Preparation and application of bacillus-sourced multi-copper oxidase LYSUF1 capable of degrading biogenic amine in fermented food

Abstract

The invention discloses preparation and application of bacillus-sourced multi-copper oxidase LYSUF1 capable of degrading biogenic amine in fermented food, and belongs to the technical field of molecular biology. The invention provides the bacillus-sourced multi-copper oxidase, and the expression of the enzyme in escherichia coli is realized. The invention also provides application of the multi-copper oxidase in degradation of biogenic amines, the multi-copper oxidase is added into fermented food, so that the biogenic amines can be effectively degraded, an enzyme library for degrading the biogenic amines can be expanded, and the safety of the fermented food is further improved.

Description

Technical Field

[0001] The invention relates to the preparation and application of a bacillus-derived multi-copper oxidase LYSUF1 capable of degrading biogenic amines in fermented food, and belongs to the technical field of molecular biology. Background Art

[0002] Biogenic amines are a class of nitrogen-containing organic substances with biological activity, which are commonly found in fermented foods. The appropriate amount of biogenic amines synthesized by the human body itself is of great help in maintaining the normal physiological functions of the body. The appropriate amount of biogenic amines (such as histamine, tyramine, etc.) synthesized endogenously by the human body can maintain normal physiological functions by regulating neurotransmitter transmission, immune response and other pathways. However, when the body ingests high concentrations of biogenic amines or specific enzymes malfunction, excessive exogenous biogenic amines can cause a series of adverse reactions. The toxicity between different biogenic amines can also show synergistic and additive effects. Therefore, it is crucial to control the content of biogenic amines.

[0003] Multi-copper oxidases are a class of copper-containing oxidases widely found in microorganisms, animals and plants. As a class of metal oxidases containing four copper ion active centers, they exhibit broad-spectrum substrate catalytic ability with their unique electron transfer mechanism, and have great potential in oxidizing certain biogenic amines. Bacterial multi-copper oxidases have broad substrate specificity and also show significant environmental tolerance advantages. This strong tolerance stems from their unique structural characteristics - the copper ion binding domain (T1-T3 cluster) resists proton attack through a dynamic hydrogen bond network, the "molecular barrier" formed by hydrophobic amino acid residues can weaken the denaturation effect of ethanol on enzyme proteins, and the surface positively charged domain reduces the interference of high salt ions on enzyme conformation through electrostatic repulsion, so it has greater advantages.

[0004] At present, there is almost no research on multi-copper oxidases with strong tolerance that can degrade biogenic amines in fermented foods. Patent CN108060142B discloses a multi-copper oxidase derived from Bacillus amyloliquefaciens, which has 30% enzyme activity remaining at 5% vol alcohol; when the ethanol concentration rises to 20%, the remaining enzyme activity is less than 5%. Patent CN109468288A discloses a multi-copper oxidase derived from Lactobacillus fermentum, which is placed in 18% sodium chloride solution for 1 hour, and the relative enzyme activity only retains 40%; and when the sodium chloride concentration is increased to 20%, the remaining enzyme activity is less than 10%. The tolerance of multi-copper oxidase to ethanol and sodium chloride is an important property that determines the application of the enzyme. Therefore, it is crucial to provide a multi-copper oxidase with strong tolerance to degrade biogenic amines in fermented foods. Summary of the invention

[0005] The object of the present invention is to solve the problem that the content of biogenic amines in existing traditional fermented foods is generally high, and to obtain a multi-copper oxidase derived from Bacillus with strong tolerance, which has strong ability to degrade biogenic amines in a variety of fermented foods.

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

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

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

[0009] The present invention also provides a gene encoding the multi-copper oxidase.

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

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

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

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

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

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

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

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

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

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

[0020] In one embodiment, the genetically engineered bacterium uses pET28a(+) as an expression vector and expresses the multicopper oxidase shown in SEQ ID NO.2.

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

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

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

[0024] The present invention also provides a method for producing multicopper oxidase, which is to inoculate the genetically engineered bacterium into a culture medium for culturing and collect the multicopper oxidase.

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

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

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

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

[0029] In one embodiment, nickel column affinity chromatography is used for the purification.

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

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

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

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

[0034] The present invention also provides the use of the multi - copper oxidase in reducing biogenic amines.

[0035] In one embodiment, the use is to contact the multi - copper 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 use includes reducing the content of biogenic amines in fermented foods.

[0038] 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.

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

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

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

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

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

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

[0045] In one embodiment, the use is to add the multi - copper oxidase to yellow rice wine to reduce the content of the main biogenic amines therein.

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

[0047] In one embodiment, the use is to add the multi - copper oxidase to cooking wine to reduce the content of the main biogenic amines therein.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0062] In one embodiment, the genetically engineered bacterium uses bacteria or fungi as host cells.

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

[0064] The present invention also provides the use of the above enzyme preparation, or the multicopper oxidase with the amino acid sequence shown in SEQ ID NO.1, or the genetically engineered bacterium expressing the multicopper 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.

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

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

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

[0068] In one embodiment, the genetically engineered bacterium uses bacteria or fungi as host cells.

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

[0070] Beneficial effects:

[0071] (1) The multicopper oxidase recombinant enzyme LYSUF1 from Bacillus cereus provided by the present invention has strong performance in degrading biogenic amines and the ability to tolerate ethanol and NaCl. When the NaCl concentration reaches 20%, the relative enzyme activity of LYSUF1 still remains 53.22%. When the alcohol content reaches 25% vol, the enzyme activity remains above 35%. It helps to achieve the degradation of biogenic amines in fermented foods.

[0072] (2) The present invention also provides the application of the multi-copper oxidase LYSUF1 in degrading biogenic amines in fermented foods. In soy sauce, this enzyme has significant degradation effects on phenethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), and tyramine (TYR). After 7 days of treatment, the degradation rate of phenethylamine is 12.19%, the degradation rate of putrescine is 6.42%, cadaverine is completely degraded (degradation rate is 100%), the degradation rate of histamine is 38.75%, and the degradation rate of tyramine is 19.38%. The total degradation rate of this enzyme in soy sauce is approximately 26.37%.

[0073] (3) The present invention also provides the application of the multi-copper oxidase LYSUF1 in degrading biogenic amines in fermented foods. In Chinese rice wine, this enzyme has degradation effects on putrescine (PUT), cadaverine (CAD), histamine (HIS), and tyramine (TYR). After 7 days of treatment, the degradation rate of putrescine is 4.75%, the degradation rate of cadaverine is 15.35%, the degradation rate of histamine is 23.57%, and the degradation rate of tyramine is 3.64%. Although the degradation rates are different, overall this enzyme helps to reduce the content of biogenic amines in Chinese rice wine. The total degradation rate of this enzyme in Chinese rice wine is approximately 7.21%.

[0074] (4) The present invention also provides the application of the multi-copper oxidase LYSUF1 in degrading biogenic amines in fermented foods. In cooking wine, this enzyme has obvious degradation effects on putrescine (PUT) and histamine (HIS), and the degradation rates are 17.27% and 24.66% respectively. The degradation rate of tyramine (TYR) is 7.63%. The total degradation rate of this enzyme in cooking wine is approximately 26.84%.

[0075] (5) The present invention also provides the application of the multi-copper oxidase LYSUF1 in degrading biogenic amines in fish sauce. In fish sauce, this enzyme has significant degradation effects on phenethylamine (PHE), histamine (HIS), and tyramine (TYR). After 7 days of treatment, the degradation rate of phenethylamine is 65.9%, histamine is completely degraded (degradation rate is 100%), and the degradation rate of tyramine is 26.97%. The total degradation rate of this enzyme in fish sauce is approximately 27.93%.

[0076] (6) The present invention also provides the application of the multi-copper oxidase LYSUF1 in degrading biogenic amines in red wine. In red wine, this enzyme has significant degradation effects on tryptamine (TRY), phenethylamine (PHE), histamine (HIS), and tyramine (TYR). After 7 days of treatment, tryptamine is completely degraded (degradation rate is 100%), the degradation rate of phenethylamine is 4.73%, the degradation rate of histamine is 19.8%, and the degradation rate of tyramine is 32.82%. The total degradation rate of this enzyme in red wine is approximately 29.11%.

[0077] (7) The present invention also provides the application of multi-copper oxidase LYSUF1 in degrading biogenic amines in low-salt soy sauce. In low-salt soy sauce, this enzyme has significant degradation effects on phenethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), tyramine (TYR), and spermidine (SPD). After 7 days of treatment, the degradation rate of phenethylamine is 56.27%, the degradation rate of putrescine is 29.99%, the degradation rate of cadaverine is 14.86%, the degradation rate of histamine is 26.71%, the degradation rate of tyramine is 39.65%, and the degradation rate of spermidine is 5.35%. The total degradation rate of this enzyme in low-salt soy sauce is approximately 29.18%.

[0078] (8) The present invention also constructs a recombinant Escherichia coli expressing multi-copper oxidase LYSUF1. 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 LYSUF1 enzyme protein. Description of the Drawings

[0079] Figure 1 Agarose gel electrophoresis verification diagram of the multi-copper oxidase gene: M: DNA Maker; 1: PCR amplification product (1566 bp).

[0080] Figure 2 Plasmid map of the multi-copper oxidase pET28a-LYSUF1 expression vector.

[0081] Figure 3 Protein electrophoresis diagram of recombinant multi-copper oxidase LYSUF1 under different induction conditions; M: PageRuler TM Prestained protein molecular weight standard, 10 to 180 kDa; 1: pET28a empty vector; 2 - 3: supernatant and precipitate of the crude enzyme solution under the induction conditions of 0.2 mM IPTG, 16 °C, 10 h; 4 - 5: supernatant and precipitate of the crude enzyme solution under the induction conditions of 0.5 mM IPTG, 20 °C, 14 h; 6 - 7: supernatant and precipitate of the crude enzyme solution under the induction conditions of 0.4 mM IPTG, 25 °C, 10 h; 8 - 9: supernatant and precipitate of the crude enzyme solution under the induction conditions of 0.2 mM IPTG, 30 °C, 10 h;

[0082] Figure 4 Protein electrophoresis diagram of the separation and purification process of recombinant multi-copper oxidase LYSUF1; from left to right are lanes M, 1, 2, M: Unstained Protein Ladder; 1: supernatant of the crude enzyme solution before purification; 2: purified protein multi-copper oxidase KCYOBN (about 63.14 kDa). Detailed Embodiments

[0083] (1) Technical terms:

[0084] 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 sweetened powder, flavored milk powder, infant milk powder and other formula milk powders, condensed milk, milk fat, cheese, ice cream, casein, milk tablets, lactose, etc.

[0085] 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 salting, smoking, drying, freezing, canning, fermentation, etc.

[0086] The "meat products" involved in the present invention refer to products made mainly from animal muscle tissues or edible internal organs 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 meat products and Western meat products; the Chinese 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, Prepared meat products, Meat cake, Salted meat; the Western meat products include cooked and smoked ham, cooked and smoked sausage, sausage products, blood sausage, fermented sausage, bacon, ham or meat enema.

[0087] 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.

[0088] 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), and spermine (SPE).

[0089] (II) Reagents

[0090] The yellow rice wine, soy sauce, low-salt soy sauce, cooking wine, fruit wine, fish sauce, etc. involved in the following examples were purchased from a certain supermarket in Wuxi, Jiangsu Province.

[0091] (III) Culture Media

[0092] LB medium: Yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, adjust the pH to 7.0 with NaOH, autoclave at 121 °C for 20 min.

[0093] TB medium: Purchased from Qingdao Haibo Company. Autoclave at 121 °C for 20 min.

[0094] (IV) Detection Methods

[0095] The biogenic amine content was detected by high performance liquid chromatography (HPLC).

[0096] Example 1: PCR Amplification of the Multicopper Oxidase Gene LYSUF1

[0097] (1) Primers were designed according to the multicopper oxidase gene (QCX93764.1) in Bacillus cereus in the NCBI database, and the DNA of Bacillus cereus LH5 preserved in our laboratory was used as a template to amplify the multicopper oxidase gene LYSUF1. The primers required for amplification are as follows:

[0098] F: 5'-ATGAAGmGATTTGTATTArCvGyhrTTACrGTCTCyryAATATyTTTAATTGC-3';

[0099] R: 5'-TTAyTCTGGCwTATTAGGAATrTTwbGrTTTGGAACATA-3'.

[0100] The PCR reaction solution was prepared 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.

[0101] (2) The PCR products were verified for the amplification results by 1.2% agarose gel electrophoresis. As Figure 1 shown, the size of the amplified sequence was the same as that of the target gene sequence, approximately 1566 bp, indicating successful amplification. After purifying the PCR products, they were sent to the company for sequencing, and the sequencing results are shown in SEQ ID NO.2.

[0102] Example 2: Construction of a genetically engineered bacterium producing multi-copper oxidase gene LYSUF1

[0103] The multi-copper oxidase gene LYSUF1 shown in SEQ ID NO.2 amplified in Example 1 was ligated to a plasmid and transformed into microbial cells to construct a genetically engineered bacterium producing multi-copper oxidase gene LYSUF1.

[0104] 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.

[0105] Optionally, the host is a bacterial cell or a fungal cell, including but not limited to Escherichia coli, Bacillus or yeast.

[0106] Taking recombinant Escherichia coli as an example, the construction process of the genetically engineered bacterium pET28a-LYSUF1 is described as follows:

[0107] (1) Digestion and ligation.

[0108] The plasmid pET-28a(+) and the gene fragment shown in SEQ ID NO.2 obtained in Example 1 were respectively double-digested with restriction endonucleases. The digestion system is as follows: 50 μL of plasmid, 2.5 μL each of restriction endonucleases BamH I and HindIII, and 5 μL of Green Buffer. The components in the digestion system were thoroughly mixed and then reacted in a 37°C metal bath for 60 min. After the double-digested gene fragment and plasmid were recovered and purified, they were mixed at a molar ratio of 3-10:1, and an equal volume of Solution I ligase was added. After thorough mixing, they were incubated in a 16°C metal bath overnight to prepare the recombinant vector pET-28a(+)-LYSUF1.

[0109] (2) Transformation.

[0110] After placing the E. coli BL21(DE3) competent cells stored at -80°C on ice for 10 min of ice bath, use a pipette to aspirate 10 μL of the ligation product to be transformed obtained in step (1), add it to the competent cells, and gently pipette and mix well. After uniform mixing, perform an ice bath for 30 min. After the ice bath ends, perform a heat shock at 42°C for 45 s, and immediately take it out and place it in ice for 2 min after completion. Then add 700 μL of LB liquid medium, at 37°C, 200 r·min -1 Shake culture for 60 min. 8000 r·min -1 Centrifuge for 1 min, discard most of the supernatant, and leave about 200 μL of supernatant to resuspend the bacteria. Spread the bacterial solution evenly on an LB solid medium plate containing 50 mg·L -1 Kanamycin, and invert and culture it in an incubator at 37°C overnight. After culturing until single colonies grow, pick them and perform PCR verification to screen positive transformants.

[0111] (3) Enzyme digestion verification.

[0112] Extract the plasmid of the recombinant bacteria, perform double enzyme digestion with BamH I and HindIII, and obtain the pET-28a(+) fragment and the target fragment LYSUF1 respectively. Send the target fragment to the company for sequencing, and the sequencing result is consistent with the target gene sequence, verifying that the recombinant bacteria E. coli BL21 / pET-28a(+)-LYSUF1 is successfully constructed. Name the recombinant enzyme expressed by this strain LYSUF1. The construction process is as Figure 2 shown.

[0113] Example 3: Induced expression and purification of recombinant enzyme LYSUF1

[0114] The specific steps are as follows:

[0115] (1) Inoculate the recombinant bacteria E. coli BL21 / pET-28a(+)-LYSUF1 constructed in Example 2 into an LB medium containing 50 mg·L -1 Kanamycin, and culture it at 37°C, 150 r·min -1 for 14 h under the condition to prepare a seed solution.

[0116] (2) Transfer the obtained seed solution to a TB fermentation medium containing 50 mg·L -1 Kanamycin at an inoculation amount of 5% (v / v), and culture it at 37°C, 160 r·min -1 until the OD 600 reaches 0.4 - 0.6, and culture it under different induction conditions to obtain a bacterial solution.

[0117] (3) Centrifuge the bacterial solution at 4°C, 12000 r·min -1Under the condition, collect the lower-layer bacterial cells after centrifugation for 10 min, add sodium phosphate buffer solution (pH 7.4) at 0.1 mol·L -1 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, with an interval of 3 s, and the disruption time is 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 LYSUF1 enzyme protein.

[0118] (4) Optimal induction conditions for recombinant enzyme LYSUF1

[0119] 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.

[0120] Collect the fermented broth after cultivation, prepare the crude enzyme solution according to the method in Example 3, and measure the catalytic activity against different biogenic amines. The results show that after optimizing different induction conditions, it is found that the band of recombinant enzyme LYSUF1 is the most obvious under the conditions of IPTG concentration of 0.4 mM, induction temperature of 25°C, and time of 10 h ( Figure 3 ).

[0121] (5) Purification of recombinant enzyme LYSUF1

[0122] Perform affinity chromatography on the supernatant obtained in step (4) using a His Trap TM HP (GE Healthcare) to purify the protein, and use an AKTA avant 25 instrument to separate and purify the target protein.

[0123] Perform gel electrophoresis analysis on the supernatant of the crude enzyme solution before purification of LYSUF1 and the protein after purification of LYSUF1 respectively. The results are as Figure 4 shown.

[0124] The results show that the molecular weight of recombinant multi-copper oxidase LYSUF1 is about 63.14 kDa. The SDS-PAGE gel electrophoresis analysis of the crude enzyme solution confirms the successful expression of the recombinant engineering bacteria. Lane 2 proves that the target protein is successfully separated and purified by the affinity chromatography nickel column to obtain pure enzyme.

[0125] (6) Enzyme Activity Assay of Multicopper Oxidase LYSUF1

[0126] Referring to the laccase assay methods disclosed in Patent 201811383747.4 and 202111359832.9, the visible light absorption method was used to determine the activity of multicopper oxidase. That is, ABTS was used as the substrate, and the activity of multicopper oxidase was calculated by detecting the amount of ABTS oxidized by the enzyme. The reaction time was 2 min, and the reaction system was 100 μL of enzyme solution, 2.9 mL of sodium citrate buffer containing 0.5 mmol / L ABTS and 1 mmol / L CuCl2. The amount of enzyme required to oxidize 1 μmol of ABTS per minute was defined as one enzyme activity unit (U). The enzyme activity calculation formula is as follows:

[0127]

[0128] In the formula: ε is the molar extinction coefficient of ABTS at 420 nm, ε = 3.6×10 4 L / (mol·cm); Δt is the reaction time (min); ΔOD is the change in absorbance at 420 nm; V1 is the total reaction volume (mL); V2 is the enzyme amount (100 μL). C is the protein concentration of the enzyme (μg / mL). Therefore, the specific enzyme activity of multicopper oxidase LYSUF1 is 497.33 U / g.

[0129] (7) Effect of NaCl on the Enzyme Activity of Recombinant Enzyme LYSUF1

[0130] The purified multicopper oxidase was mixed with NaCl solutions at different final concentrations, incubated for 1 h, and the enzyme activity was measured. Taking the enzyme activity measured without adding NaCl as 100%, its relative enzyme activity was measured to determine the effect of NaCl on the enzyme activity.

[0131] Table 1 Effect of NaCl on the Enzyme Activity of Recombinant Enzyme LYSUF1

[0132]

[0133] The results showed that low concentrations of NaCl would activate the enzyme activity of multicopper oxidase LYSUF1. As the concentration of NaCl increased, the enzyme activity decreased. When the NaCl concentration reached 12%, the enzyme activity still exceeded 80%. When the NaCl concentration reached 16%, the enzyme activity still exceeded 72%; when the NaCl concentration reached 20%, the relative enzyme activity still remained at 53.22%, showing strong salt tolerance.

[0134] In the prior art, a multi-copper oxidase derived from Lactobacillus sakei was prepared with the application number 202111359832.9. In a 500 mM (about 2.92%) NaCl system, the remaining enzyme activity was only 1.11%, and its tolerance was inferior to that of the present invention. In the application number 201711444305.1, the remaining enzyme activity of the multi-copper oxidase of Bacillus amyloliquefaciens did not seem to exceed 10% when the NaCl concentration was 20%. It can be seen that the NaCl tolerance of the multi-copper oxidase LYSUF1 provided by the present invention is superior to that of similar enzymes reported in the prior art.

[0135] (8) Effect of ethanol on the enzyme activity of recombinant enzyme LYSUF1

[0136] The purified multi-copper oxidase was mixed with ethanol solutions of different concentrations, incubated for 1 h, and the enzyme activity was measured. Taking the enzyme activity measured without adding ethanol as 100%, its relative enzyme activity was measured to determine the effect of ethanol on the enzyme activity.

[0137] Table 2 Effect of ethanol on the enzyme activity of recombinant enzyme LYSUF1

[0138]

[0139] The results showed that with the increase of ethanol concentration, the enzyme activity decreased. When the ethanol concentration was about 5% vol, the relative enzyme activity remained 82.24%; when the ethanol concentration was about 15% vol, the relative enzyme activity of LYSUF1 remained above 60%; when the alcohol content reached 25% vol, the enzyme activity of LYSUF1 remained above 35%, indicating strong ethanol tolerance.

[0140] In the prior art, for the multi-copper oxidase from Bacillus amyloliquefaciens in Patent Application No. 201711444305.1, when the alcohol content is 5% vol, the remaining enzyme activity is approximately 30%. For the multi-copper oxidase prepared from Weissella cibaria in Patent Application No. 201811383747.4 in a 15% vol system, the remaining enzyme activity is less than 20%. For the novel multi-copper oxidase prepared from Lactobacillus fermentum in Patent Application No. 201811433209.1 in a 15% vol system, the remaining enzyme activity is less than 25%. For the multi-copper oxidase prepared from Lactobacillus sakei in Patent Application No. 202111359832.9 at an alcohol content of 20% vol, the remaining enzyme activity is less than 30%. For the multi-copper oxidase from Lactobacillus hilgardii capable of degrading biogenic amines prepared in Patent Application No. 202410100856.X, at 20% vol, except for tyramine and phenethylamine, there is almost no enzyme activity. It can be seen that the ethanol tolerance of the above-disclosed multi-copper oxidases is inferior to that of the multi-copper oxidase LYSUF1 of the present invention.

[0141] Example 4: Preparation of the multi-copper oxidase LYSUF1 enzyme preparation

[0142] The purified multi-copper oxidase LYSUF1 in Example 3 was mixed with an enzyme stabilizer to prepare an enzyme preparation containing multi-copper oxidase LYSUF1. 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.

[0143] Example 5: Application of the multi-copper oxidase LYSUF1 in commercially available soy sauce

[0144] Soy sauce is mainly brewed from soybeans or black beans, wheat or bran, and salt through processes such as oil production and fermentation. The composition of soy sauce is relatively complex, the total biogenic amine content of the sample ranges from 10 - 1898.17 mg / L, the salt content of high-salt soy sauce is approximately 18.5 - 20.5 g NaCl / 100 mL, and the pH is about 4.4 - 4.6. NaCl and acidity limit the progress of the enzymatic reaction.

[0145] The recombinant multi-copper oxidase LYSUF1 prepared in Example 3 was ultrafiltered and concentrated to adjust the protein concentration to 150 μg / ml, and then added to commercially available soy sauce to make the enzyme concentration in soy sauce 75 μg / mL. It was left standing at room temperature (25°C) for 7 days. The results showed that the multi-copper oxidase LYSUF1 could effectively reduce the biogenic amine content in soy sauce.

[0146] In soy sauce, this enzyme has significant degradation effects on phenethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), and tyramine (TYR). As shown in Table 3, after 7 days of treatment, the degradation rate of phenethylamine is 12.19%, the degradation rate of putrescine is 6.42%, cadaverine is completely degraded (degradation rate is 100%), the degradation rate of histamine is 38.75%, and the degradation rate of tyramine is 19.38%. The total degradation rate of this enzyme in soy sauce is approximately 26.37%.

[0147] Table 3 Biogenic amine content (mg / L) in soy sauce before and after adding LYSUF1 and the degradation rate (%) of corresponding biogenic amines

[0148]

[0149] Example 6: Application of multi-copper oxidase LYSUF1 in Chinese rice wine

[0150] Chinese rice wine is a fermented wine brewed mainly from rice, millet, corn, wheat, water, etc., with the addition of koji and / or some enzyme preparations, yeast and other saccharification and fermentation agents. The alcohol content is about 14% vol - 20% vol. Some organic acids are produced during the brewing process, and its pH value is about 3.5 - 4.5. The total biogenic amine content varies. The relatively high concentration of ethanol and high acidity in Chinese rice wine are very unfavorable for the catalytic reaction of enzymes. Therefore, controlling the biogenic amine content in Chinese rice wine by enzymatic method has more stringent conditions. The specific steps are as follows:

[0151] Adjust the protein concentration of the recombinant multi-copper oxidase LYSUF1 to 150 μg / ml and add it to Chinese rice wine at a final concentration of 75 μg / mL. React at room temperature of about 28°C for 7 days. The control group is a commercially available Chinese rice wine (18% vol) with the enzyme replaced by PBS buffer. The results are shown in Table 4.

[0152] In Chinese rice wine, this enzyme has a degradation effect on putrescine (PUT), cadaverine (CAD), histamine (HIS), and tyramine (TYR). After 7 days of treatment, the degradation rate of putrescine is 4.75%, the degradation rate of cadaverine is 15.35%, the degradation rate of histamine is 23.57%, and the degradation rate of tyramine is 3.64%. Although the degradation rates are different, overall this enzyme helps to reduce the biogenic amine content in Chinese rice wine. The total degradation rate of this enzyme in Chinese rice wine is approximately 7.21%. It is proved that the multi-copper oxidase LYSUF1 derived from Bacillus cereus has certain advantages in degrading histamine and cadaverine in Chinese rice wine containing ethanol. Application No. 202410100856.X prepared a multi-copper oxidase LHMCO derived from Lactobacillus hilgardii that can degrade biogenic amines 1614 The degradation rates of degrading histamine and cadaverine are 5.76% and 4.82% respectively, and the effect is inferior to that of the present invention.

[0153] Table 4 Biogenic amine content (mg / L) of yellow rice wine before and after adding LYSUF1 and the degradation rate (%) of the corresponding biogenic amines

[0154]

[0155] Example 7: Application of multi-copper oxidase LYSUF1 in cooking wine

[0156] "Cooking wine" is the name of the wine used for cooking, brewed by adding yellow rice wine, with a low alcohol concentration of less than 15%. Cooking wine contains both ethanol and NaCl, which is very unfavorable for the catalytic reaction of enzymes. The specific steps are as follows:

[0157] Adjust the protein concentration of the recombinant multi-copper oxidase LYSUF1 to 150 μg / ml and add it to cooking wine at a final concentration of 75 μg / mL, and react at room temperature of about 28°C for 7 days. The control group is a commercially available cooking wine with the enzyme replaced by PBS buffer. The results are shown in Table 5.

[0158] In cooking wine, this enzyme has obvious degradation effects on putrescine (PUT) and histamine (HIS), and the degradation rates are 17.27% and 24.66% respectively. The degradation rate of tyramine (TYR) is 7.63%. The total degradation rate of this enzyme in cooking wine is about 26.84%.

[0159] Table 5 Biogenic amine content (mg / L) of cooking wine before and after adding LYSUF1 and the degradation rate (%) of the corresponding biogenic amines

[0160]

[0161] Example 8: Application of multi-copper oxidase LYSUF1 in fish sauce

[0162] Fish sauce is a fish soy sauce product made from fish by-products or fish viscera as raw materials, adding 30% - 40% (mass fraction) of salt and fermenting in 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 this process also produces a large amount of biogenic amines, and the biogenic amine content of fish sauce products on the market varies. The specific steps are as follows:

[0163] Adjust the protein concentration of the recombinant multi-copper oxidase LYSUF1 to 150 μg / ml and add it to fish sauce at a final concentration of 75 μg / mL, and react at room temperature of about 28°C for 7 days. The control group is a commercially available fish sauce with the enzyme replaced by PBS buffer. The results are shown in Table 6.

[0164] In fish sauce, the enzyme has significant degradation effects on phenethylamine (PHE), histamine (HIS), and tyramine (TYR). After 7 days of treatment, the degradation rate of phenethylamine is 65.9%, histamine is completely degraded (degradation rate is 100%), and the degradation rate of tyramine is 26.97%. The total degradation rate of the enzyme in fish sauce is approximately 27.93%.

[0165] Table 6 Biogenic amine contents (mg / L) in fish sauce before and after adding LYSUF1 and the degradation rates (%) of corresponding biogenic amines

[0166]

[0167] Example 9: Application of multi-copper oxidase LYSUF1 in red wine

[0168] The volume fraction of ethanol in wine is generally above 13%, which not only increases the difficulty of BA degradation in wine but also significantly enhances the toxicity of BA. The specific steps are as follows: Adjust the protein concentration of the recombinant multi-copper oxidase LYSUF1 to 150 μg / ml, add it to red wine at a ratio of 1:1, and react at room temperature of about 28°C for 7 days. The control group is red wine with the enzyme replaced by PBS buffer. The results are shown in Table 7.

[0169] In red wine, the enzyme has significant degradation effects on tryptamine (TRY), phenethylamine (PHE), histamine (HIS), and tyramine (TYR). After 7 days of treatment, tryptamine is completely degraded (degradation rate is 100%), the degradation rate of phenethylamine is 4.73%, the degradation rate of histamine is 19.8%, and the degradation rate of tyramine is 32.82%. The total degradation rate of the enzyme in red wine is approximately 29.11%. It shows the advantage of LYSUF1 derived from Bacillus cereus in degrading biogenic amines in fermented alcoholic beverages.

[0170] Table 7 Biogenic amine contents (mg / L) in red wine before and after adding LYSUF1 and the degradation rates (%) of corresponding biogenic amines

[0171]

[0172] Example 10: Application of multi-copper oxidase LYSUF1 in commercially available soy sauce 2

[0173] Soy sauce is mainly brewed from soybeans or black beans, wheat or bran, and salt through processes such as oil production and fermentation. The composition of soy sauce is relatively complex, and the range of the total biogenic amine content in the sample is 10 - 1898.17 mg / L. The salt content of low-salt soy sauce is approximately 8 g NaCl / 100 mL. The pH is about 4.4 - 4.6. NaCl and acidity limit the progress of the enzymatic catalytic reaction.

[0174] The recombinant multicopper oxidase LYSUF1 prepared in Example 3 was ultrafiltered and concentrated to adjust the protein concentration to 150 μg / ml, and then added to commercially available low-salt soy sauce so that 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 the multicopper oxidase LYSUF1 could effectively reduce the biogenic amine content in low-salt soy sauce.

[0175] As shown in Table 8, in low-salt soy sauce, this enzyme had significant degradation effects on phenethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), tyramine (TYR) and spermidine (SPD). After 7 days of treatment, the degradation rate of phenethylamine was 56.27%, the degradation rate of putrescine was 29.99%, the degradation rate of cadaverine was 14.86%, the degradation rate of histamine was 26.71%, the degradation rate of tyramine was 39.65%, and the degradation rate of spermidine was 5.35%. The total degradation rate of this enzyme in low-salt soy sauce was approximately 29.18%.

[0176] Table 8 Biogenic amine content (mg / L) in low-salt soy sauce before and after adding LYSUF1 and the degradation rate (%) of corresponding biogenic amines

[0177]

[0178]

[0179] Example 11: Application of multicopper oxidase LYSUF1 in fermented sausage

[0180] By mass, take 65 - 80% lean meat and 20 - 35% fat. Wash and remove bones, tendons, muscle membranes, lymph, blood vessels, diseased and damaged parts. Separate the fat and lean and cut them into 4 - 5 cm meat pieces. Put the lean meat and about 5 - 8% ice chips into a chopper and chop for 1 - 3 min. Based on the mass of pork, add 0.01 - 0.15% sodium nitrite, 2 - 3% salt, 0.2 - 0.3% compound phosphate, and 0.05 - 0.06% sodium ascorbate. Spices, pepper, garlic, chili, and nutmeg are 0.2% - 0.3% of the raw meat. The recombinant multicopper oxidase LYSUF1 prepared in Example 3 was ultrafiltered and concentrated to adjust the protein concentration to 150 μg / ml, added to the sausage raw materials, chopped for 1 - 2 min, then add the fat and about 5 - 8% ice chips, and chop for 4 - 6 min. After marinating, stuff it into sausage casings. After the sausage stuffed into the casings was marinated at 4 °C for 12 h, raise the temperature to 30 °C and ferment until the pH decreased to about 5.1, and mature at 14 - 16 °C for 1 - 10 days. Compared with the fermented sausage without adding multicopper oxidase LYSUF1, the results showed that the multicopper oxidase LYSUF1 could effectively reduce the biogenic amine content in the fermented sausage.

[0181] At present, the research on the degradation of biogenic amines in fermented food systems by multi-copper oxidases is almost blank. This application lays the technical foundation for the catalytic degradation of biogenic amines by this multi-copper 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 conditions of high ethanol content.

[0182] Comparative Example 1: Multi-copper oxidase LHMCO 1614 Degradation of biogenic amines in high-salt soy sauce

[0183] The specific implementation method refers to Example 5, with the difference that the multi-copper oxidase LYSUF1 of the present invention is replaced by the multi-copper oxidase LHMCO derived from Lactobacillus hilgardii in Patent CN202410100856.X 1614 , and the degradation rate of the enzyme on high-salt soy sauce was investigated. The results showed that it was the multi-copper oxidase LHMCO 1614 , and the degradation rate of biogenic amines in high-salt soy sauce was 19.70% (Table 9). Therefore, LYSUF1 derived from Bacillus has better application effects in high-salt systems such as soy sauce.

[0184] Table 9 Content (mg / L) of soy sauce before and after adding LHMCO 1614 and the degradation rate (%) of the corresponding biogenic amines

[0185]

[0186] 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. A multi-copper oxidase LYSUF1 capable of 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 multi-copper oxidase activity.

2. A gene encoding the multi-copper oxidase according to claim 1.

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

4. A recombinant microbial cell expressing the multi-copper oxidase according to claim 1.

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

2.

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

8. A method for preparing the multi-copper oxidase LYSUF1 according to claim 1, characterized in that, Cultivate the recombinant microbial cell according to claim 4 or the genetically engineered bacterium according to claim 5 or 6 in a medium for a period of time, and collect the multi-copper oxidase LYSUF1.

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

10. Application of the multi-copper oxidase LYSUF1 according to claim 1 in reducing biogenic amines.

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

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

13. An enzyme preparation containing the multi-copper oxidase LYSUF1 according to claim 1.

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

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