Flavin-Containing Monoamine Oxidase MAO6sh Capable of Degrading Biogenic Amines and Application Thereof

Abstract

The present disclosure discloses a flavin-containing monoamine oxidase MAO6.sup.sh capable of degrading biogenic amines and an application thereof, belonging to the technical field of molecular biology. The present disclosure provides a monoamine oxidase derived from Saccharopolyspora hirsuta, and achieves the expression of the monoamine oxidase in Escherichia coli. The present disclosure further provides an application of the monoamine oxidase in degradation of biogenic amines. Tryptamine, phenylethylamine and cadaverine can be effectively degraded by adding the monoamine oxidase to commercially available Huangjiu, the degradation rate is 33.76% or above, and the safety of fermented food is further improved.

Claims

1. A genetically engineered bacterium, wherein Escherichia coli BL21 (DE3) is used as a host to express a monoamine oxidase shown in SEQ ID NO:1.

2. The genetically engineered bacterium according to claim 1, wherein E. coli BL21 (DE3) is used as a host.

3. The genetically engineered bacterium according to claim 2, wherein pET28a(+) is used as an expression vector to express the monoamine oxidase gene shown in SEQ ID NO:3.

4. A method for preparing a monoamine oxidase, wherein the genetically engineered bacterium according to claim 1 is cultured in a culture medium for a period of time, and the monoamine oxidase is collected.

5. The method according to claim 4, wherein the method involves collecting bacterial cells from a cell culture fluid, and crushing the cells to obtain a crude enzyme solution containing the monoamine oxidase.

6. The method according to claim 5, wherein the method further involves purifying the crude enzyme solution.

7. A method for reducing a content of biogenic amines in food, wherein a monoamine oxidase shown in SEQ ID NO:1 is added to food to degrade the biogenic amines in the food.

8. The method according to claim 7, wherein the food is fermented food; and the fermented food comprises fermented vegetables and alcoholic beverages.

9. The method according to claim 8, wherein the fermented food is table vinegar, soy sauce, Huangjiu, or fermented sausage.

10. The method according to claim 8, wherein the biogenic amine comprises one or more of tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine.

11. The method according to claim 7, wherein the method comprises adding the monoamine oxidase obtained by fermentation of a genetically engineered bacterium that expresses the monoamine oxidase to the food and allowing the mixture to react at 20-25 C. for 24-48 hours.

12. The method according to claim 7, wherein the method comprises adding the monoamine oxidase to Huangjiu and allowing the mixture to react at a room temperature of 25-28 C. for 24-48 hours.

Description

BRIEF DESCRIPTION OF FIGURES

[0066] FIG. 1 shows an agarose gel electrophoresis verification diagram of a monoamine oxidase gene; M: DNA Maker; 1: PCR amplification product (1302 bp).

[0067] FIG. 2 shows a plasmid profile of an expression vector of a monoamine oxidase pET28a-MAO6.sup.sh.

[0068] FIG. 3 shows an electrophoretogram of protein separation and purification processes of a recombinant monoamine oxidase MAO6.sup.sh; M: Unstained Protein Ladder; 1: pET28a-empty expression vector; 2. crude enzyme solution supernatant of monoamine oxidase pET28a-MAO6.sup.sh induced at 16 C.; 3. crude enzyme solution supernatant of monoamine oxidase pET28a-MAO6.sup.sh induced at 20 C.; 4. crude enzyme solution supernatant of monoamine oxidase pET28a-MAO6.sup.sh induced at 28 C.; 5: crude enzyme solution supernatant of monoamine oxidase pET28a-MAO6.sup.sh induced at 37 C.; 6: purified enzyme solution of monoamine oxidase pET28a-MAO6.sup.sh induced at 16 C.; 7. crude enzyme solution precipitate of monoamine oxidase pET28a-MAO6.sup.sh induced at 16 C.; 8: crude enzyme solution precipitate of monoamine oxidase pET28a-MAO6.sup.sh induced at 20 C.; 9: crude enzyme solution precipitate of monoamine oxidase pET28a-MAO6.sup.sh induced at 37 C.

[0069] FIG. 4 shows the ability of a monoamine oxidase MAO6.sup.sh for degrading different biogenic amines before and after purification. Putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenylethylamine (PHE), tryptamine (TRY), spermine (SPE), and spermidine (SPD).

[0070] FIG. 5 shows the relative enzyme activity of a monoamine oxidase MAO6.sup.sh at different temperatures. Putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenylethylamine (PHE), tryptamine (TRY), spermine (SPE), and spermidine (SPD).

[0071] FIG. 5 and FIG. 6 show the temperature stability of a monoamine oxidase MAO6.sup.sh. Putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenylethylamine (PHE), tryptamine (TRY), spermine (SPE), and spermidine (SPD).

[0072] FIG. 7 shows the relative enzyme activity of a monoamine oxidase MAO6.sup.sh at different pH values. Putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenylethylamine (PHE), tryptamine (TRY), spermine (SPE), and spermidine (SPD).

[0073] FIG. 8 shows the pH stability of a monoamine oxidase MAO6.sup.sh. Putrescine (PUT), tyramine (TYR), histamine (HIS), cadaverine (CAD), phenylethylamine (PHE), tryptamine (TRY), spermine (SPE), and spermidine (SPD).

[0074] FIG. 9 shows the relative enzyme activity of a monoamine oxidase MAO6.sup.sh under different ethanol conditions. Putrescine (PUT), cadaverine (CAD), and tryptamine (TRY).

[0075] FIG. 10 shows a phylogenetic tree diagram of a monoamine oxidase gene MAO6.sup.sh

DETAILED DESCRIPTION

(1) Technical Terms:

[0076] The dairy products involved in the present disclosure refer to foods made from animal milk as a raw material through different processing methods, and the types include but are not limited to pasteurized milk, sterilized milk, blended milk, fermented milk, whole milk powder, skimmed milk powder, whole milk powder with sugar, flavored milk powder, infant milk powder and other formula milk powder, condensed milk, milk fats, cheeses, ice creams, casein, milk slices, lactose, etc.

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

[0078] The meat products involved in the present disclosure refer to products made from animal muscle tissues or edible internal organs as main raw materials through various processing methods. According to the national standard for meat and meat product terms (GB/T 19480-2009), meat products are divided into two 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, air-dried sausage, fresh sausage products, smoked and fresh sausage, semi-dry sausage, dry sausage, prepared meat products, meat cake, and 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 sausage.

[0079] The fermented foods involved in the present disclosure refer to foods made by fermentation of microorganisms (such as bacteria, yeast, or fungi). 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, table vinegar, fermented soy beans, Huangjiu, beer, wine, etc.

[0080] The biogenic amines involved in the present disclosure refer to low-molecular-weight organic compounds having biological activity and containing amino groups, including but not limited to tryptamine (TRY), phenylethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), tyramine (TYR), spermidine (SPD), and spermine (SPE).

(2) Reagent

[0081] The Huangjiu and other products involved in the following examples were purchased from a supermarket in Wuxi, Jiangsu Province.

(3) Culture Medium

[0082] An LB culture medium was formed by 5 g/L yeast extract, 10 g/L tryptone and 10 g/L sodium chloride by adjusting the pH to 7.0 with NaOH, and was sterilized under a high pressure at 121 C. for 10 min.

[0083] A TB culture medium was purchased from Qingdao Hopebio company, and was sterilized under a high pressure at 121 C. for 10 min.

(4) Detection Method

[0084] The content of biogenic amines was detected by high-performance liquid chromatography (HPLC).

[0085] Enzyme activity measurement: The activity of a biogenic amine oxidase was measured by an indirect measurement method of catalase. The amine oxidase acts on biogenic amines to degrade the biogenic amines into corresponding aldehydes and hydrogen peroxide. Under the presence of peroxidase, hydrogen peroxide reacts with 4-aminoantipyrine and 2,4,6-tribromo-3-hydroxybenzoic acid to generate a quinone dye. The product has a maximum absorption value at 510 nm, and the magnitude of the activity of the amine oxidase was linearly related to the color intensity of the product within a certain range, so that the activity of the amine oxidase can be measured by measuring the change in A510.

[0086] The reaction was carried out in a 96-well plate. The reaction system included 20 l of enzyme solution (80 ug.Math.mL.sup.1) and 100 l of prepared solution (including 200 mmol.Math.L.sup.1 potassium phosphate buffer with a pH of 7.6, 1.5 mmol.Math.L.sup.1 4-aminoantipyrine, and 1 mmol.Math.L.sup.1 2,4,6-tribromo-3-hydroxybenzoic acid). To initiate the reaction, 20 L of biogenic amine solution (10 mmol.Math.L.sup.1) and 70 L of peroxidase (1.4 mg.Math.mL.sup.1) were added, the absorbance was measured at 510 nm, the reaction temperature was 37 C., and the reaction time was 10 min. The change in absorbance of 0.01 per minute was defined as one enzyme activity unit (U).

[0087] Definition of specific activity of monoamine oxidase (U/mg): The enzyme activity per milligram of protein.

Example 1

PCR Amplification of Monoamine Oxidase Gene MAO6.SUP.sh

[0088] Specific steps were as follows: [0089] (1) Primers were designed according to the monoamine oxidase gene (WP_150070586.1) in Saccharopolyspora hirsuta in an NCBI database, and the DNA of Saccharopolyspora hirsuta T14 preserved in the laboratory was used as a template to amplify the monoamine oxidase gene MAO6.sup.sh.

[0090] The primers required for amplification were as follows:

TABLE-US-00001 F: 5-ctagctagcATGGACTCCTWCGACGTCGTSGTCA TCGGTGCCGGrTTCGCCGG-3; and R: 5-CAAGCTTTCAGCCTCGSGGGCCGCGCAGGGCGTC CTGCACGGCCCTGGAGGCGCGCAGGCCGCTC-3.

[0091] A PCR reaction solution was prepared according to the requirements of a reaction system of TaKaRa LA Taq. The PCR amplification system was as follows: pre-denaturation at 98 C. for 10 s, annealing at 75 C. for 30 s, and extension at 72 C. (1 min.Math.kb.sup.1) for 35 cycles. [0092] (2) The DNA of Saccharopolyspora hirsuta T14 was used as a template for PCR amplification, and the amplification results of PCR products were verified by 1.2% agarose gel electrophoresis. As shown in FIG. 1, the amplified sequence size was the same as the target gene sequence size, approximately 1302 bp, indicating successful amplification. The PCR products were purified and then sent to the company for sequencing, and the sequencing results were shown in SEQ ID NO. 2 (with enzyme digestion sites Nhe I and Hind III at both ends of the sequence).

Example 2

Construction of Genetically Engineered Bacterium Capable of Producing Monoamine Oxidase Gene MAO6.SUP.sh

[0093] The amine oxidase gene MAO6.sup.sh amplified in Example 1 was linked to plasmids so as to be transformed to microbial cells to construct a genetically engineered bacterium capable of producing the monoamine oxidase gene MAO6.sup.sh.

[0094] 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.

[0095] Optionally, the host was a bacterial cell or a fungal cell, including but not limited to E. coli, Bacillus, or yeast.

[0096] Taking recombinant E. coli as an example, the construction process of the genetically engineered bacterium pET28a-MAO6.sup.sh was described as follows:

(1) Obtaining of Target Fragment

[0097] The DNA of the strain Saccharopolyspora hirsuta T14 preserved in the laboratory was used as a template to complete PCR amplification by combining primers and whole genome DNA together, and the PCR reaction system and the amplification procedure were the same as those described in Example 1. The gel of the PCR product with correctly verified bands was carefully cut, and the gel was recovered and purified to obtain the monoamine oxidase gene fragment MAO6.sup.sh. After sequencing, the nucleotide sequence of the gene was determined to be as shown in SEQ ID NO. 3.

(2) Enzyme Digestion and Linkage

[0098] The plasmid pET-28a(+) and the target fragment obtained in step (1) were subjected to double enzyme digestion respectively with restriction enzymes NheI and HindIII. The enzyme digestion system was as follows: 50 L of plasmid, 2.5 l of restriction enzyme Nhe I and 2.5 L of restriction enzyme HindIII, and 5 l of FastDigest Green Buffer. The components in the enzyme digestion system were fully mixed uniformly, and then, the mixture was placed in a metal bath at 37 C. for reacting for 60 min. After double enzyme digestion, the gene fragment and the plasmid were recovered and purified, and then mixed in a molar ratio of (3-10):1. An equal volume of Solution I ligase was added and fully mixed uniformly, and then, the mixture was incubated overnight in a metal bath at 16 C. to prepare a recombinant vector pET28a-MAO6.sup.sh.

(3) Transformation

[0099] After E. coli BL21 (DE3) competent cells preserved at 80 C. were placed in an ice bath for 10 min, 10 L of linkage product to be transformed, obtained in step (2), was sucked with a pipette and added to the competent cells and gently blown and sucked and mixed uniformly, and then, the mixture was placed in an ice bath for 30 min. After the ice bath, the mixture was subjected to thermal shock at 42 C. for 45 s and then immediately taken out and placed in ice for 2 min. Then, 700 L of LB liquid culture medium was added for shaking culture at 37 C. for 60 min at 200 r.Math.min.sup.1. Centrifugation was carried out for 1 min at 8000 r.Math.min.sup.1, most of the supernatant was discarded, and about 200 L of supernatant was retained to resuspend the bacterial cells. The bacterial solution was uniformly coated on an LB solid culture medium plate containing 50 mg.Math.L.sup.1 kanamycin, the plate was inverted and cultured overnight in an incubator at 37 C. until a single colony grows up and was picked, and then, PCR verification was carried out for screening positive transformants.

(4) Enzyme Digestion Verification

[0100] The plasmid of the recombinant bacterium was extracted and subjected to NheI and HindIII double enzyme digestion to respectively obtain a pET-28a(+) fragment of 5369 bp and a target fragment of 1302 bp. The target fragment was sent to the company for sequencing, and the sequencing results were consistent with the target gene sequence, verifying the successful construction of the recombinant bacterium E. coli BL21/pET28a-MAO6.sup.sh. The recombinant enzyme expressed by this strain was named as MAO6.sup.sh. The construction process was shown in FIG. 2.

Example 3

Induced Expression and Purification of Recombinant Enzyme MAO6.SUP.sh

[0101] Specific steps were as follows: [0102] (1) The recombinant bacterium E. coli BL21/pET28a-MAO6.sup.sh constructed in Example 2 was inoculated into an LB culture medium containing 50 mg.Math.L.sup.1 kanamycin, and cultured at 37 C. for 14 h at 150 r.Math.min.sup.1 to prepare a seed solution. [0103] (2) The obtained seed solution was transferred into a TB fermentation culture medium containing 50 mg.Math.L.sup.1 kanamycin at an inoculation volume of 5% (v/v), and cultured at 37 C. at 160 r.Math.min.sup.1 until the OD600 was 0.4-0.6. The IPTG with a final concentration of 0.5 mmol.Math.L.sup.1 was added and cultured at 16 C., 20 C., 28 C. and 37 C. respectively for 16 h at 160 rpm to obtain bacterial solutions.

[0104] The enzyme yield of the monoamine oxidase in the bacterial solution was measured, and the results showed that 17.5 mg of MAO6.sup.sh enzyme protein was contained in every 100 mL of bacterial solution with the OD600 of 1 induced at 16 C.

[0105] The activity of the monoamine oxidase was measured, and the results showed that at 16 C., the specific enzyme activities of the monoamine oxidase in the crude enzyme solution for tryptamine, putrescine and cadaverine were 13.94 U/mg, 15.66 U/mg and 14.70 U/mg respectively. When the induction temperature was slightly higher, more inclusion bodies were produced (as shown in FIG. 3), leading to a decrease in enzyme activity. [0106] (3) The bacterial solutions were respectively centrifuged at 4 C. for 10 min at 12000 r.Math.min.sup.1, and then, lower-layer bacterial cells were collected; and 0.1 mol.Math.L.sup.1 phosphate buffer saline with a pH of 7.4 was added to resuspend the bacterial cells, and then, the bacterial cells were collected by centrifugation. The above-mentioned steps were repeated twice. An ultrasonic cell crusher was used to crush the bacterial cells under ultrasonic conditions of 400 W, working for 2 s at an interval of 3 s, and a crushing time of 1 h. After crushing, the supernatant and precipitate were collected by centrifugation at 4 C. at 12000 r.Math.min.sup.1, filtered through a 0.22 M filter membrane, and stored at a low temperature for future use. [0107] (4) Purification of Recombinant Enzyme MAO6.sup.sh

[0108] The supernatant obtained in step (3) was processed by an affinity

[0109] chromatography column HisTrap HP (GE Healthcare) to purify the protein, and an AKTA avant 25 instrument was used to separate and purify the target protein.

[0110] The supernatant of the crude enzyme solution before MAO6.sup.sh purification and the protein after MAO6.sup.sh purification were respectively analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and the results were shown in FIG. 3. The molecular weight of the recombinant monoamine oxidase MAO6.sup.sh was about 46.7 kDa. The crude enzyme solution was analyzed by SDS-PAGE, confirming the successful expression of the recombinant engineering bacterium. The lane 6 proves that the pure enzyme of the target protein was successfully obtained through the separation and purification of an affinity chromatography nickel column. Although induced by multiple conditions, when the recombinant bacterium E. coli BL21/pET28a-MAO6.sup.sh expresses the enzyme gene, inclusion bodies easily occur. The inclusion body phenomenon can be observed in lanes 7, 8, and 9.

Example 4

Preparation of Enzyme Preparation of Monoamine Oxidase MAO6.SUP.sh

[0111] The monoamine oxidase MAO6.sup.sh purified in Example 3 was mixed with an enzyme stabilizer to prepare an enzyme preparation containing the monoamine oxidase MAO6.sup.sh. The stabilizer refers to a substance that can protect the stability of enzymes during production, storage and use to prevent enzyme inactivation or degradation, including but not limited to saccharides, polyols, proteins, polymers, metal ions, etc.

Example 5

Degradation of Different Biogenic Amines by Recombinant Enzyme MAO6.SUP.sh

[0112] The catalytic ability of the monoamine oxidase MAO6.sup.sh before and after purification was measured respectively using a single biogenic amine as a substrate. Systems containing putrescine, tyramine, histamine, cadaverine, phenylethylamine, tryptamine, spermine or spermidine with a concentration of 10 mmol/L were prepared respectively, the pure enzyme in Example 3 was added to the reaction system after adjusting the protein concentration (80 ug/mL, 20 uL) for reacting for 30 min at 37 C., and the results were shown in

[0113] FIG. 4. The MAO6.sup.sh has better effects on substrates, namely putrescine, tryptamine and cadaverine. The MAO6.sup.sh has a specific enzyme activity of 161.59 U/mg for putrescine, 149.16 U/mg for tryptamine, and 146.05 U/mg for cadaverine.

Example 6

Degradation of Biogenic Amines by Recombinant Enzyme MAO6.SUP.sh .at Different Reaction Temperatures, and Temperature Stability

[0114] The reaction system was the same as that of Example 5, except that the temperature of the reaction system was changed to 25 C., 30 C., 35 C., 40 C., 45 C., 50 C., 55 C. and 60 C. respectively, the enzyme activity of the recombinant enzyme MAO6.sup.sh under different temperature reaction conditions was measured, the relative enzyme activity at each temperature was calculated respectively using a single biogenic amine as a substrate at the highest enzyme activity of 100%, and the results were shown in FIG. 5. The optimal temperature for the MAO6.sup.sh for the most suitable substrate was approximately 45-50 C. When the temperature was within the range of 25-45 C., the enzyme activity decreased rapidly with the increasing temperature.

[0115] After the enzyme solution purified in Example 3 was respectively incubated for 30 min in water baths at 15 C., 30 C., 35 C., 40 C., 45 C., 50 C., 55 C. and 60 C., the enzyme activity was measured according to the standard enzyme reaction system to determine the stable temperature range thereof, and the results were shown in FIG. 6. When the incubation temperature exceeds 45 C., about 30% of residual enzyme activity can be retained, indicating that the monoamine oxidase has certain heat resistance. When the incubation temperature was 50 C., the residual enzyme activity for putrescine was 36.66%, the residual enzyme activity for cadaverine was 30.55%, and the residual enzyme activity for tryptamine was 26.34%.

Example 7

Degradation of Biogenic Amines by Recombinant Enzyme MAO6.SUP.sh .at Different Reaction pH Values, and pH Stability

[0116] The reaction system was the same as that of Example 5, except that the pH of the reaction system was respectively adjusted to 3.0-9.0, the enzyme activity of the recombinant enzyme MAO6.sup.sh under different pH reaction conditions was measured, the relative enzyme activity at each temperature was calculated respectively using a single biogenic amine as a substrate at the highest enzyme activity of 100%, and the results were shown in FIG. 7. When putrescine was used as a substrate, the optimal pH for the MAO6.sup.sh was about 5.0-6.0. When the pH was within the range of 3.0-5.0, the enzyme activity decreased rapidly with the increase of the pH. However, under alkaline conditions, the tolerance was stronger than the acid tolerance.

[0117] After the recombinant enzyme solution purified in Example 3 was diluted by an appropriate multiple, the enzyme was incubated at 45 C. for 30 min under different pH ambient conditions, the enzyme activity was measured, and the results were shown in FIG. 8. The monoamine oxidase MAO6.sup.sh was more stable under neutral to alkaline conditions.

Example 8

Influence of Ethanol with Different Concentrations on Monoamine Oxidase MAO6.SUP.sh

[0118] The reaction system was the same as that of Example 5, except that ethanol with different concentrations (0-20% vol) was added respectively. The results showed that the recombinant monoamine oxidase MAO6.sup.sh inhibited the enzyme activity of each biogenic amine to different degrees. When the concentration of ethanol was lower than 10% vol, the relative enzyme activities of the monoamine oxidase MAO6.sup.sh for putrescine (29.44%), tryptamine (30.27%) and cadaverine (35.78%) were about 30%. When the concentration of ethanol was about 15 vol %, the relative enzyme activities of the monoamine oxidase MAO6.sup.sh for putrescine, tryptamine and cadaverine were 9.57%, 11.21% and 13.67% respectively (FIG. 9).

[0119] The above results indicate that the monoamine oxidase MAO6.sup.sh has a good catalytic degradation ability for putrescine, tryptamine and cadaverine in a system containing a low-concentration ethanol solution. This characteristic provides a good foundation for the application in degradation of alcoholic beverages containing low-concentration ethanol (such as beer and Huangjiu) or systems rich in putrescine, tryptamine and cadaverine.

Example 9

Application of Monoamine Oxidase MAO6.SUP.sh .in Commercially Available Huangjiu

[0120] The alcohol content of the commercially available Huangjiu is about 14%-20% vol, the brewing process will produce some organic acids, the pH value of the commercially available Huangjiu is about 3.5-4.5, and the total content of biogenic amines is about 80-150mg/L. The ethanol with a higher concentration and high acidity of the Huangjiu are very unfavorable for the catalytic reaction of enzymes, so the enzymatic control of the content of biogenic amines in the Huangjiu requires more stringent conditions. Specific steps were as follows:

[0121] The recombinant monoamine oxidase MAO6.sup.sh prepared in Example 3 was subjected to ultrafiltration and concentration to adjust the protein concentration to 200 g/ml, and then added to the Huangjiu in a ratio of 1:1 for reacting at a room temperature of about 28 C. for 48 h. The control group was commercially available Huangjiu (18% vol) without enzymes, and the total content of biogenic amines in the control group was 136.2 mg.Math.L.sup.1.

[0122] The results were shown in Table 1.

TABLE-US-00002 TABLE 1 Degradation rates of different biogenic amines by recombinant enzyme Biogenic amine Tryptamine Putrescine Phenylethylamine Cadaverine Histamine Tyramine Spermidine Spermine Degradation 54.07 33.76 5.78 43.78 21.44 19.31 8.51 7.75 rate %

[0123] The results showed that the commercially available Huangjiu contains 8 types of biogenic amines, the total content of biogenic amines was 136.2 mg.Math.L.sup.1, and tryptamine and cadaverine were the two most important biogenic amines therein. After calculation, the recombinant monoamine oxidase MAO6.sup.sh has the degradation rate for total biogenic amines in the commercially available Huangjiu of 25.56%, and has the degradation rates for tryptamine, putrescine and cadaverine of 54.07%, 33.76% and 43.78% respectively.

Example 10

Application of Monoamine Oxidase MAO6.SUP.sh .in Commercially Available Low-Salt Soy Sauce

[0124] The soy sauce is mainly brewed from soybeans or black beans, wheat or bran, and table salt through processes such as oil making and fermentation. The components of the soy sauce are relatively complex, the range of the total content of biogenic amines in a sample is 41.18-1898.17 mg/L, the salt content of ordinary soy sauce is about 12 g NaCl/100 mL, and the salt content of low-salt soy sauce is about 8 g NaCl/100 mL. The pH is about 4.4-4.6. The NaCl and acidity limit the progress of the enzymatic catalytic reaction.

[0125] The recombinant monoamine oxidase MAO6.sup.sh prepared in Example 3 was subjected to ultrafiltration and concentration to adjust the protein concentration to 200 ug/ml, and then added to the commercially available low-salt soy sauce for standing at a room temperature (25 C.) for 24-48 h. The results showed that the monoamine oxidase MAO6.sup.sh can effectively reduce the content of biogenic amines in the low-salt soy sauce.

Example 11

Application of Monoamine Oxidase MAO6.SUP.sh .in Table Vinegar

[0126] The table vinegar is a sour seasoning produced by various fermentations, and the content of the acetic acid contained in the table vinegar varies and generally ranges from 5% to 8%. The total amount of biogenic amines in different brands of table vinegar varies greatly, and the total amount of biogenic amines can reach up to 229.98 mg/L.

[0127] The recombinant monoamine oxidase MAO6.sup.sh prepared in Example 3 was subjected to ultrafiltration and concentration to adjust the protein concentration to 200 g/ml, and then added to the commercially available table vinegar for standing at a room temperature (25 C.) for 24-48 h. The results showed that the monoamine oxidase MAO6.sup.sh can effectively reduce the content of biogenic amines in the table vinegar.

Example 12

Application of Monoamine Oxidase MAO6.SUP.sh .in Fermented Sausage

[0128] 65-80% of lean meat and 20-35% of fat meat were taken by mass and cleaned, and bones, tendons, myolemma, lymph nodes, blood vessels, lesions and injury sites were removed. The fat meat and the lean meat were separated and cut into 4-5 cm meat pieces. The lean meat and about 5-8% of flake ice were placed in a chopper and chopped for 1-3 min. Based on the mass of pork, 0.01-0.15% of sodium nitrite, 2-3% of table salt, 0.2-0.3% of compound phosphate, and 0.05-0.06% of sodium ascorbate were added. Spices, pepper, garlic, chili, and nutmeg were 0.2-0.3% of the raw meat. The recombinant monoamine oxidase MAO6.sup.sh prepared in Example 3 was subjected to ultrafiltration and concentration to adjust the protein concentration to 200 g/ml, and then added to the sausage raw material for chopping for 1-2 min, and then, the fat meat and about 5-8% of flake ice were added for chopping for 4-6 min. The sausage was pickled and then poured into the sausage casing. The sausage poured into the sausage casing was pickled at 4 C. for 12 h, then heated to 30 C., fermented until the pH decreased to about 5.1, and matured at 14-16 C. for 1-10 d. Compared with the fermented sausage without the monoamine oxidase MAO6.sup.sh, the results showed that the monoamine oxidase MAO6.sup.sh can effectively reduce the content of biogenic amines in the fermented sausage.

[0129] At present, there is almost no research on degradation of biogenic amines in fermented food systems by the monoamine oxidase. The monoamine oxidase provided in the present disclosure and the method for degrading biogenic amines in the food industry lay a technical foundation of the monoamine oxidase for catalytic degradation of biogenic amines in relatively harsh systems such as Huangjiu, cooking wine and fruit wine containing alcohols and acids, and have greater potential for other systems with higher content of tryptamine and cadaverine.

[0130] Although the present disclosure has been disclosed above with preferred examples, it is not intended to limit the present disclosure. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present disclosure. Therefore, the scope of protection of the present disclosure should be defined by the claims.