacting it at 25–28°C for at least 24 hours or at least 48 hours.

[0047] In one embodiment, the biogenic amine includes, but is not limited to, one or more of tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine.

[0048] The present invention provides an enzyme preparation for degrading biogenic amines, wherein the enzyme preparation contains a monoamine oxidase with an amino acid sequence as shown in SEQ ID NO.1.

[0049] In one embodiment, the enzyme preparation further contains a stabilizer that protects the stability of the enzyme during production, storage and use, and prevents enzyme inactivation or degradation.

[0050] In one embodiment, the stabilizer includes, but is not limited to, sugars, polyols, proteins, polymers, metal ions, etc.

[0051] This invention provides a method for degrading biogenic amines in an environmental system. The method involves using the monoamine oxidase or genetically engineered bacteria expressing the monoamine oxidase to degrade the biogenic amines. The amino acid sequence of the monoamine oxidase is shown in SEQ ID NO.1. The environmental system is a non-biological in vivo environment.

[0052] In one embodiment, the environmental system includes, but is not limited to, dairy products, fish products, meat products, and fermented foods.

[0053] In one embodiment, the environmental system includes, but is not limited to, fermented foods such as rice wine, soy sauce, vinegar, low-sodium soy sauce, cooking wine, fruit wine, shrimp paste, and fish sauce.

[0054] In one embodiment, the genetically engineered bacteria uses bacteria or fungi as host cells.

[0055] In one embodiment, the biogenic amine includes, but is not limited to, one or more of tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine.

[0056] The present invention also provides the application of the above-mentioned enzyme preparation or the monoamine oxidase with the above-mentioned amino acid sequence as shown in SEQ ID NO.1 or the genetically engineered bacteria expressing the monoamine oxidase with the amino acid sequence as shown in SEQ ID NO.1 in the preparation of products that degrade biogenic amines in an environmental system.

[0057] The present invention also provides the application of the above-mentioned enzyme preparation or the monoamine oxidase with the above-mentioned amino acid sequence as shown in SEQ ID NO.1 or the genetically engineered bacteria expressing the monoamine oxidase with the amino acid sequence as shown in SEQ ID NO.1 in the preparation of fermented foods.

[0058] In one embodiment, the fermented food includes, but is not limited to, fermented vegetables and alcoholic beverages.

[0059] In one embodiment, the fermented food includes, but is not limited to, rice wine, soy sauce, vinegar, low-sodium soy sauce, cooking wine, fruit wine, shrimp paste, and fish sauce.

[0060] In one embodiment, the genetically engineered bacteria uses bacteria or fungi as host cells.

[0061] The present invention also provides the application of the above-mentioned enzyme preparation or the monoamine oxidase with the above-mentioned amino acid sequence as shown in SEQ ID NO.1 or the genetically engineered bacteria expressing the monoamine oxidase with the amino acid sequence as shown in SEQ ID NO.1 in the food field for the degradation of biogenic amines.

[0062] Beneficial effects:

[0063] (1) The monoamine oxidase recombinant enzyme from Polysaccharidus sacchariformis provided by the present invention has a strong ability to degrade biogenic amines. The enzyme also has certain pH stability and temperature stability, and can tolerate a certain concentration of ethanol, which helps to achieve the degradation of biogenic amines in alcoholic beverages.

[0064] (2) The present invention also provides the monoamine oxidase MAO7. sh Application in the degradation of biogenic amines in fermented foods. This enzyme demonstrated the ability to degrade tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine in 18% vol commercially available rice wine within 48 hours, achieving degradation rates of 22.18%, 14.25%, 47.60%, 25.36%, 13.67%, 8.53%, 13.65%, and 0.71%, respectively. It particularly exhibited potential for degrading putrescine and cadaverine, contributing to further improvements in the safety of fermented foods.

[0065] (3) This invention also constructs a monoamine oxidase MAO7 expression system. sh Recombinant Escherichia coli, through 200 r·min at 20℃ -1 After 16 hours of induction culture under the specified conditions, the enzyme was ultrasonically disrupted, and the protein content of the collected crude enzyme solution was determined. The results showed that 100 mL of bacterial solution with an OD of 1 contained 50 mg of MAO7. sh Enzyme protein. Attached Figure Description

[0066] Figure 1Agarose gel electrophoresis verification image of the monoamine oxidase gene: M: DNA Marker; 1: PCR amplification product (1293bp).

[0067] Figure 2 Monoamine oxidase pET28a-MAO7 SH Plasmid map of the expression vector.

[0068] Figure 3 Recombinant monoamine oxidase MAO7 SH Electrophoresis diagram of the protein separation and purification process; M: Unstained Protein Ladder; 1: Crude enzyme supernatant before purification; 2: Protein monoamine oxidase MAO7 after purification. SH (Approximately 47 kDa).

[0069] Figure 4 Monoamine oxidase (MAO7) before and after purification SH Degradation capacity for different biogenic amines: putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenylethylamine (PHE), tryptamine (TRY), spermine (SPE), and spermidine (SPD).

[0070] Figure 5 and 6 Monoamine oxidase MAO7 at different temperatures SH The relative enzyme activities of putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenylethylamine (PHE), tryptamine (TRY), spermine (SPE), and spermidine (SPD).

[0071] Figure 6 Monoamine oxidase MAO7 SH Temperature stability; putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenylethylamine (PHE), tryptamine (TRY), spermine (SPE), and spermidine (SPD).

[0072] Figure 7 Monoamine oxidase MAO7 at different pH values SH The relative enzyme activities of putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenylethylamine (PHE), tryptamine (TRY), spermine (SPE), and spermidine (SPD).

[0073] Figure 8 Monoamine oxidase MAO7 SH pH stability. Putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenylethylamine (PHE), tryptamine (TRY), spermine (SPE), and spermidine (SPD).

[0074] Figure 9 Monoamine oxidase MAO7 under different ethanol conditions SH Relative residual enzyme activity; putrescine (PUT), cadaverine (CAD). Detailed Implementation

[0075] (I) Technical Terminology:

[0076] The "dairy products" involved in this invention refer to foods made from animal milk through different processing methods. These types include, but are not limited to, pasteurized milk, sterilized milk, flavored milk, fermented milk, whole milk powder, skim milk powder, sweetened whole milk powder, flavored milk powder, infant formula and other formula milk powders, condensed milk, milk fats, cheeses, ice cream, casein, milk tablets, lactose, etc.

[0077] The "fish products" involved in this invention refer to products made from fish meat or certain organs of fish as the main raw materials through various processing methods; the processing methods include pickling, smoking, drying, freezing, canning, fermentation, etc.

[0078] The "meat products" involved in this invention refer to products made primarily from animal muscle tissue or edible offal through various processing methods. According to the national standard for meat and meat product terminology (GB / T 19480-2009), meat products are divided into two main 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, cured sausage, dried sausage, fresh sausage product, smoked and fresh sausage, semi-dry sausage, dry sausage, prepared meat products, and meat cake. cake), salted meat; the Western-style meat products include smoked ham, smoked sausage, sausage products, blood sausage, fermented sausage, bacon, ham or meat sausage.

[0079] The "fermented foods" involved in this invention are foods made through the fermentation process of microorganisms (such as bacteria, yeast, or fungi). The types of fermented foods include, but are not limited to, alcoholic beverages, fish and shrimp paste, fruit wine, soy sauce, yogurt, cheese, fermented rice wine, pickles, soy sauce, vinegar, fermented black beans, rice wine, beer, and wine.

[0080] The "biogenic amines" involved in this invention are low molecular weight organic compounds containing amino groups that have biological activity, including but not limited to tryptamine (TRY), phenylethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), tyramine (TYR), spermidine (SPD), and spermine (SPE).

[0081] (II) Reagents

[0082] The rice wine and other ingredients mentioned in the following examples were purchased from a supermarket in Wuxi City, Jiangsu Province.

[0083] (III) Culture Medium

[0084] LB medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, pH adjusted to 7.0 with NaOH, autoclaved at 121℃ for 20 min.

[0085] TB culture medium: purchased from Qingdao Haibo Company. Autoclaved at 121℃ for 20 minutes.

[0086] (iv) Testing methods

[0087] The content of biogenic amines was detected by high performance liquid chromatography (HPLC).

[0088] Enzyme activity assay: The activity of biogenic amine oxidase was determined using an indirect method based on catalase. Amine oxidase acts on biogenic amines, degrading them into the corresponding aldehydes and hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide reacts with 4-aminoantipyrrolidone and 2,4,6-tribromo-3-hydroxybenzoic acid to form quinone dye. This product has a maximum absorbance at 510 nm. The activity of amine oxidase is linearly related to the intensity of the product color within a certain range. Therefore, the activity of amine oxidase can be determined by measuring the change in A510.

[0089] The reaction was carried out in a 96-well plate, and the reaction system consisted of 10 μL of enzyme solution (200 mg·L⁻¹). -1 ), 100 μL of prepared solution (containing 200 mmol·L⁻¹) -1 Potassium phosphate buffer solution, pH 7.6, 1.5 mmol / L -1 4-Aminoantipyridine, 1 mmol·L -1 2,4,6-Tribromo-3-hydroxybenzoic acid was added to initiate the reaction, followed by the addition of 20 μL of a biogenic amine solution (10 mmol·L⁻¹).-1 ) and 70 μL peroxidase (1.4 mg·mL) -1 The absorbance was measured at 510 nm, the reaction temperature was 37 °C, and the reaction time was 30 min. One unit of enzyme activity (U) was defined as a change in absorbance of 0.01 μL per minute.

[0090] The definition of specific activity of monoamine oxidase (U / mg): the enzyme activity contained in one milligram of protein.

[0091] Example 1: Monoamine oxidase gene MAO7 SH PCR amplification

[0092] (1) Primers were designed based on the monoamine oxidase gene (WP_150067525.1) of Saccharopolyspora hirsuta in the NCBI database. Using the DNA of Saccharopolyspora hirsutaT14 preserved in our laboratory as a template, the monoamine oxidase gene MAO7 was amplified. SH The primers required for amplification are as follows:

[0093] F: 5'-ctagctagcATGGATCGyTACGACGsGGTCGTCATCGGyGCsGGyTTCGCCGG-3';

[0094] R: 5'-caagcttTCACTkCTCGGCCGCCAGGATCTCCGC-3'.

[0095] The PCR reaction solution was prepared according to the requirements of the TaKaRa LA Taq reaction system. The PCR amplification system was as follows: 98℃ pre-denaturation for 10 s, 70℃ annealing for 30 s, and 72℃ extension (1 min·kb). -1 Repeat 35 times.

[0096] (2) Using Saccharopolyspora hirsuta T14 DNA as a template, PCR amplification was performed. The PCR products were verified by 1.2% agarose gel electrophoresis. Figure 1 The amplified sequence shown is the same size as the target gene sequence, approximately 1293 bp, indicating successful amplification. The PCR product was purified and sent to the company for sequencing. The sequencing results are shown in SEQ ID NO.2 (the sequence contains restriction enzyme sites NheⅠ and HindⅢ at both ends).

[0097] Example 2: Monoamine oxidase gene MAO7 sh Construction of genetically engineered bacteria

[0098] The amine oxidase gene MAO7 amplified in Example 1sh The plasmid was ligated and transformed into microbial cells to construct the monoamine oxidase gene MAO7. sh Genetically engineered bacteria.

[0099] Optionally, the plasmids include, but are 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(+), and pET30a(+); the Duet series vectors include pRSFDuet-1 and pCDFDuet-1; and the pTrc series vectors include pTrc99a.

[0100] Optionally, the host is a bacterial cell or a fungal cell, including but not limited to Escherichia coli, Bacillus, or yeast.

[0101] Taking recombinant Escherichia coli as an example, the genetically engineered bacterium pET28a-MAO7 sh The construction process is described as follows:

[0102] (1) Obtain the target segment.

[0103] Using DNA from the strain *Saccharopolyspora hirsuta* T14 preserved in our laboratory as a template, primers and whole-genome DNA were used to perform PCR amplification. The PCR reaction system and amplification procedure were the same as described in Example 1. The PCR product gel, after verifying the correct bands, was carefully cut, recovered, and purified. The monoamine oxidase gene fragment MAO7 was obtained. SH Sequencing confirmed the nucleotide sequence of the gene as shown in SEQ ID NO.3.

[0104] (2) Enzyme digestion and ligation.

[0105] The plasmid pET-28a(+) and the target fragment obtained in step (1) were double-digested with restriction endonucleases Nhe I and HindIII, respectively. The digestion system was as follows: 50 μL plasmid, 2.5 μL each of restriction endonucleases Nhe I and HindIII, and 5 μL Green Buffer. The components in the digestion system were thoroughly mixed and then incubated in a 37°C metal bath for 60 min. After double digestion, the gene fragment and plasmid were recovered and purified, then mixed in a molar ratio of 3–10:1. An equal volume of Solution I ligase was added, and the mixture was thoroughly mixed and incubated overnight in a 16°C metal bath to prepare the recombinant vector pET-28a(+)-MAO7. SH .

[0106] (3) Transformation.

[0107] After placing E. coli BL21(DE3) competent cells stored at -80℃ on ice for 10 min, 10 μL of the ligation product obtained in step (2) was pipetted into the competent cells and gently mixed by pipetting. After thorough mixing, the cells were incubated on ice for 30 min. After the ice bath, the cells were heat-shocked at 42℃ for 45 s, and immediately removed and placed on ice for 2 min. Then, 700 μL of LLB liquid culture medium was added, and the cells were incubated at 37℃ for 200 rpm. -1 Incubate with shaking for 60 minutes at 8000 rpm. -1 Centrifuge for 1 min, discard most of the supernatant, and resuspend the bacterial cells in approximately 200 μL of supernatant. Spread the bacterial suspension evenly on a medium containing 50 mg·L⁻¹ of supernatant. -1 On LB solid medium plates containing kanamycin, invert the plates and incubate overnight at 37°C. After the plates have grown, pick single bacteria and perform PCR verification to screen for positive transformants.

[0108] (4) Enzyme digestion verification.

[0109] The plasmid of the recombinant bacteria was extracted and double-digested with Nhe I and HindII to obtain a 5369 bp pET-28a(+) fragment and a 1293 bp target fragment, respectively. The target fragment was sent to a company for sequencing, and the sequencing results were consistent with the target gene sequence, verifying the recombinant E. coli BL21 / pET-28a(+)-MAO7. SH Construction successful. The recombinase expressed by this strain is named MAO7. SH The construction process is as follows: Figure 2 As shown.

[0110] Example 3: Recombinase MAO7 SH Induced expression and purification

[0111] The specific steps are as follows:

[0112] (1) The recombinant E. coli BL21 / pET-28a(+)-MAO7 constructed in Example 2 SH Inoculate with a solution containing 50 mg / L -1 In LB medium containing kanamycin, at 37°C and 150 rpm -1 Seed culture was prepared by culturing under the specified conditions for 14 hours.

[0113] (2) The obtained seed culture was transferred to a solution containing 50 mg·L⁻¹ at an inoculum volume of 5% (v / v). -1 In TB fermentation medium, kanamycin was fermented at 37°C and 160 rpm. -1Under the specified conditions, culture until the OD600 reaches 0.4-0.6, then add [a specific ingredient] to a final concentration of 0.5 mmol·L⁻¹. -1 IPTG at 20℃, 200 r·min -1 After culturing for 16 hours under the specified conditions, the bacterial culture was obtained.

[0114] (3) Incubate the bacterial solution at 4℃ and 12000 r·min -1 Under the specified conditions, after centrifugation for 10 min, the lower layer of cells was collected, and 0.1 mol·L⁻¹ was added. -1 The bacterial cells were resuspended in sodium phosphate buffer (pH 7.4) and collected by centrifugation. This step was repeated twice. The bacterial cells were then disrupted using an ultrasonic cell disruptor under the following conditions: 400W, 2 seconds on, 3 seconds off, for a total disruption time of 30 minutes. After disruption, the cells were incubated at 4°C and 12000 rpm. -1 The supernatant was collected by centrifugation, filtered through a 0.22 μM filter membrane, and stored at low temperature for later use. The protein content of the collected crude enzyme solution was determined, and the results showed that 100 mL of bacterial culture with an OD of 1 contained 50 mg of MAO7. sh Enzyme protein.

[0115] (4) Recombinase MAO7 SH Purification

[0116] The supernatant obtained in step (3) was subjected to HisTrap affinity chromatography. TM HP (GE Healthcare) purified the protein using an AKTA avant 25 instrument to separate and purify the target protein.

[0117] MAO7 SH Crude enzyme supernatant and MAO7 before purification SH The purified protein was analyzed by gel electrophoresis, and the results are as follows: Figure 3 As shown.

[0118] The results showed that recombinant monoamine oxidase MAO7 SH The molecular weight is approximately 47 kDa. SDS-PAGE gel electrophoresis analysis of the crude enzyme solution confirmed successful expression by the recombinant engineered bacteria. Lane 2 demonstrates that the target protein was successfully purified using affinity chromatography with a nickel column to obtain the pure enzyme.

[0119] Example 4: Monoamine oxidase MAO7 sh Preparation of enzyme preparations

[0120] The monoamine oxidase MAO7 purified in Example 3 sh Mixed with an enzyme stabilizer, a monoamine oxidase (MAO6) was prepared. shThe enzyme preparation. The stabilizer refers to a substance that can protect the stability of the enzyme during production, storage, and use, and prevent enzyme inactivation or degradation, including but not limited to sugars, polyols, proteins, polymers, and metal ions.

[0121] Example 5: Recombinase MAO7 SH Degradation of different biogenic amines

[0122] Using a single biogenic amine as a substrate, the monoamine oxidase (MAO7) was measured before and after purification. SH The catalytic ability was assessed. Systems containing 10 mmol / L of putrescine, tyramine, histamine, cadaverine, phenethylamine, tryptamine, spermine, or spermidine were prepared. The purified enzyme solution from Example 3 (with an enzyme protein concentration of 200 mg / L) was then added. -1 The enzyme was added to the reaction system to achieve a final concentration of 10 mg / L. The reaction was carried out at 37°C for 30 minutes, and enzyme activity was measured at 510 nm. The results are as follows: Figure 4 As shown, compared to the other 6 biogenic amines, the pure enzyme MAO7 SH It was even more effective against the substrates putrescine and cadaverine, with specific enzyme activities reaching 49.19±6.6 U / mg and 43.62±4.58 U / mg, respectively.

[0123] Example 6: Recombinase MAO7 SH Degradation of biogenic amines at different reaction temperatures and their stability at different temperatures.

[0124] The reaction system was the same as in Example 5, except that the reaction temperature was changed to 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, and 60℃, and the recombinant enzyme MAO7 was measured under different temperature reaction conditions. SH The enzyme activity was calculated, with the highest enzyme activity taken as 100%, and the relative enzyme activity was calculated at various temperatures using a single biogenic amine as the substrate. The results are as follows: Figure 5 As shown, MAO7 SH The optimal temperature for the optimal substrate putrescine is approximately 50°C. When the temperature is in the range of 25-50°C, the enzyme activity decreases as the temperature increases, and the enzyme activity decreases rapidly.

[0125] The purified enzyme solution from Example 3 was incubated in water baths at 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, and 60°C for 30 minutes each. Following the standard enzyme reaction system, enzyme activity was measured to determine its stable temperature range. The results are shown in [Figure number missing]. Figure 6 When the temperature exceeds 50℃, the stability of the enzyme decreases.

[0126] Example 7: Recombinase MAO7 SH Degradation of biogenic amines at different reaction pH and enzyme stability under different pH conditions

[0127] The reaction system was the same as in Example 5, except that the pH of the reaction system was adjusted to be between 3 and 9, and the recombinant enzyme MAO7 was measured under different pH reaction conditions. SH The enzyme activity was calculated, with the highest enzyme activity taken as 100%, and the relative enzyme activity was calculated at various temperatures using a single biogenic amine as the substrate. The results are as follows: Figure 7 As shown, when putrescine is used as a substrate, MAO7 SH The optimal pH is about 6. When the pH is in the range of 3 to 6, the enzyme activity decreases as the pH increases, and the enzyme activity decreases rapidly.

[0128] The recombinant enzyme solution purified in Example 3 was diluted appropriately, and the enzyme was incubated at 50°C for 30 min under different pH conditions. The enzyme activity was then measured, and the results are shown below. Figure 8 In general, enzymes are most stable under neutral conditions (pH 7-8). The lower the pH, the less stable the enzyme; under highly acidic conditions, some enzyme activity is lost.

[0129] Example 8: Effects of different concentrations of ethanol on monoamine oxidase MAO7 SH Impact

[0130] The reaction system was the same as in Example 5, except that different concentrations of ethanol (0-20 vol%) were added. The results showed that the recombinant monoamine oxidase MAO7 in the reaction system... SH The enzyme activity of all biogenic amines was inhibited to varying degrees. When the ethanol concentration was below 10 vol%, the activity of monoamine oxidase MAO7 was significantly reduced. SH The relative enzyme activities against putrescine (42.14%) and cadaverine (35.24%) exceeded 30%, and when the ethanol concentration did not exceed 15 vol%, the monoamine oxidase MAO7... SH The relative enzyme activity against putrescine (18.27%) and cadaverine (13.58%) exceeded 10%.

[0131] The monoamine oxidase MAO7 SH It exhibits excellent catalytic ability for putrescine and cadaverine in solutions containing low concentrations of ethanol, a characteristic that provides a solid foundation for its application in the degradation of putrescine and cadaverine in alcoholic beverages containing low concentrations of ethanol.

[0132] Example 9: Monoamine oxidase MAO7 SH Application in Shaoxing wine

[0133] Yellow rice wine is a fermented wine made primarily from rice, millet, sorghum, corn, wheat, and water, through the addition of koji (fermentation starter) and / or some enzyme preparations and yeast as saccharifying and fermenting agents. Its alcohol content is approximately 14% vol to 20% vol. The brewing process produces some organic acids, with a pH value of approximately 3.5-4.5, and a total biogenic amine content of approximately 80-150 mg / L. The high concentration of ethanol and high acidity of yellow rice wine are highly unfavorable for enzymatic catalytic reactions; therefore, controlling the biogenic amine content of yellow rice wine requires more stringent conditions. The specific steps are as follows:

[0134] The recombinant monoamine oxidase MAO7 SH The protein concentration was adjusted to 200 μg / ml and added to rice wine at a 1:1 ratio. The reaction was carried out at room temperature (approximately 28°C) for 48 hours. The control group consisted of commercially available rice wine (18% vol) with the enzyme replaced by PBS buffer. The results are shown in Table 1.

[0135] Table 1. Degradation rates of different biogenic amines by recombinant enzymes

[0136]

[0137] The results showed that commercially available rice wine contained eight biogenic amines, with a total biogenic amine content of 136.2 mg / L. -1 Putrescine and cadaverine are two of the main biogenic amines, and the recombinant enzyme recombinant monoamine oxidase MAO7 is also present. SH The degradation rate of total biogenic amines in commercially available rice wine was 26.52%, with the highest degradation rates for putrescine and cadaverine, at 47.60% and 25.36%, respectively.

[0138] Example 10: Monoamine oxidase MAO7 sh Application in commercially available low-sodium soy sauce

[0139] Soy sauce is mainly made from soybeans or black beans, wheat or wheat bran, and salt through processes such as oil extraction and fermentation. The composition of soy sauce is relatively complex; the total biogenic amine content of samples ranges from 41.18 to 1898.17 mg / L. Regular soy sauce has a salt content of approximately 12 g NaCl / 100 mL, while low-sodium soy sauce has a salt content of approximately 8 g NaCl / 100 mL. The pH is approximately 4.4–4.6. NaCl and acidity limit the progress of enzyme-catalyzed reactions.

[0140] The recombinant monoamine oxidase MAO7 prepared in Example 3 was used. sh Ultrafiltration concentration was adjusted to a protein concentration of 200 ug / ml, and then added to commercially available low-sodium soy sauce. The mixture was allowed to stand at room temperature (25°C) for 24–48 hours. Results showed that monoamine oxidase (MAO7) was reduced. sh It can effectively reduce the content of biogenic amines in low-sodium soy sauce.

[0141] Example 11: Monoamine oxidase MAO7sh Applications in vinegar

[0142] Vinegar is an acidic condiment produced by various fermentations. The amount of acetic acid it contains varies, generally ranging from 5% to 8%. The total amount of biogenic amines varies greatly among different brands of vinegar, and can be as high as 229.98 mg / L.

[0143] The recombinant monoamine oxidase MAO7 prepared in Example 3 was used. sh Ultrafiltration concentration was adjusted to a protein concentration of 200 ug / ml, and the mixture was added to commercially available vinegar. The mixture was allowed to stand at room temperature (25°C) for 24–48 hours. Results showed that monoamine oxidase (MAO7) was reduced. sh It can effectively reduce the content of biogenic amines in vinegar.

[0144] Example 12: Monoamine oxidase MAO7 sh Application in fermented sausages

[0145] By weight, take 65-80% lean meat and 20-35% fat. Wash and remove bones, tendons, muscle membranes, lymph nodes, blood vessels, diseased and damaged parts. Separate the lean and fat meat and cut into 4-5cm pieces. Put the lean meat and about 5-8% ice chips into a meat chopper and chop for 1-3 minutes. Based on the weight 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 added at 0.2%-0.3% of the raw meat. The recombinant monoamine oxidase MAO7 prepared in Example 3 is used. sh Ultrafiltration concentration is used to adjust the protein concentration to 200 ug / ml, which is then added to the sausage raw materials. The mixture is chopped for 1-2 minutes, followed by the addition of fat and approximately 5-8% ice chips, and chopped for another 4-6 minutes. After marinating, the mixture is stuffed into casings. The sausages are then marinated at 4℃ for 12 hours, followed by increasing the temperature to 30℃ and fermentation until the pH drops to approximately 5.1. They are then matured at 14-16℃ for 1-10 days. This process is different from the process without added monoamine oxidase (MAO7). sh Compared to fermented sausages, the results showed that monoamine oxidase MAO7... sh It can effectively reduce the content of biogenic amines in fermented sausages.

[0146] Currently, research on the degradation of biogenic amines in fermented food systems by monoamine oxidase is almost nonexistent. This application lays the technical foundation for the catalytic degradation of biogenic amines in relatively demanding systems containing alcohol and acid, such as rice wine, cooking wine, and fruit wine. It also has greater potential for other systems with higher levels of putrescine and cadaverine.

[0147] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A monoamine oxidase capable of degrading biogenic amines, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. The gene encoding the monoamine oxidase of claim 1.

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

4. Recombinant microbial cells expressing the monoamine oxidase of claim 1.

5. A genetically engineered bacterium, characterized in that, Using Escherichia coli as a host, the monoamine oxidase shown in SEQ ID NO. 2 is expressed.

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, the monoamine oxidase gene shown in SEQ ID NO.3 was expressed.

7. The method for constructing the genetically engineered bacteria of claim 6, characterized in that, The gene sequence shown in SEQ ID NO.2 or SEQ ID NO.3 is ligated to the vector and transformed into E. coli cells.

8. A method for preparing the monoamine oxidase of claim 1, characterized in that, The recombinant microbial cells of claim 4 or the genetically engineered bacteria of any one of claims 5 to 6 are cultured in a culture medium for a period of time, and the monoamine oxidase is collected.

9. The method according to claim 8, characterized in that, The method involves collecting bacterial cells from a cell culture medium and breaking the cells to obtain a crude enzyme solution containing monoamine oxidase.

10. The method according to claim 9, characterized in that, The crude enzyme solution was also purified.

11. The application of the monoamine oxidase according to claim 1 in reducing biogenic amines in the food industry.

12. The application according to claim 11, characterized in that, The application is to reduce the content of biogenic amines in fermented foods; the fermented foods include fermented vegetables or alcoholic beverages.

13. The application according to claim 11 or 12, characterized in that, The biogenic amines include one or more of tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine.

14. An enzyme preparation for degrading biogenic amines, characterized in that, The enzyme preparation contains monoamine oxidase with an amino acid sequence as shown in SEQ ID NO.

1.

15. A method for degrading biogenic amines in an environmental system, characterized in that, Biogenic amines are degraded by contacting the monoamine oxidase of claim 1, the recombinant microbial cells of claim 4, or any of the genetically engineered bacteria of claims 5-6 with biogenic amines in the environment.

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