CN113667654B

CN113667654B - Preparation method and application of recombinant halophilic archaea histamine oxidase

Info

Publication number: CN113667654B
Application number: CN202110593340.XA
Authority: CN
Inventors: 侯靖; 杨晓妍; 崔恒林
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2021-05-28
Filing date: 2021-05-28
Publication date: 2022-07-22
Anticipated expiration: 2041-05-28
Also published as: CN113667654A

Abstract

The invention belongs to the field of genetic engineering, and relates to a preparation method and application of recombinant halophilic archaea histamine oxidase; the method comprises the following steps: firstly, a coding gene hod is obtained based on the genome of halobios (Halovivax sp.) WLHSJ27-1_Hvv(ii) a Hod is prepared by molecular cloning_HvvConnecting to a vector to construct a recombinant plasmid; transforming the recombinant plasmid into an archaeohalophilic bacteria host to obtain a transformed recombinant host cell, culturing the transformed recombinant host cell in an Hv-YPC liquid culture medium to a stable period, centrifugally collecting the thallus at a low temperature, carrying out heavy suspension in a cell lysate, and collecting a supernatant for recombinant protein purification through ultrasonic crushing and centrifugation; purifying by nickel affinity chromatography and gel filtration chromatography to obtain high purity histamine oxidase (Hod)_Hvv；Hod_HvvCan catalyze various amines, is high-temperature resistant and resistant to various metal ions, and has excellent enzymatic properties.

Description

Preparation method and application of recombinant halophilic archaea histamine oxidase

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a preparation method and application of recombinant halophilic archaea histamine oxidase.

Background

Histamine is a low molecular nitrogenous organic base formed by decarboxylation of histidine by histamine decarboxylase, and a small amount of histamine regulates various physiological functions in the human body. Ingestion of excessive amounts of histamine (8-40 mg) results in acute poisoning, with typical symptoms including rash, erythema, sweating, nausea, vomiting, diarrhea, mouth burning, swelling of the tongue and face, headache, respiratory distress, palpitations, and hypotension; symptoms of histamine intoxication may last for several hours or a day, and in rare cases, for several days. The problem of excessive histamine is widely existed in high-salt foods such as fish sauce, shrimp paste, soy sauce and the like.

There are three main methods for the control of histamine in food products: physical, chemical and biological methods. The physical method mainly controls the histamine-producing microorganisms in the food through low-temperature storage, ultrahigh-pressure treatment and irradiation treatment of the food, and the method is simple to operate and expensive. The chemical method comprises adding chemical additives or spicery into food to control amine-producing microorganism in food, thereby achieving histamine control effect. Such methods have the disadvantage of having a large impact on the flavour and nutrition of the food product. The biological method is carried out by adding microorganism or enzyme capable of degrading histamine into food. At present, the types of microorganisms capable of degrading histamine are more, but the safety cannot be controlled. The biological enzyme method has good degradation effect on histamine and no toxicity on products, but the currently reported histamine degrading enzymes have few types and poor tolerance on salt.

Histamine-degrading enzymes are mainly of two classes, histamine oxidase and histamine dehydrogenase. The histamine oxidase related by the invention is derived from halophilic archaea, and pure enzyme is obtained by using methods such as heterologous expression and affinity chromatography, so that the expression amount is large, and the subsequent purification method is simple. The halophilic archaea histamine oxidase has wide adaptability to NaCl and temperature, and can be used for degrading histamine in high-salt foods such as fish sauce and shrimp paste.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a preparation method of recombinant halophilic archaea histamine oxidase.

The invention obtains a novel histamine oxidase gene hod based on the genome of halophilic archaea (Halovivax sp.) WLHSJ27-1_HvvThe nucleotide sequence is shown as SEQ ID No.1, and the gene size is 2037 bp; TatP is used for predicting and finding that the enzyme has no Tat signal peptide, is an intracellular enzyme and consists of 678 amino acid residues, and the amino acid sequence of the enzyme is shown as SEQ ID No. 2.

In order to achieve the above object, the present invention comprises the steps of:

the invention relates to a preparation method of novel halophilic archaea histamine oxidase, which specifically comprises the following steps:

(1) construction of pTA04-hod_HvvRecombinant expression plasmid:

firstly, a novel histamine oxidase gene hod is obtained based on the genome of halophilic archaea (Halovivax sp.) WLHSJ27-1_Hvv(ii) a Designing a primer containing an enzyme cutting site according to a target gene, and amplifying a target fragment hod by using a conventional PCR (polymerase chain reaction) technology_Hvv(ii) a And hod is obtained by molecular cloning_HvvConnecting to pTA04 carrier to construct recombinant plasmid;

(2) transformation of the recombinant plasmid Halofax volcanii:

transforming the recombinant plasmid obtained in the step (1) into a halophilic archaea host to obtain a transformed recombinant host cell; the halophilic archaea host is Halofarax volcanii; carrying out shake-flask culture on the transformed recombinant host cell in an Hv-YPC liquid culture medium, carrying out low-temperature centrifugation to collect thalli for later use after the cell culture reaches a stable period;

(3)Hod_Hvvand (3) purification:

collecting the thalli in the step (2) in cell lysate for resuspension, carrying out ultrasonic cell disruption after resuspension, and centrifugally collecting supernatant for recombinant protein purification; firstly, purifying the supernatant by adopting nickel column affinity chromatography to obtain an enzyme solution containing imidazole, and then removing the imidazole in the enzyme solution by utilizing gel filtration to obtain high-purity histamine oxidase recorded as Hod_Hvv。

Preferably, the halophilic archaea (Halovivax sp.) WLHSJ27-1 in the step (1) is preserved in China general microbiological culture Collection center with the strain preservation number of CGMCC 22177, and the preservation date is 2021, 4 months and 12 days.

Preferably, hod described in step (1)_HvvThe nucleotide sequence is shown in SEQ ID No.1, and the gene size is 2037 bp.

Preferably, the restriction sites in step (1) are selected from restriction sites of restriction enzymes EcoR I and BamHI.

Preferably, the Halofax volcanii in step (2) is obtained from the Japanese Collection of Microorganisms (JCM) under strain number JCM 8879.

Preferably, the composition of the Hv-YPC medium in the step (2) is (per liter): 5.0g yeast extract, 1.0g soybean peptone, 1.0g acid hydrolyzed casein, 4.2g KCl, MgSO₄·7H₂O 33.0g，MgCl₂·6H₂O 30.0g，NaCl 144.0g，1M Tris-HCl(pH 8.0)12.0mL，CaCl₂0.33g (pre-dissolved in water first, added after dissolution), the above components were dissolved in 1L distilled water, pH was adjusted to 7.5, and sterilization was performed at 115 ℃ for 30 min.

Preferably, the temperature of the shake flask culture in the step (2) is 37 ℃ and the rotation speed is 180 rpm.

Preferably, the low temperature in the step (2) is 0-4 ℃.

Preferably, the specific operation of obtaining the mature enzyme in the step (3) is as follows: equilibrating the nickel column with 10 bed volumes of cell lysate, and then binding the supernatant to the nickel column 3 times; and eluting the hybrid protein by using a buffer solution I with the volume 15 times of that of the column bed, and eluting the target protein by using a buffer solution II with the volume 5 times of that of the column bed to obtain the pure enzyme.

Preferably, the cell lysate component is 2M NaCl, 20mM Tris-HCl, pH 8.0.

Preferably, the buffer I component is 2M NaCl, 20mM Tris-HCl, 40mM imidazole, pH 8.0.

Preferably, the buffer II component is 2M NaCl, 20mM Tris-HCl, 250mM imidazole, pH 8.0.

Preferably, the mobile phase used in the gel filtration chromatography in step (3) is a cell lysate.

The histamine oxidase prepared by the invention can be used for degrading histamine in various high-salt foods such as fish sauce, shrimp paste, pickled food and the like.

The present study uses histamine phosphate as a substrate and measures its reaction product H₂O₂Content determination Hod_HvvThe method comprises the following steps:

dissolving 100mM histamine phosphate substrate in distilled water, collecting 60 μ L system of 5 μ g enzyme solution, 5 μ L substrate and enzyme activity determination buffer solution, reacting at 37 deg.C for 30min, stopping reaction on ice, adding 60 μ L detection solution into the system, reacting for 30s, and analyzing with nucleic acid protein analyzer DU800^TMDetermination of the Absorbance A₅₉₅. Enzyme activity determination buffer solution is used as a blank control instead of enzyme solution. Preparation of H₂O₂Calculating enzyme activity by using a content standard curve. One unit of enzyme activity is defined as the amount of enzyme required to produce l μ g histamine per minute under specific conditions. Hod with histamine phosphate as substrate measured at 37 deg.C and pH 8.0 with 2M NaCl_HvvThe specific enzyme activity is 23.6U/mg.

Histamine phosphate as substrate, Hod_HvvThe optimum NaCl concentration is 2M within the range of 0.07-4M NaCl, and the activity of the compound can still reach 100% after 4 hours under the NaCl concentrations of 0.5M, 2M and 4M; the optimal reaction temperature is 60-65 ℃ within the range of 30-80 ℃, the activity of 100% is still maintained after 4 hours at 30 ℃, and the enzyme activity reaches 3 hours after 50 ℃400 percent; within the pH range of 6.0-9.0, the optimum pH is 6.5, and the activity of 100% can be still maintained after 4h under the conditions of pH 6.0, 7.5 and 9.0. Can resist various metal ions, Sr²⁺Promoting enzyme activity; can degrade various biogenic amines, and has highest enzyme activity when histamine is used as a substrate. Maximum reaction rate V with histamine as substrate_max30.83. mu.g/min/mg, kinetic constant K_m0.5321 mM.

Preferably, the assay buffer in step (b) is 2M NaCl, 20mM Tris-HCl, pH 8.0.

Preferably, the detection solution in the step (A) is 5mM 3-methyl-2-benzothiazolone hydrazone hydrochloride, 2.5mM m-diaminobenzoic acid and 0.067mg/mL horseradish peroxidase.

The invention has the advantages and technical effects that:

(1) the invention relates to a novel histamine oxidase gene obtained by screening from halophilic archaea (Halovivax sp.) WLHSJ27-1, the gene has low consistency with other published histamine oxidases from other microorganisms, and no research on halophilic archaea histamine oxidase is available at present, the invention can fill the blank of research on halophilic archaea histamine oxidase, so that the novel gene hod for encoding the halophilic archaea histamine oxidase_HvvAnd halophilic archaea histamine oxidase Hod_HvvAll have high research value.

(2) Histamine oxidase Hod_HvvThe catalyst has excellent enzymatic properties and can catalyze various amines; high temperature resistance, the optimal reaction temperature is 60-65 ℃, and the reaction is stable at 30 ℃ and 50 ℃; the tolerance to NaCl and pH is good, and the stability is good from low salt to high salt, and from neutral to alkaline; can resist various metal ions. Histamine oxidase with excellent properties can be applied to various high-salt foods with high histamine content, such as fish sauce, shrimp paste and the like, to degrade histamine.

Drawings

FIG. 1 shows hod of the present invention_HvvRecombinant plasmid double restriction map in example 2.

FIG. 2 shows Hod of the present invention_HvvSDS-PAGE electrophorogram in example 3.

FIG. 3 shows Hod of the present invention_HvvEnzyme activity assay in the examples4H₂O₂Content standard curve.

FIG. 4 shows Hod in the present invention_HvvOptimal reaction temperature profile in example 5.

FIG. 5 shows Hod in the present invention_HvvPlot of the optimum response NaCl concentration in example 5.

FIG. 6 shows Hod in the present invention_HvvpH profile of the optimal reaction in example 5.

FIG. 7 shows Hod in the present invention_HvvThermal stability profile in example 6.

FIG. 8 shows Hod in the present invention_HvvNaCl stability profile in example 6.

FIG. 9 shows Hod in the present invention_HvvpH stability profile in example 6.

FIG. 10 shows different pairs Hod of metal ions in example 6 of the present invention_HvvGraph of the effect of catalytic activity.

FIG. 11 shows Hod under different substrates in example 7 of the present invention_HvvAnd (4) a catalytic activity diagram.

FIG. 12 shows histamine as substrate Hod in example 8 of the present invention_HvvKinetic profiles.

Detailed Description

The invention is further described with reference to the drawings and the following description of specific embodiments.

Halophilic archaea (Halovivax sp.) WLHSJ27-1 was isolated from the autonomous region of inner Mongolia, the Shaohai Hai of Uolanzi, for 2019, 12 months and 20 days.

Example 1:

novel gene hod encoding histamine oxidase of archaeophilus halophilus_HvvObtaining;

based on the whole genome, open reading frame prediction and gene annotation results of a strain of halophilic archaea (Halovivax sp.) WLHSJ27-1 (isolated by the inventor and preserved in China general microbiological culture Collection center, CGMCC 22177 preservation date: 2021.4.12), a suspected histamine oxidase gene is screened. Hod obtained by database alignment analysis_HvvThe size of the gene is 2037bp, and the nucleotide sequence is shown as SEQ ID No. 1. The encoded protein has a size of 678 amino acid residuesThe amino acid sequence is shown as SEQ ID No. 2.

The identity of the sequences to the known histamine oxidase amino acid sequences in the database was aligned by means of Blastp (http:// blast. ncbi. nlm. nih. gov.). Hod_HvvThe similarity with histamine oxidase of Arthrobacter globiformis is 39.91%, the similarity with histamine oxidase of Arthrobacter crystalloid is 38.61%, and the homology is more than 30%. Multiple sequence alignment of the predicted suspected histamine oxidase with the currently published histamine oxidase sequence using Genedoc revealed Hod_HvvThe conserved amino acid sites in (A) are consistent with the histamine oxidase which has been published at present.

In conclusion, Hod_HvvIs a novel copper-containing histamine oxidase from halophilic archaea.

Example 2:

hod_Hvvconstructing and expressing recombinant expression plasmids;

the gene hod obtained by the present invention_HvvCloning to an expression vector to construct a recombinant expression strain. Obtaining gene open reading frame sequence based on ORF analysis of NCBI ORF Finder, designing and amplifying hod_HvvGene upstream primer hod_HvvEcoR I-F (5'-ATAGAATTCGCAGAAAGCCACACGCGA-3') and downstream primer hod_Hvv-BamH I-R (5'-ATAGGATCCGTCGTCGTCGGCGTCCGC-3'), PCR amplification to obtain the target fragment (2037 bp).

Constructing an expression plasmid by adopting a restriction enzyme digestion cloning method, namely, using a restriction enzyme EcoR I and BamH I double digestion PCR product, connecting the purified fragment with a plasmid pTA04 subjected to EcoR I and BamH I double digestion to obtain a recombinant plasmid pTA04-hod_HvvThe 3' end of the insert contained 6 XHis tag. CaCl₂Transformation method into E.coli DH5 alpha, ampicillin resistance screening positive clones. The plasmid extraction kit is adopted to extract the plasmid of the positive clone, and the double enzyme digestion verification is carried out on the positive recon (figure 1, wherein M is Marker, and

lanes

1 and 2 are plasmids pTA04-hod_Hvv-1、pTA04-hod_Hvv-2,

lanes

3 and 4 are plasmids pTA04-hod_Hvv-1、pTA04-hod_Hvv-2 double cleavage results) and sequenced by Shanghai. Correct recombinant expression plasmidTransforming Halofax volcanii expression strain to construct recombinant expression strain; the transformant which is successfully transformed is the transformed recombinant host cell;

inoculating the successfully transformed transformant into 200mL of Hv-YPC liquid culture medium according to the inoculation amount of 1% (v/v), placing the medium in a shaker at 37 ℃ for 200rpm, carrying out shake-flask culture until the medium is in a stationary phase, and carrying out low-temperature centrifugation to collect thalli.

The above restriction enzyme, T4 DNA ligase, was purchased from New England Biolabs; the kit was purchased from Axygen, USA.

Example 3:

recombinant protein Hod_HvvPurifying;

resuspending the thallus collected in the example 2 in cell lysate, and ultrasonically crushing to obtain a crude enzyme solution, wherein the His-tag label contained in the target protein can be combined with nickel, so the target protein is purified by nickel column affinity chromatography; eluting the hybrid protein by using low-concentration imidazole and eluting the target protein by using high-concentration imidazole, and specifically comprises the following steps:

(1) resuspending the thallus with 25mL of cell lysate (2M NaCl, 20mM Tris-HCl, pH 8.0), and carrying out ultrasonic treatment in ice bath for 10-15 min under the ultrasonic conditions of 200W of power, 3s of ultrasonic treatment and 5s of stopping. Centrifuging twice after ultrasonic treatment, and taking supernatant for later use.

(2) The column was equilibrated with 10 bed volumes of lysine buffer.

(3) The supernatant was run through the column and repeated three times to ensure that the protein of interest was well bound to the nickel column.

(4) The heteroproteins were eluted using 15 bed volumes of buffer I (2M NaCl, 20mM Tris-HCl, 40mM imidazole, pH 8.0).

(5) The target protein was eluted using 5 bed volumes of buffer II (2M NaCl, 20mM Tris-HCl, 250mM imidazole, pH 8.0) and the eluate was collected and stored at 4 ℃ until use.

(6) The eluate was concentrated in a Millipore ultrafiltration tube having a molecular weight cut-off of 10kDa to give a concentrated solution, and the protein concentration was 1mg/mL or more as measured by the Bradford method.

(7) The concentrated solution was subjected to gel filtration chromatography (HiTrap)^TMDesaling (5mL)) was used to remove imidazoleThe mobile phase was cell lysate (2M NaCl, 20mM Tris-HCl, pH 8.0) to give histamine oxidase, noted Hod_Hvv。

(8) The resultant histamine oxidase was detected by SDS-PAGE (FIG. 2, M: protein Prestained Marker, lane 1: protein after passing through desalting column).

Example 4 (performance test):

Hod_Hvvand (3) enzyme activity determination: dissolving 100mM histamine phosphate substrate in distilled water, collecting 60 μ L system of 5 μ g enzyme solution, 5 μ L substrate and enzyme activity determination buffer solution, reacting at 37 deg.C for 30min, stopping reaction on ice, adding 60 μ L detection solution into the system, reacting for 30s, and analyzing with nucleic acid protein analyzer DU800^TMDetermination of the Absorbance A₅₉₅. An enzyme activity determination buffer solution is used as a blank control to replace an enzyme solution, and 3 groups are arranged in parallel. Measurement of H₂O₂The enzyme activity was calculated using a content standard curve (FIG. 3). One unit of enzyme activity is defined as the amount of enzyme required to produce l μ g histamine per minute under specific conditions. Hod with histamine phosphate as substrate, measured at 37 ℃ and pH 8.0 with 2M NaCl_HvvThe specific enzyme activity is 23.6U/mg.

Example 5:

Hod_Hvvanalyzing the optimal reaction condition;

measuring Hod with histamine phosphate as substrate and 5 μ g enzyme solution_HvvThe optimum reaction conditions of (1).

The temperature gradients were set at 30, 40, 50, 55, 60, 65, 70, 80 ℃ and the enzyme activities at different temperatures were measured as in example 4. The relative enzyme activity was calculated with the highest enzyme activity as 100%. The optimal reaction temperature is 60-65 ℃ (figure 4).

The optimum NaCl determination range is 0.07-4M, and enzyme activity determination buffer solutions (20mM Tris-HCl, pH 8.0) of 0.07, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4M NaCl are respectively prepared. The enzyme activity was measured at the optimum temperature of 60 ℃ by the method of example 4. The relative enzyme activity was calculated with the highest enzyme activity as 100%. The optimum reaction NaCl was 2M (FIG. 5).

The optimum pH measurement range is 6-9, and 0.1M K is respectively allocated₂HPO₄-KH₂PO₄buffer, adjusting pH to 6.0, 6.5, 7.0,7.5; 0.1M Tris-HCl buffer, adjusting pH to 7.5, 8.0, 8.5, 9.0; the enzyme activity was measured at the optimum temperature of 60 ℃ and the optimum NaCl 2M by the method of example 4. The relative enzyme activity was calculated with the highest enzyme activity as 100%. The optimum reaction pH was 6.5 (FIG. 6).

Example 6:

Hod_Hvvanalyzing the enzymatic stability;

using histamine phosphate as substrate, measuring Hod with 5 μ g enzyme solution under optimum reaction conditions_HvvStability of (2).

The thermal stability is achieved by placing the enzyme solution at 30 deg.C, 50 deg.C, and 70 deg.C for 0, 1, 2, 3, and 4h respectively. The enzyme activity was measured under the optimum conditions by the method of example 4, and the relative enzyme activity was calculated with the enzyme activity at 0h as 100%. The enzyme activity still keeps 100 percent of activity after 4 hours at the temperature of 30 ℃; at 50 ℃, the enzyme activity reaches 400% after 3 hours, and slightly decreases after 4 hours; after 1h at 70 ℃ the enzyme activity was completely lost (FIG. 7).

The specific operation of NaCl stability is to place the enzyme solution in enzyme activity determination buffer solution (20mM Tris-HCl, pH 8.0) containing 0.5, 2, 4M NaCl for 0min, 10min, 30min, 1h, 2h, 3h, 4h, respectively. The enzyme activity was measured in the same manner as in example 4. The relative enzyme activity was calculated with the enzyme activity at 0min under 2M NaCl as 100%. Under 0.5M and 2M, the enzyme activity of 100% is still kept after 4h, and when the enzyme solution is placed under 4M NaCl, the enzyme activity is lower initially, but the enzyme activity is increased along with the increase of time until the enzyme activity is stable after 30 min. Indicating that the enzyme has good NaCl stability (fig. 8).

The specific operation of pH stability is: the enzyme solutions were separately placed at a pH of 6.0K₂HPO₄-KH₂PO₄buffer, pH 7.5Tris-HCl buffer, pH 9.0Tris-HCl buffer, standing for 0h, 1h, 2h, 3h, 4 h. The enzyme activity was measured in the same manner as in example 4. The relative enzyme activity was calculated with the enzyme activity at 0h at pH 6.0 as 100%. Under the conditions of pH 6.0, 7.5 and 9.0, the enzyme activity of 100 percent is still maintained after 4 hours, which indicates that the enzyme has better pH stability (figure 9).

The metal ion resistance is specifically operated as follows: KCl and MgCl were added to the reaction system of application example 4₂、SrCl₂、NiSO₄、CoCl₂、BaCl₂The solution was made to have a final metal ion concentration of 10 mM. The enzyme activity was measured by the method of example 4; wherein, con refers to a control group, the reaction without adding metal ions is used as a control group, and the enzyme activity is 100 percent. Of the metal ions tested, Sr²⁺、K⁺Has promoting effect on enzyme activity; mg (Mg)²⁺Has no obvious influence on enzyme activity; ba²⁺、Co²⁺、Ni²⁺The enzyme activity is inhibited, but 50% of the activity can be still maintained, and the enzyme has better metal ion tolerance (figure 10).

Example 7:

hod under different substrates_HvvThe enzyme activity of (1).

Using different types of biogenic amine as substrates, taking 5 mu g of enzyme solution to determine Hod under the optimal reaction condition_HvvThe enzyme activity of (2).

The specific operation is as follows: 100mM of histamine, tryptamine, cadaverine, spermine, putrescine and tyramine are respectively prepared. The enzyme activity was measured in the same manner as in example 4. The enzyme activity with histamine as substrate was set to 100%, and the relative enzyme activity was calculated. The enzyme has the highest enzyme activity on histamine, and tyramine is the second enzyme; 30% of enzyme activity on tryptamine, cadaverine and putrescine; but has no enzyme activity to spermine. Indicating that the enzyme has broad substrate specificity (FIG. 11).

Example 8:

Hod_Hvvenzymatic kinetic analysis of (2).

Using histamine phosphate as substrate, taking 5 mu g enzyme liquid to test Hod under optimum reaction condition_HvvThe enzyme activity of (2);

the specific operation is as follows: final histamine concentrations of 0.05, 0.15, 0.2, 0.25, 0.3, 0.5, 0.8, 1, 1.5, 2mM were set in each system. Under the optimum conditions, enzyme activities under different substrates were measured by the method of example 4, an enzyme kinetics curve was constructed using GraphPad Prism 7, and an enzyme kinetics constant K was calculated according to Michael-Menten equalisation_mAnd a maximum reaction rate V_max. Maximum reaction rate V of the obtained reaction substrate_max30.83. mu.g/min/mg, kinetic constant K_m0.5321mM (FIG. 12).

Description of the drawings: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, while the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and within the scope of the following claims.

Sequence listing

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Asp Val Gly Glu Ala Ser Leu Cys Ile Lys Ile Glu Leu Ala Glu Pro

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Glu Lys Glu Ala Leu Arg Ala Tyr Asp Asp Gly Gly Glu Arg Pro Asp

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Arg Arg Ala Leu Leu Val Ile Arg Asn Ser Ser Asp Gly Lys Thr Leu

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Glu Ala Val Val Ser Leu Glu Glu Ala Ser Val Val Ser Thr Glu Glu

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Val Asn Gly Gln Pro Ser Ile Ala Ile Glu Glu Phe Ile Ala Cys Glu

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Glu Thr Val Lys Arg Asn Glu Glu Trp Gln Ala Ala Leu His Asp Arg

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Gly Ile Glu Asn Thr Asp Arg Ala Met Val Asp Pro Trp Ser Val Gly

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His Glu Phe Val Pro Glu Asp Val Asp Arg Ser Arg Arg Leu Ala His

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Gly Leu Thr Phe Val Arg Pro Ser Glu Asp Asp Gly Asp Glu Gly Tyr

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Ala His Pro Val Ser Gly Leu His Thr Phe Val Asp Leu Asp Arg Met

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Glu Val Val Lys Val Val Asp Tyr Gly Pro Pro Asp Glu Asp Ser Pro

195 200 205

Ile Pro Pro Glu Glu Met Ala Tyr Arg Glu Gly Asp Val Glu Leu Arg

210 215 220

Asp Asp Leu Thr Ala Tyr Asn Val Asp Gln Pro Asp Gly Pro Ser Trp

225 230 235 240

Ser Val Asp Gly His Lys Leu Glu Trp Gln Asn Trp His Met Arg Val

245 250 255

Gly Trp Thr Gln Arg Glu Gly Leu Val Leu Tyr Asp Val Gly Tyr Glu

260 265 270

Asp Asp Gly Glu Val Arg Ser Ile Ile Asp Arg Ala Ser Cys Ala Glu

275 280 285

Met Ser Val Pro Tyr Gly Asp Gln Asn Ile Asn Asp Arg Phe Lys Asn

290 295 300

Ala Met Asp Val Gly Glu Tyr Asn Ile Gly Arg Leu Ala Lys Ser Leu

305 310 315 320

Thr Asn Gly Cys Asp Cys Leu Gly His Met His Tyr Trp Asp Ala Val

325 330 335

Met Asn Thr Ala Ala Gly Glu Pro Asn Val Leu Glu Asn Ala Ile Cys

340 345 350

Leu His Glu Glu Asp Asn Gly Thr Leu Trp Glu Arg Ser Asp Trp Arg

355 360 365

Thr Glu Thr Asp Glu Val Arg Arg Arg Arg Arg Leu Val Val Ser Phe

370 375 380

Val Ala Ala Val Gly Asn Tyr Asp Tyr Ile Phe Asn Trp Tyr Phe Tyr

385 390 395 400

Gln Asp Ala Ser Met Glu Val Glu Val Arg Leu Thr Gly Ile Asp Ser

405 410 415

Val Ser Ala Val Gly Pro Asp Glu Asp Pro Asp Gly Tyr Gly Glu Leu

420 425 430

Leu Ala Pro Gln Leu Asn Gly Pro Ile His Gln His Phe Phe Asn Phe

435 440 445

Arg Leu Asp Met Asn Val Asp Asp Gly Pro Asn Ser Leu Tyr Arg Val

450 455 460

Gln Asn Glu Gln Val Pro Ile Gly Pro Gly Gly Leu Asp Pro Met Gly

465 470 475 480

Glu Ala Asp Glu Arg Thr Ala Asn Pro Gly Gly Gln Ala Phe Tyr Ala

485 490 495

Lys Arg Glu Gln Leu Ser Ser Glu Ser Ala Ala Lys Asp Leu Ile Asp

500 505 510

Pro Leu Lys Gly Arg Tyr Trp Gln Val Ile Asn Pro Ala Glu Glu Asn

515 520 525

Tyr Leu Gly Lys Pro Thr Gly Tyr Lys Leu Val Pro Gly Gly Asn Val

530 535 540

Glu Ala Ala Met Gln Pro Glu Ser Ser Val Met Lys Arg Ser Gly Phe

545 550 555 560

Ile Gln Tyr His Leu Trp Ala Thr Pro Phe Arg Glu Asp Glu Arg Phe

565 570 575

Pro Ala Gly Thr Tyr Pro Asn Gln His Pro Gly Gly Ala Gly Leu Pro

580 585 590

Ala Trp Thr Glu Ala Asp Arg Asn Leu Glu Glu Glu Asp Leu Val Leu

595 600 605

Trp Tyr Thr Leu Gly Val Asn His Val Thr Arg Pro Glu Asp Trp Pro

610 615 620

Ile Leu Ser Val Gln Val Tyr Ser Phe Thr Leu Gln Pro Val Asn Phe

625 630 635 640

Phe Asp Gly Ser Pro Ala Met Asp Val Pro Pro Gln His Ala Ile Glu

645 650 655

Gly Gln Asp Val Pro Gly His Gly Asp His Cys Ala Thr Asp Ala Gly

660 665 670

Ala Asp Ala Asp Asp Asp

675

Claims

1. A preparation method of recombinant halophilic archaea histamine oxidase is characterized by comprising the following specific steps:

(1) construction of pTA04-hod _HvvRecombinant expression plasmid:

firstly, based on halophilic archaeaHalovivax Obtaining a histamine oxidase gene from the genome of sp, WLHSJ27-1hod _Hvv(ii) a Designing a primer containing an enzyme cutting site according to a target gene, and amplifying a target fragment by using a conventional PCR (polymerase chain reaction) technologyhod _Hvv(ii) a And by molecular cloning techniquehod _HvvConnecting to pTA04 vector to construct recombinant plasmid;

said halophilic archaeaHalovivaxsp, WLHSJ27-1 is preserved in China general microbiological culture collection center with the preservation number of CGMCC 22177 and the preservation date of 2021, 4 months and 12 days;

the above-mentionedhod _HvvThe nucleotide sequence is shown as SEQ ID No.1, and the gene size is 2037 bp; the restriction enzyme site is selected from restriction enzymeEcoR I andBam(iii) a cleavage site of HI;

(2) transformation of recombinant plasmidsHaloferax volcanii：

Transforming the recombinant plasmid obtained in the step (1) into a halophilic archaea host to obtain a transformed recombinant host cell; the halophilic archaea host isHaloferax volcanii(ii) a Carrying out shake-flask culture on the transformed recombinant host cell in an Hv-YPC liquid culture medium, carrying out low-temperature centrifugation to collect thalli for later use after the cell culture reaches a stable period;

（3）Hod_Hvvand (3) purification:

collecting the thalli in the step (2) in cell lysate for resuspension, carrying out ultrasonic cell disruption after resuspension, and centrifugally collecting supernatant for recombinant protein purification; firstly, purifying supernate by adopting nickel column affinity chromatography to obtain imidazole-containing enzyme solution; then removing imidazole in the enzyme solution by gel filtration to obtain high purityDuchenyamine oxidase, noted Hod_Hvv。

2. The method for preparing a recombinant halophilic archaea histamine oxidase according to claim 1, wherein the step (2) is performed in the step (2)Haloferax volcaniiPurchased from the Japanese Collection of microorganisms under the strain accession number JCM 8879.

3. The method for producing recombinant halophilic archaea histamine oxidase according to claim 1, wherein the composition of said Hv-YPC medium in step (2) is as follows, in terms of per liter: 5.0g of yeast extract, 1.0g of soybean peptone, 1.0g of acid casein hydrolysate, 4.2g of KCl and MgSO₄·7H₂O 33.0 g，MgCl₂·6H₂O 30.0 g，NaCl 144.0 g，1 M、pH 8.0 Tris-HCl 12.0 mL，CaCl₂0.33g, adding distilled water to a constant volume of 1L, adjusting pH to 7.5, and sterilizing at 115 deg.C for 30 min.

4. The method for preparing recombinant halophilic archaea histamine oxidase according to claim 1, wherein the temperature in the shake flask culture in the step (2) is 37 ℃, and the rotation speed is 180 rpm; the low temperature is 0-4 ℃.

5. The method for preparing recombinant halophilic archaea histamine oxidase according to claim 1, wherein the specific operation of obtaining the imidazole-containing enzyme solution in step (3) is as follows: equilibrating the nickel column with 10 bed volumes of cell lysate, and then binding the supernatant to the nickel column 3 times; and eluting the hybrid protein by using a buffer solution I with the volume 15 times of that of the column bed, and eluting the target protein by using a buffer solution II with the volume 5 times of that of the column bed to obtain the imidazole-containing enzyme solution.

6. The method of claim 5, wherein the cell lysate fraction is 2M NaCl, 20mM Tris-HCl, pH 8.0; the buffer solution I comprises 2M NaCl, 20mM Tris-HCl and 40mM imidazole, and has the pH value of 8.0; the buffer II comprises 2M NaCl, 20mM Tris-HCl, 250mM imidazole and pH 8.0.

7. The process for preparing recombinant halophilic archaea histamine oxidase according to claim 1, wherein a mobile phase used in the gel filtration chromatography in the step (3) is a cell lysate; the cell lysate component is 2M NaCl, 20mM Tris-HCl, pH 8.0.

8. Use of the recombinant halophilic archaea histamine oxidase prepared according to the method of any one of claims 1 to 7 for degrading histamine in fish gravy, shrimp paste, and pickled food.