Application of laccase AmLac in biogenic amine degradation

Abstract

The invention belongs to the technical field of biology, and particularly discloses application of laccase AmLac in degradation of biogenic amines. The amino acid sequence of the laccase is shown as SEQ ID NO.1. The laccase has different degrees of degradation effects on the biogenic amines such as histamine, spermidine, tyramine, phenylethylamine and putrescine, and the degradation rates of the histamine and the putrescine both reach 100%. The enzyme is stable in enzymatic property, has alcohol tolerance and acid tolerance, has certain adaptive capacity to food matrixes, and can effectively degrade biogenic amines in wine and reduce potential hazards of the biogenic amines to human bodies, so that the safety and stability of products are improved. Therefore, the AmLac has a wide application prospect in the food industry, and especially when the AmLac is used for treating food containing high biogenic amine, the safety quality of the food can be remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological proteases and specifically discloses a laccase Am Application of Lac in degradation of biogenic amines. Background Art

[0002] Biogenic Amines (BAs) are a class of nitrogen-containing small molecule organic substances widely found in fermented foods, the most common of which are tyramine, putrescine, cadaverine, and histamine. Biogenic amines play an important role in the human body. They can regulate gene expression by changing the structure of DNA and adjusting signal transduction pathways, thereby maintaining normal physiological functions. However, once the human body ingests more than 100 mg of biogenic amines, it will cause severe poisoning and strong side effects, such as migraine, diarrhea, and vomiting, which are extremely harmful to the human body.

[0003] Biogenic Amines (BAs) are one of the main adverse metabolites in wine. Currently, more than 20 BAs have been detected in wine, including putrescine, histamine, tyramine, cadaverine, tryptamine, β-phenylethylamine, spermine, spermidine, methylamine, ethylamine, isopentylamine, etc. Among them, histamine, tyramine and putrescine have the highest content and the most extensive research. Histamine is recognized as the most toxic BA in wine. Many countries have clearly defined the content of histamine in wine, among which Germany and the Netherlands are the most stringent, with a limit of 2 mg / L. In wine, tyramine can promote the release of norepinephrine in the body, and its toxicity is second only to histamine. A moderate amount of tyramine is absorbed by the intestine and oxidized and decomposed by monoamine oxidase. Excessive intake will cause accumulation, thereby causing adverse reactions such as migraine. Putrescine is the most common BA in wine and can be detected in almost all wines. Although its toxicity is lower than that of histamine, it can inhibit the activity of related amine oxidases (AOs), reduce the decomposition of histamine and tyramine by AOs, and lead to the accumulation of histamine and tyramine content and enhanced toxicity. Therefore, the large accumulation of putrescine can also cause indirect damage to the body and cause discomfort. Like putrescine, BAs such as cadaverine, spermine, and spermidine have no direct toxic effects, but under certain conditions, their presence can also react with nitrite to accumulate carcinogenic nitroso. Therefore, strict control of the types and contents of BAs in wine has a positive effect on brewing healthy and high-quality wines.

[0004] At present, enzymatic degradation of biogenic amines in food is the most feasible, which not only does not damage food nutrition, but also does not produce new toxic substances. The existing biogenic amine degradation enzymes are mainly divided into three categories: amine oxidase, amine dehydrogenase and multi-copper oxidase. Among them, amine oxidase and amine dehydrogenase do not have high requirements for substrate specificity, and the types of degraded amines are single and inefficient; amine oxidases include primary amine oxidase, diamine oxidase, and monoamine oxidase. Among them, primary amine oxidase can only degrade phenylethylamine, with an optimal pH of 7.0, diamine oxidase only works on diamines, especially histamine, with an optimal pH of 8.5, and monoamine oxidase can degrade tyramine and tryptamine, with an optimal pH of 6.0-7.0. Therefore, the common problems of enzymes that currently degrade fermented foods are: 1) Few types of biogenic amines are degraded; 2) The efficiency of degrading biogenic amines is low; 3) The optimal pH is neutral, which is not suitable for use in acidic fermented foods, and the enzymes used in wine also need to be resistant to alcohol. Therefore, it is necessary to find an enzyme that can efficiently degrade multiple biogenic amines at one time and is not easily affected by the food matrix for use in fermented foods. Summary of the invention

[0005] In view of these defects, the present invention provides a laccase Am Application of Lac in the degradation of biogenic amines Am Lac can degrade histamine, putrescine, spermidine, tyramine and phenylethylamine at the same time, and the degradation rate of histamine and putrescine reached 100%. The enzyme has stable enzymatic properties, alcohol tolerance and acid tolerance, has a certain adaptability to food matrices, and can play a role in the degradation of biogenic amines in fermented foods.

[0006] To achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a laccase Am Application of laccase in the degradation of biogenic amines, the amino acid sequence of the laccase is shown in SEQ ID NO.1, and the biogenic amines include one or more combinations of histamine, putrescine, spermidine, tyramine and phenylethylamine.

[0007] Amino acid sequence SEQ ID NO.1: MAEDKNTINDYFFYEPAFNTHDGGYVPLEEPNIPEQKLKIPEALKPDKETDTDMYYTVIAEAGESQIMPGQKTKTWGYNQPLLGKTIIFKRGKNIHVTVKNELSELTTYHWHGLNVPGDIDGGCHTPV YPGESKVLNFKCDQPAATLWLHAHPCPSTAEQVWHGLATAVVVKDEHESSLPLPRNYGVDDIPLILQDRKFHEDNQWDYHADYDPDGVQGPTAMINGTVNPYFDVTTQKVRFRILDGANRREWRLHFS DNLSFTQVAGDGSLLEHPVKFTHLMLTCAERAEIVVDFSDYKPGDEVTLYSDDVPIVHFRIHEFAKDDSTLPDTLFKLNAPEVTPDTPVHKIVMSGMDEEVAINDKKFNMQRIDSKQTLGSCEIWEVT NSNDCDGGMIHPYHMHGCQFQVLSRNGKAPYPNETGLKDTIAVDPNETVKIKVWFDHTGVFMYHCHIIEHEDGGMMAQIEVQDPKDPQKFNLMDMDTLMNAFAKERGVKPSELNLGGMECYDKMNMKM.

[0008] Laccase Am Lac has a high efficiency in degrading five biogenic amines (histamine, putrescine, spermidine, tyramine and phenylethylamine). The degradation rates of histamine and putrescine are both 100%, and the degradation rates of spermidine, tyramine and phenylethylamine are 95.09%, 84.95% and 78% respectively. It can be used for the removal of biogenic amines.

[0009] The laccase Am Lac is suitable for acidic conditions, with the optimum enzyme activity at pH 4.5, and can maintain more than 80% enzyme activity in the pH range of 4.5-5.0.

[0010] Preferably, the laccase Am Application of Lac in degradation of biogenic amines in fermented foods or low-alcohol foods.

[0011] Low alcohol content refers to an alcohol content of less than 20% v / v which is generally recognized in the art. Am Lac is suitable for low alcohol conditions and has good enzyme activity in the alcohol content range of 0-20% (v / v).

[0012] More preferably, the laccase Am Application of Lac in degradation of biogenic amines in wine.

[0013] Laccase provided by the present invention Am Lac can adapt to the matrix of wine and can play a role in degrading biogenic amines in the finished wine or in the production process. It has a high efficiency in degrading histamine, putrescine and tyramine, which are the most abundant biogenic amines in wine.

[0014] In a second aspect, the present invention also provides a method for reducing the content of biogenic amines in wine, characterized in that the method is to add a laccase with an amino acid sequence as shown in SEQ ID NO.1 to the wine or during the wine production process. Am Lac.

[0015] Preferably, the laccase Am Lac is available in the form of enzyme liquid or enzyme powder.

[0016] More preferably, the laccase is added to the wine in an amount of 2000 U / mL.

[0017] In a third aspect, the present invention provides a genetically engineered bacterium, wherein the genetically engineered bacterium expresses a laccase having an amino acid sequence as shown in SEQ ID NO.1. Am Lac.

[0018] Preferably, the genetically engineered bacteria contains a gene with a nucleotide sequence as shown in SEQ ID NO.2; the method for constructing the genetically engineered bacteria is: connecting the nucleotide sequence as shown in SEQ ID NO.2 with an expression vector to construct a recombinant expression vector, and transforming the recombinant expression vector into a host cell to construct the genetically engineered bacteria.

[0019] Nucleotide sequence SEQ ID NO.2:

[0020] More preferably, the host is Escherichia coli.

[0021] More preferably, the expression vector is a pET series plasmid.

[0022] More preferably, the expression vector is pET-28a.

[0023] In a fourth aspect, the present invention provides the use of the above-mentioned genetically engineered bacteria in the degradation of biogenic amines.

[0024] In a fifth aspect, the present invention provides a method for producing the above-mentioned laccase Am Lac method, the above genetically engineered bacteria are fermented and cultured and then the laccase in the fermentation liquid or the bacterial cell is extracted. Am Lac. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 This is a PCR verification diagram of the target band in the Rosetta-pET-28a(+)-MCO engineered probiotic constructed in Example 1; wherein lane M is a marker; lanes 1 and 2 are target bands; Figure 2 The figure is the SDS-PAGE electrophoresis diagram of the purified expression product of the recombinant strain Rosetta-pET-28a(+)-MCO; wherein lane M is a marker; lane 1 is a crude enzyme solution; lane 2 is a 20 mmol / L eluent; lane 3 is a 50 mmol / L eluent; and lane 4 is a purified sample; Figure 3 Laccase Am Relative enzyme activity of Lac under different pH conditions; Figure 4 The effect of ethanol concentration on laccase Am Effect of Lac enzyme activity. DETAILED DESCRIPTION

[0027] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] The term "transformation" refers to the process by which a host cell acquires new genetic markers as a result of the incorporation of foreign DNA.

[0029] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0030] The E. coli expression vector pET-28a(+) and strain Rosetta (DE3) are commercially available.

[0031] The molecular biology experimental methods not specifically described below are all carried out in accordance with the specific methods listed in the book "Molecular Cloning Experiment Guide" (3rd edition) by J. Sambrook, or in accordance with the kits and product instructions.

[0032] Example 1 Construction of the recombinant strain Rosetta-pET-28a(+)-MCO This embodiment provides a method for expressing laccase Am A recombinant strain of Lac and a preparation method thereof.

[0033] Lactobacillus mikimotoi ( Apilactobacillus micheneri ) genome as a template to obtain the target gene fragment by PCR.

[0034] Primers: Upstream: ATGGCACTTGAAAAATTTGC; Downstream: TTACTGCTTTTCTGTGACGTC.

[0035] PCR reaction program: pre-denaturation at 95°C for 3 min, then the following cycles: denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min30 s; after 34 cycles, extension at 72°C for 10 min and insulation at 4°C.

[0036] Obtaining laccase Am The Lac gene has an open reading frame sequence (ORF) of 1536 bp in length, encoding 512 amino acids, and its nucleotide sequence is shown in SEQ ID NO.2.

[0037] Laccase AmThe Lac gene was digested and connected to the pET-28a(+) plasmid to obtain the recombinant vector pET-28a(+)-MCO, which was then transformed into Escherichia coli Rosseta(DE3) to construct the recombinant strain Rosetta-pET-28a(+)-MCO. The recombinant strain was screened on an ampicillin plate and identified by PCR sequencing to obtain positive bacteria transformed with the pET-28a(+)-MCO plasmid. The verification results are shown in Figure 1 .

[0038] Example 2 This embodiment provides a laccase Am Lac method.

[0039] The recombinant strain Rosetta-pET-28a(+)-MCO provided in Example 1 was activated overnight and inoculated into 100 mL LB culture medium. After shaking culture at 37°C and 220 rpm for about 2.5 h, 0.1 mM IPTG and CuCl2 solution were added and the culture was induced in a shaker at 16°C and 220 rpm for 21 h.

[0040] After induction, centrifuge at 4°C, 6500 rpm for 30 min, collect the cells, resuspend the cells in PBS buffer with pH 7.0, centrifuge at 4°C, 6500 rpm for 30 min, discard the supernatant, repeat washing the cells three times, and finally resuspend the cells in PBS buffer, ultrasonically disrupt, centrifuge at 4°C, 6500 rpm for 10 min, collect the supernatant after the cells are disrupted, and obtain the crude enzyme solution; the crude enzyme solution uses a nickel ion affinity chromatography column (Ni2+-NTA) to purify the recombinant protein through the NGC protein purification system, and collect 20 mM, 50 mM and 200 mM imidazole solutions respectively; the 200 mM imidazole solution is ultrafiltered and desalted with a cutoff tube (10 kDa) to obtain the purified sample. The purification results of the target protein were detected by SDS-PAGE electrophoresis, as shown in Figure 2 Lane 4 is the recombinantly expressed laccase Am The target band of Lac has a molecular weight of approximately 58.1 kDa.

[0041] Example 3 This example provides laccase Am Determination of the ability of Lacase to degrade biogenic amines. The ability of laccase to degrade biogenic amines was tested using high performance liquid chromatography. The specific method is as follows: (1) Biogenic amine standard stock solutions: Prepare the five biogenic amines (histamine, putrescine, tyramine, spermidine and phenylethylamine) into standard solutions with a concentration of 1 mg / mL using deionized water and store at -20°C.

[0042] (2) Sample preparation: Purified laccase AmLac was added to 1 mL of a mixed amine solution containing five biogenic amines to be degraded at a final enzyme activity of 2000 U / mL. The concentration of the five biogenic amines was 40 mg / L. The mixture was shaken at 28°C and 150 rpm for 48 h, then boiled for 10 min, cooled and derivatized. The content of biogenic amines was determined by high performance liquid chromatography (HPLC). A biogenic amine solution containing the same mass of high temperature inactivated enzyme was used as the control group. The degradation rate of laccase on biogenic amines was determined by comparing the peak graph of the standard product of the biogenic amine solution with the inactivated enzyme. The results are shown in Table 1.

[0043] Derivatization treatment method: 1 mL of the sample to be tested is sequentially added with 2 mol / L NaOH solution, saturated NaHCO3 buffer and 10 mg / mL dansyl chloride derivatization solution (dissolved in acetone, prepared and used immediately), with volumes of 200 μL, 300 μL and 2 mL respectively. After mixing the added reagents, place them in a 60°C water bath, protect from light and react for 30 min. After heating, add 100 μL of ammonia water and dilute to 5 mL with acetonitrile. Take the supernatant and filter it through a 0.22 μm sterile filter membrane, and place it in a brown vial for testing.

[0044] Table 1 Laccase Am Degradation rate of Lac on biogenic amines

[0045] “ND” stands for no degradation of biogenic amines.

[0046] As shown in Table 1, laccase Am Lac has excellent degradation effects on five biogenic amines (histamine, putrescine, spermidine, tyramine and phenylethylamine), showing a broad-spectrum biogenic amine degradation activity. The degradation rates of histamine and putrescine by the enzyme were both 100%, indicating that it has a complete degradation effect on these two common biogenic amines; the degradation rates of spermidine, tyramine and phenylethylamine were 95.09%, 84.95% and 78% respectively, also showing good degradation ability. Laccase Am The diverse degradation properties of Lac give it a wide range of application potential in the removal of biogenic amines.

[0047] Example 4 Enzymatic properties of laccase (1) Effect of pH on laccase Am Effect of Lac enzyme activity Mixed amine solutions with pH values ​​of 3.0, 3.5, 40, 4.5, 5.0, 5.5 and 6.0 were prepared using citrate buffers at different pH values ​​to determine the activity of laccase under different pH conditions. AmThe enzyme activity of Lac in degrading biogenic amines was measured after derivatization at 28°C, 150 rpm for 48 h and then boiling for 10 min. The enzyme activity under different pH conditions was measured, and the highest enzyme activity was taken as 100%. Figure 3 As shown, the enzyme activity is highest at pH 4.5, and when the pH is 4.0-5.5, the enzyme activity can still be maintained at more than 50% of the enzyme activity at the optimal pH.

[0048] (2) Effect of ethanol on laccase Am Effect of Lac enzyme activity Preparation of ethanol buffer with different concentrations: Use deionized water to prepare ethanol with volume fractions of 2.5%, 5%, 10%, 15% and 20%, respectively, and adjust the above solutions to the required pH value of 4.5 with 0.1 mol / L acetic acid and sodium acetate.

[0049] Laccase Am The enzyme activity of Lac was measured after being incubated at 4°C for 2 hours in the above-mentioned ethanol buffer with different volume fractions (ethanol was not added to the control group), and the enzyme activity measured without adding ethanol was 100%. Laccase enzyme activity determination method: ABTS was used as the substrate, and the enzyme activity of the enzyme was calculated according to the degree of oxidation of ABTS.

[0050] The 500 μL reaction system includes 480 μL sodium dihydrogen phosphate-citrate buffer (containing 1 mmol / L ABTS, 1mmol / L CuCl2, pH 4.5) and 20 μL enzyme solution. After reacting in a 40°C water bath for 10 min, 500 μL of anhydrous ethanol is added to terminate the reaction and the absorbance value at a wavelength of 420 nm is measured. Activity is defined as the amount of enzyme that oxidizes 1 μmol of substrate per minute. Enzyme activity is calculated as follows: Enzyme activity (U / L) = (A×V) / (ε×t×V0); Wherein: A is the absorbance at 420 nm wavelength; V is the total volume of the solution / μL; ε is the molar absorption coefficient; t is the reaction time / min; V0 is the volume of laccase added / μL.

[0051] The results are as follows Figure 4 As shown, laccase Am Lac has good tolerance to ethanol. When the ethanol concentration is less than 10%, the relative enzyme activity reaches 80%; when the ethanol concentration is 20%, the relative enzyme activity is 75%.

[0052] Example 5 Laccase Am Degradation of biogenic amines in wine by Lac The purified laccase AmLac (provided in Example 2) was added to 1 mL of a certain brand of commercially available wine at a final enzyme activity of 2000 U / mL and mixed. After being evenly mixed, it was placed in a shaker at 28°C, shaken at 150 rpm for 48 hours, boiled for 10 minutes, cooled and derivatized, and the content of biogenic amines was determined by high performance liquid chromatography (HPLC). A biogenic amine solution containing the same mass of high temperature inactivated enzyme was used as a blank control group. The results are shown in Table 2.

[0053] Table 2 Content of biogenic amines in wine

[0054] “--*” means that spermidine was not detected in the commercially available wine.

[0055] The results show that the laccase provided by the present invention Am Lac can effectively degrade biogenic amines in wine, significantly reducing the content of major biogenic amines (histamine, putrescine, tyramine and phenylethylamine) in wine, especially histamine, putrescine and tyramine, which are almost completely degraded.

[0056] It can be seen that the laccase involved in the present invention Am Lac has good adaptability to wine matrix and can efficiently degrade biogenic amines during the wine production process or in the finished product, controlling the content of potentially harmful biogenic amines in wine, and effectively reducing its potential risks to human health, making it an ideal choice for optimizing the quality of alcoholic beverages, especially wine.

[0057] In summary, the laccase involved in the present invention Am The diverse degradation characteristics and enzymatic properties of Lac make it have a wide range of application potential in the removal of biogenic amines, especially for the quality control of food and fermented products. Am Lac can effectively reduce the content of biogenic amines and reduce their potential harm to the human body, thereby improving the safety and stability of the product. Am Lac has broad application prospects in the food industry, especially when processing foods containing high levels of biogenic amines, it can significantly improve the safety quality of food.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A laccase Am The application of Lac in the degradation of biogenic amines is characterized by: The laccase Am The amino acid sequence of Lac is shown in SEQ ID NO.1, and the biogenic amines include one or more combinations of histamine, putrescine, spermidine, tyramine and phenylethylamine.

2. The use according to claim 1, characterized in that: The laccase Am Application of Lac in degradation of biogenic amines in fermented foods or low-alcohol foods.

3. The use according to claim 2, characterized in that: The laccase Am Application of Lac in degradation of biogenic amines in wine.

4. A method for reducing the content of biogenic amines in wine, characterized in that: Adding the laccase with the amino acid sequence shown in SEQ ID NO.1 to the wine or during the wine production process Am Lac.

5. The method according to claim 4, characterized in that The laccase Am Lac is available in the form of enzyme liquid or enzyme powder.

6. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria express a laccase whose amino acid sequence is shown in SEQ ID NO.1 Am Lac.

7. The genetically engineered bacterium according to claim, characterized in that: The genetically engineered bacteria contains a gene with a nucleotide sequence as shown in SEQ ID NO.2; the method for constructing the genetically engineered bacteria is: connecting the nucleotide sequence as shown in SEQ ID NO.2 with an expression vector to construct a recombinant expression vector, and transforming the recombinant expression vector into a host cell to construct the genetically engineered bacteria.

8. The genetically engineered bacterium according to claim 7, characterized in that: The host is Escherichia coli, and the expression vector is a pET series plasmid; preferably, the expression vector is pET-28a.

9. Use of the genetically engineered bacteria according to any one of claims 6 to 8 in the degradation of biogenic amines.

10. A laccase producing an amino acid sequence as shown in SEQ ID NO.1 Am Lac method, characterized in that Extracting laccase from the fermentation liquid or the bacterial cell after fermentation culture of the genetically engineered bacteria according to any one of claims 6 to 8 Am Lac.

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