CN118256459A - Recombinase and application thereof in degradation of aflatoxin B1Application in (a) - Google Patents

Abstract

The invention discloses a recombinase, the amino acid sequence of which is shown as SEQ ID NO. 1, the nucleotide sequence of a coding gene is shown as SEQ ID NO. 2, and the recombinase belongs to a multi-copper oxidase superfamily. The expression vector is constructed and prokaryotic expression is carried out to obtain the recombinase, and the enzymatic characteristics of the recombinase are researched, so that the recombinase can be found to degrade AFB ₁ efficiently. The invention enriches the types of AFB ₁ degrading enzymes, provides better choice for solving the pollution of agricultural products and feeds, and simultaneously provides theoretical basis for clarifying the action mechanism of the Fenton fiber microzyme and reasonably applying microorganism and enzyme preparations.

Description

Recombinase and application thereof in degradation of aflatoxin B 1

Technical Field

The invention relates to a recombinase for degrading aflatoxin B ₁ and application thereof, belonging to the field of biology.

Background

Aflatoxin B ₁(Aflatoxin B₁,AFB₁) is the most widely polluted and extremely toxic mycotoxin in foods and feeds, and can cause economic loss and seriously harm the health of people and animals. Animals ingest AFB ₁ -contaminated feed, resulting in the remaining of its metabolites in eggs, milk products and meats, which are harmful to human health. AFB ₁ has hepatotoxicity, immunotoxicity, genotoxicity, mutagenicity, etc., and liver and kidney are main target organs, and long-term low-level contact with AFB ₁ can cause chronic toxicity and even liver cancer.

Currently, the detoxification methods of AFB ₁ mainly include a physical detoxification method, a chemical detoxification method and a biological detoxification method. The physical removal of AFB ₁ mainly comprises adsorption, heating, ultraviolet irradiation, solvent extraction, and the like. The chemical detoxication method mainly uses chemical additives to destroy the toxic structure of AFB ₁, so that the AFB ₁ is decomposed into low-toxicity or nontoxic compounds, and is mainly divided into an alkali treatment method and an oxidation treatment method. The physicochemical detoxification method has the defects of poor detoxification effect, influence on the taste of the feed, reduction of the nutritional value of the feed and the like. The biological detoxification method has mild action conditions, good specificity, high detoxification efficiency, no pollution to the environment and capability of destroying the chemical structure of the AFB ₁, and has become the main trend of the detoxification research of the AFB ₁.

The microbial degradation of AFB ₁ has short time and low cost, and does not affect the nutrition quality of the feed, so the microbial degradation is the first choice in the feed industry. AFB ₁ degrading bacteria such as Cellulomyces tenuis (Cellulosimicrobium funkei), tetracoccus halophilus (Tetragenococcus halophilus), stenotrophomonas (Stenotrophomonas acidaminiphila) and Bacillus licheniformis (Bacillus licheniformis) can effectively degrade AFB ₁, but the strains separated from the nature can be pathogenic bacteria or conditional pathogenic bacteria, and the living bacteria preparation is directly used as a feed additive or a food additive, which may cause biosafety problem or refusal of eating by animals, and is not suitable for practical production. The enzyme secreted by the microorganism in the growth process is utilized to change the structure of AFB ₁ for degradation, and the possible ways of degrading AFB ₁ mainly involve ring cleavage, hydrolysis, decarboxylation or deamination, so that compared with the microorganism, the enzyme is convenient to use and has been widely accepted in the food and feed industries.

The multiple copper oxidase superfamily can oxidize a variety of substrates, such as lignin-rich aromatic compounds, polyphenols, metal ions, ascorbate ions, siderophores, pigments, and the like, using molecular oxygen as an electron acceptor. All the amino acid sequences of the multicopper oxidases comprise a 10kDa-20kDa copper-reducing protein-like domain, and the multicopper oxidases can be divided into enzymes comprising 2 domains, 3 domains and 6 domains, depending on the number of copper-reducing protein-like domains, which include laccase (EC 1.10.3.2), ascorbate oxidase (EC 1.10.3.3), nitrite reductase (EC 1.7.2.1), bilirubin oxidase (EC 1.3.3.5), and metallooxidase (EC 1.16.3.1), etc. Laccase has been reported to have a good effect of degrading AFB ₁, and is widely used in related enzyme preparations. However, there is still a lack of AFB ₁ degrading enzymes suitable for industrial production.

In summary, to solve the problem of pollution of AFB ₁ in agricultural products, feed materials and feeds, it is necessary to screen more enzymes capable of safely and effectively degrading AFB ₁, so as to enrich the variety of AFB ₁ degrading enzymes, provide theoretical basis for enzyme preparation utilized in actual production, and reduce economic loss of the aquaculture industry.

Disclosure of Invention

The first object of the present invention is a novel aflatoxin B ₁ degrading enzyme.

The Fennella has very high degradation activity of aflatoxin B ₁, and the applicant screens an enzyme capable of efficiently degrading aflatoxin B ₁ from the Fennella by a molecular biological means, and the enzyme belongs to a multi-copper oxidase superfamily. Further, the applicant optimizes the sequence of the multi-copper oxidase and constructs an expression vector, and prokaryotic expression is carried out to obtain the recombinase, so that the activity of the recombinase in degrading aflatoxin B ₁ is verified.

The amino acid sequence of the recombinase is shown as SEQ ID NO. 1.

The nucleotide sequence of the gene for encoding the recombinase is shown as SEQ ID NO.2.

The invention further provides a method for preparing the recombinase, which comprises the following steps:

(1) The gene sequence of the recombinant enzyme is connected with a vector plasmid in an enzyme digestion way, so as to obtain an expression vector containing a target gene;

(2) Transforming competent cells by using the obtained expression vector to obtain recombinant bacteria for expressing the target protein;

(3) And separating and purifying the recombinant enzyme from the recombinant bacteria.

The invention also provides application of the recombinase, or the coding gene thereof, or the expression vector thereof or the recombinant bacterium in degradation of aflatoxin B ₁.

The invention further provides a method for degrading aflatoxin B ₁, which comprises the following steps: the recombinant enzyme is added into a polluted sample of aflatoxin B ₁ for enzymatic reaction.

Preferably, the temperature of the enzymatic reaction is 30 ℃ to 50 ℃ and the pH is neutral or slightly alkaline.

The invention also provides an enzyme preparation for degrading aflatoxin B ₁, which contains the recombinase.

The beneficial effects of the invention are as follows:

The invention separates, identifies and re-obtains a new AFB ₁ degrading enzyme from the Fennella, enriches the variety of the AFB ₁ degrading enzyme, provides better choice for solving the pollution of agricultural products and feeds, and simultaneously is beneficial to elucidating the action mechanism of the Fennella and provides theoretical basis for the application of microorganisms and enzyme preparations.

Drawings

Fig. 1: molecular docking results of AFB ₁ and the Fenton fiber microzyme multi-copper oxidase, and a left graph is a 3D graph after the multi-copper oxidase is docked with AFB ₁; the right panel shows 2D interactive images of the docking of AFB ₁ ligand with multiple copper oxidases.

Fig. 2: the MW is the molecular weight of the protein Marker as a result of the expression test of the recombinant multi-copper oxidase; Is an uninduced strain; NPE is the supernatant protein; DPE is inclusion body protein; the No.1 strain is escherichia coli BL21 (DE 3); no.2 strain is E.coli T7E.

Fig. 3: SDS-PAGE detection result of recombinant multicopper oxidase.

Fig. 4: effect of enzyme concentration of different recombinant multicopper oxidase on degradation effects of AFB ₁.

Fig. 5: effect of different reaction pH on the effect of recombinant multicopper oxidase on degradation of AFB ₁.

Fig. 6: effect of different reaction temperatures on the effect of recombinant multicopper oxidase on the degradation of AFB ₁.

Fig. 7: the linear equation of dynamics of the recombinant multicopper oxidase degradation AFB ₁.

Fig. 8: influence of metal ions on the effect of recombinant multicopper oxidase on the degradation of AFB ₁.

Detailed Description

The technical scheme of the invention is further described in detail below with reference to specific embodiments. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Various modifications and equivalent substitutions will occur to those skilled in the art based on the following examples, and are also considered to be within the scope of the present invention. The experimental procedures, which are not specified in the following examples, are generally carried out according to conventional conditions or instructions or according to the procedures suggested in the operating manual provided by the manufacturer.

Key materials and description:

Fender fiber microzyme: the Fennella SLAQ strain is purchased from China Center for Type Culture Collection (CCTCC) No. M2013564, and is disclosed in patent document CN 103725636A.

PET-28b-3C vector plasmid: purchased from beijing bang allied biogenic technologies limited.

Coli BL21 (DE 3) strain: the E.coli expression strain is most commonly available from Shanghai ze Biotechnology Inc.

Coli T7E strain: purchased from Beijing village allied biogenic gene technology Co., ltd, is an E.coli BL21 enhanced strain.

Example 1 screening test for aflatoxin B ₁ degrading enzyme in Cellularomyces fens

(1) Culturing the Fender fiber microzyme in LB broth to prepare extracellular fluid and intracellular fluid respectively, then taking each component to react with AFB ₁ (10 mug/mL), extracting AFB ₁ after the reaction is finished, and measuring the degradation rate of AFB ₁. The results showed that the degradation rate of extracellular fluid to AFB ₁ was 82.07% and the degradation rate of intracellular fluid to AFB ₁ was 6.19%, indicating that the component degrading AFB ₁ in the Cellularomyces fimbriae was mainly extracellular.

(2) After the extracellular fluid is added with proteinase K or is subjected to heat denaturation treatment, the efficiency of degrading AFB ₁ by the extracellular fluid is obviously reduced, and the ingredient which plays the role of degrading AFB ₁ in the extracellular fluid is presumed to be protein. And then sequentially carrying out gradient centrifugation, separation and concentration on extracellular fluid by using a 30kDa, 10kDa and 3kDa ultrafiltration tube, and carrying out co-culture on each component obtained by concentration and AFB ₁ again and measuring the degradation rate, wherein the effect of degrading AFB ₁ by the component with the molecular weight of 10kDa-30kDa is found to be significantly higher than that of other components. Continuing to examine the degradation activity of the component with the molecular weight of 10kDa-30kDa on AFB ₁ under the conditions of different time, temperature, pH and metal ion addition, the component is found to accord with the enzymatic characteristics, and extracellular fluid is presumed to degrade AFB ₁ through enzymatic reaction.

(3) Extracting DNA of the Fennella, carrying out library construction and bioinformatics analysis on the DNA, and obtaining annotation results of the Fennella genes according to the sequencing results.

(4) And (3) performing SDS-PAGE electrophoresis detection on the 10kDa-30kDa extracellular fluid component obtained by separation and purification in the step (2), identifying active proteins, and predicting the physicochemical properties such as hydrophobicity, molecular formula, isoelectric point, stability and the like of the obtained protein amino acid sequence through an online website (https:// web. Expasy org/protparam /). 81 proteins were detected in total, and the 81 proteins were classified according to their enzymatic reactivity to give 56 enzymes belonging to the major classes of oxidoreductase, transferase, isomerase, synthetase and hydrolase 5, respectively, with the most proteins belonging to the classes of oxidoreductase and hydrolase. According to the literature referred in the prior art, the oxidoreductase is closely related to the degradation of AFB ₁, and the molecular docking result shows that a multi-copper oxidase is probably a key enzyme for degrading AFB ₁ in 13 proteins of the oxidoreductase class, and the enzyme codes 970 amino acids, and the protein has a molecular weight of 98.46kDa and better stability.

TABLE 1 class of oxidoreductase and physicochemical Properties of the extracellular fluid fraction of the 10kDa-30kDa of Cellularomyces fens

Note that: * Representing the predicted physicochemical properties of the online website. A instability factor greater than 40 indicates that the protein is unstable.

(5) The secondary structure of AFB ₁ was obtained by PubChem (https:// pubchem. Ncbi. Lm. Nih. Gov /), the multicopper oxidase homology was modeled using the protein modeling platform of the subject group, and the binding site between the target protein and AFB ₁ was modeled using MOE software (version number 2019.0102). As can be seen from FIG. 1, there is a hydrogen bond force between AFB ₁ and Arg794 of the multicopper oxidase, the hydrogen bond length isArene hydrogen bond acting force exists between Trp798 and the hydrogen bond is as long asIt was demonstrated that these two amino acid residues are likely the key amino acids for binding of the multicopper oxidase to AFB ₁. In addition, AFB ₁ has an S value of-7.40 and an RMSD value of 0.80 for the butt joint with the multicopper oxidase. Therefore, the AFB ₁ and the multicopper oxidase have better butt joint binding force.

(6) In order to evaluate the homology of the amino acid sequence of the Fenton fiber microzyme, the amino acid sequence of the Fenton fiber microzyme is compared with the amino acid sequence of laccase reported in a literature and directly compared in NCBI database, and the test has low homology of the amino acid sequence of the multi-copper oxidase and the laccase. In addition, the amino acid sequences of 100 proteins from different strains containing the domains of the multicopper oxidase were aligned, wherein the amino acid sequence homology of the multicopper oxidase of this test with the protein (Cellulosimicrobium cellulans) containing the domains of the multicopper oxidase was highest, up to 99.90%.

TABLE 2 alignment of amino acid sequence homology of Fender fiber microzyme multicopper oxidase

Note that: only the first ten with a high degree of amino acid sequence homology match are shown.

Example 2 sequence optimization and recombinant expression of a multicopper oxidase

1. Amino acid sequence optimization of multiple copper oxidases

The resulting multicopper oxidase amino acid sequences from this experiment were predicted via an online website for signal peptide cleavage sites (https:// services. Healthcare. Dtu. Dk/services. PhpSignal P-5.0), glycosylation sites (https:// services. Healthcare. Dtu. Dgy-1.0) and transmembrane domains (https:// services. Healthcare. Dk/services/TMHMM-2.0 /). Considering both bioinformatics analysis and information on homologous proteins (accession number: UTT 60473.1), we cut Val547-Pro970 as the target amino acid sequence and carried the His tag on the N-terminus for better expression of the multicopper oxidase in E.coli supernatant.

2. Primer design, enzyme digestion and connection carrier of recombinant multi-copper oxidase genes

(1) According to the plasmid map of the Escherichia coli pET-28b-3C, a primer and a restriction enzyme site are designed, wherein the two ends of the primer comprise BamHI restriction enzyme sites (5 '-GGATCC-3')/HindIII restriction enzyme sites (5 '-AAGCTT-3'), and the primer is designed as follows:

The six bases underlined in the following ：5'-GGATCCatgggcagcagccatcatcatcatcatcatagcagcggcctggaagttctgttccaggggcccggatccGTGGCAACAGGTCGTACC-3'( of the upstream primer represent BamHI cleavage sites and the lower case base represents His tag

The downstream primers were as follows: (underlined three bases represent stop codons, italics six bases represent HindIII cleavage sites)

(2) Synthesizing target genes of the multi-copper oxidase by a DNA synthesizer;

(3) 2% agarose gel electrophoresis and fragment gel recovery:

A, preparing 2% agarose gel (1 g agarose is added into 50mL TAE), slightly mixing, and heating in a microwave oven for 2min-2.5min until the mixture is clear and transparent;

B, standing for 1-3 min, and adding 5 mu L Goldview nucleic acid dye after the solution is slightly cooled, and uniformly mixing;

C, slowly pouring the solution into a gel preparation tank, inserting a gel comb, standing for 15-20 min, putting the gel into an electrophoresis tank after the agarose gel is completely solidified, and adding electrophoresis liquid until the gel is completely immersed;

D, spot-adding DNA sample mixed with 5X reducibility Loading buffer and DNA MARKER, voltage 140V, electrophoresis for 20min;

E, rapidly cutting off a strip of the multi-copper oxidase under an ultraviolet analyzer to prevent mutation caused by ultraviolet;

f, placing the cut agarose gel with the target band into a 1.5mL centrifuge tube without enzyme and weighing;

G, adding an equal amount of Binding Buffer (XPZ) according to 1G/mL; water bath at 55-60 ℃ until the gel is completely melted; transferring the melted solution into HiBind DNA Column, and sleeving HiBind DNA Column on a waste liquid collecting pipe; centrifuging at room temperature at maximum rotation speed (13000 r/min) for 1min, and discarding the waste liquid;

h, adding 300 mu L of Binding Buffer (XPZ), centrifuging at the maximum rotation speed at room temperature for 1min (13000 r/min), and discarding the waste liquid;

I, adding 700 mu L SPW Wash Buffer to elute Binding Buffer, centrifuging at the maximum rotation speed of room temperature (13000 r/min) for 2min, and discarding waste liquid; repeating the elution twice; centrifuging at room temperature at maximum rotation speed (13000 r/min) for 2min, and drying HiBind DNA Column;

J, sleeving HiBind DNA Column on a new enzyme-free 1.5mL centrifuge tube, adding 15 mu L of DEPC water, standing for 1min to dissolve DNA, and centrifuging at a maximum rotation speed of (13000 r/min) for 1min at room temperature; repeating the elution twice; the purified DNA solution was stored in a refrigerator at-20 ℃.

(4) The target gene fragment is cloned into a pET-28b-3C vector by BamHI/HindIII, and the specific steps are as follows:

A, constructing an enzyme digestion system, as shown in the following table:

	Additive amount
		Upstream: bamHI enzyme	1μL
Downstream: hindIII enzyme	1μL
		10×buffer	25μL
Recovery of product DNA from gel	2μg
		DEPC water	Make up to 50 mu L

And B, placing the reaction system in a PCR instrument to react for 15min at 37 ℃.

C, the connection system is as follows:

and D, placing the reaction system in a PCR instrument to react for 15min at 37 ℃.

3. Plasmid transformed expression strains

(1) Adding proper amount of plasmid (1-2 μL) into competent cells (E.coli BL21 (DE 3) strain No.1 and E.coli T7E strain No. 2) prepared in advance;

(2) Placing on ice for 30min, and performing heat shock in a water bath at 42 ℃ for 90s;

(3) Adding a proper amount of antibiotic-free LB liquid medium, and incubating for 40min at 37 ℃ and 150 r/min;

(4) The mixture was inoculated with a loop to LB solid plates with the corresponding resistance and incubated overnight in a 37℃incubator.

4. Expression test of recombinant enzyme

(1) Selecting a monoclonal from the culture dish to 3mL of corresponding resistant LB culture medium, and culturing for 3-4 h at 37 ℃ and 200 r/min;

(2) Taking 1.5mL of bacterial liquid to a new 15mL centrifuge tube, adding 1.5 mu L of Isopropyl thiogalactoside (Isopropyl-beta-D-thiogalactoside, IPTG) with a final concentration of 1mmol/L, and performing induction culture at 37 ℃ and 200r/min for 4h; 1.5 mu L of IPTG is added into the residual 1.5mL of bacterial liquid, the temperature is 16 ℃, and the induction culture is carried out at 200r/min for overnight;

(3) Collecting the bacterial liquid after the induction into a 1.5mL centrifuge tube, centrifuging at 10000r/min for 1min, and discarding the supernatant; 1mL of 1 XPBS was added to the pellet to sonicate the cells;

(4) Centrifuging at 10000r/min for 1min, collecting 100 μl of supernatant labeled "NPE", discarding redundant supernatant, adding 100 μl of 1 XPBS (containing 8mmol/L urea) into the precipitate, and re-suspending to obtain "DPE";

(5) To the sample, 25. Mu.L of 5 Xreducing Loading buffer was added, and after boiling for 10 minutes, SDS-PAGE was performed.

The recombinant enzyme is expressed in both supernatant (NPE) and inclusion bodies (DPE), and the optimal expression condition is that the Escherichia coli strain No.2 (Escherichia coli T7E strain) is cultured for 4 hours at 37 ℃, and the supernatant is centrifugally taken to amplify and express the purified recombinant enzyme.

5. Purification of recombinant enzymes

(1) Lysing the cells with an appropriate amount of PBS (pH 7.5), 10% glycerol and 1mmol/L phenylmethylsulfonyl fluoride (Phenylmethylsulfonyl fluoride, PMSF), mixing well, and lysing with an ultrasonic breaker for 20min; centrifuging at 12000r/min for 10min, separating supernatant (supernatant protein mixture to be purified), and retaining precipitate;

(2) Adding a certain amount of treated Ni resin into the supernatant mixed solution, placing the mixture in a shaking table at 4 ℃ for incubation for 30min, collecting the resin, and purifying according to the following steps:

(3) According to the purification result, the target protein is collected for dialysis, the concentration of the detected protein is 1100 mug/mL, the SDS-PAGE detection result is shown in figure 3, and the molecular weight of the recombinant expressed multi-copper oxidase is about 55 kDa.

EXAMPLE 3 investigation of the enzymatic Properties of recombinant multiple copper oxidases

The effect of different concentrations of recombinase on degrading AFB ₁ was verified, while the effects of different incubation temperatures, different pH, enzyme kinetic parameters and different metal ions were determined.

(1) Preparation of different concentrations of recombinase: the purified recombinase (1100. Mu.g/mL) was diluted with PBS to 5.00. Mu.g/mL, 2.50. Mu.g/mL, 1.00. Mu.g/mL, 0.75. Mu.g/mL, 0.50. Mu.g/mL, 0.25. Mu.g/mL, 0.10. Mu.g/mL, 0.05. Mu.g/mL, 0.025. Mu.g/mL, and the mixture was reacted in the absence of light at 37℃for 24 hours using PBS as a control group to determine the degradation rate of AFB ₁.

(2) Treatment at different incubation temperatures: AFB ₁ was added to the diluted recombinase to a final concentration of 1. Mu.g/mL, and the mixture was incubated at 28℃at 37℃at 40℃at 50℃at 60℃at 70℃at 80℃at 90℃at 100℃respectively, and the mixture was reacted in the absence of light for 24 hours with PBS as a control group to measure the degradation rate of AFB ₁.

(3) Treatment at different pH: and respectively regulating the pH value of the recombinase to 2, 3, 4, 5, 6, 7 and 8 by using citrate buffer solution and sodium hydrophosphate buffer solution, taking PBS as a control group, carrying out light-shielding reaction for 24 hours at 37 ℃, and measuring the degradation rate of the AFB ₁.

(4) Determination of enzyme kinetics Km and Vmax values: the recombinase Km and Vmax values were determined by double reciprocal mapping. The equation is as follows:

[ S ] is the substrate concentration, which is 0.20. Mu.g/mL, 0.50. Mu.g/mL, 1.00. Mu.g/mL, 1.50. Mu.g/mL, 2.50. Mu.g/mL. mu.L of recombinase (0.05. Mu.g/mL) and 100. Mu.L of AFB ₁ at different concentrations were added and incubated at 37℃for 1h. After the completion of the culture, the degradation rate of AFB ₁ was measured.

Definition: reaction rate = AFB ₁ degradation/(reaction volume x reaction time). In this test, the reaction rate=afb ₁ degradation/(1 ml×1 h), km, vmax were calculated.

(5) Different metal ion treatments: FeSO₄、ZnSO₄、CuSO₄、FeCl₃、CaCl₂、NiCl₂、MgCl₂、MnSO₄ serving as the sole metal ion additive is added into a degradation reaction system to enable the final concentration to be 10mmol/L, 0.05 mug/mL recombinase which is free of metal ions and sterile PBS and has the same final concentration of AFB ₁ is used as a control, and the degradation rate of AFB ₁ is measured by carrying out light-proof reaction for 24 hours at 37 ℃.

As can be seen from FIG. 4, the degradation rate of AFB ₁ by the recombinase was significantly lower (P < 0.05) than that of the other 7 groups (enzyme concentration of 5.00. Mu.g/mL, 1.00. Mu.g/mL, 0.75. Mu.g/mL, 0.50. Mu.g/mL, 0.10. Mu.g/mL, 0.05. Mu.g/mL or 0.025. Mu.g/mL) at recombinase concentrations of 0.25. Mu.g/mL and 2.50. Mu.g/mL. Considering the practical application cost of the recombinase, the subsequent experiments all adopt the recombinase with the concentration of 0.05 mug/mL to co-culture with AFB ₁ for subsequent researches.

As can be seen from fig. 5, the rate of degradation of AFB ₁ by the recombinase at pH 7 and 8 was significantly higher (P < 0.05) than that of the other 5 groups (pH 2,3,4, 5 or 6).

As can be seen from FIG. 6, the degradation rate of AFB ₁ by the recombinase was significantly lower (P < 0.05) than that of the other 3 groups (incubation temperature 37 ℃, 40 ℃ or 50 ℃) at 60 ℃ and 70 ℃.

As can be seen from fig. 7, the double reciprocal method is used to plot, the linear equation is y=1.165x+1.3343, and r ² is 0.9929. As a result of calculation, the Km value of the Michaelis constant of the recombinase to AFB ₁ was 0.87. Mu.g/mL, and the maximum reaction rate Vmax was 0.75. Mu.g/(mL. Times.h).

As can be seen from fig. 8, the addition of Fe ²⁺、Ni²⁺、Mn²⁺ and Zn ²⁺ significantly (P < 0.05) inhibited the effect of the recombinase on degradation of AFB ₁ compared to the control group (the recombinase group without added metal ions).

Claims (10)

1. A recombinant enzyme has an amino acid sequence shown in SEQ ID NO. 1.

2. A gene encoding the recombinase of claim 1, wherein the nucleotide sequence is shown in SEQ ID NO. 2.

3. An expression vector comprising the gene of claim 2.

4. A recombinant bacterium comprising the recombinase of claim 1.

5. A method for preparing the recombinase of claim 1, comprising the steps of:

(1) The gene sequence of the recombinant enzyme is connected with a vector plasmid in an enzyme digestion way, so as to obtain an expression vector containing a target gene;

(2) Transforming competent cells by using the obtained expression vector to obtain recombinant bacteria for expressing the target protein;

(3) And separating and purifying the recombinant enzyme from the recombinant bacteria.

6. The method for producing a recombinase according to claim 5, wherein: the competent cells were E.coli T7E cells.

7. The use of the recombinase of claim 1, or a coding gene thereof, or an expression vector thereof or recombinant bacteria for degrading aflatoxin B ₁.

8. A method for degrading aflatoxin B ₁, which is characterized by comprising the following steps: the recombinant enzyme of claim 1 is added to a contaminated sample of aflatoxin B ₁ for enzymatic reactions.

9. The method for degrading aflatoxin B ₁ according to claim 8, wherein: the temperature of the enzymatic reaction is 30-50 ℃, and the pH value is neutral or alkalescent.

10. An enzyme preparation for degrading aflatoxin B ₁ comprising the recombinase of claim 1.