A laccase mutant, its preparation and use

Abstract

The application discloses a laccase mutant, and preparation and application thereof, and solves the technical problem of providing an enzyme mutant with higher enzyme activity and a complex formed by the enzyme mutant and bacillus subtilis with higher enzyme stability. The application specifically discloses a protein, which is any one of the following: B1) a protein with an amino acid sequence shown in sequence 3; B2) a protein with more than 80% identity with the protein shown in B1) and with the same function, which is obtained by substitution, deletion and / or addition of amino acid residues of the protein in B1); and B3) a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of the protein in B1) or B2). The protein can be used alone to degrade zearalenone and aflatoxin B1, or the protein can be mixed with bacillus subtilis to form a complex, so that the stability of the enzyme can be improved, and the complex can be used in industrial production.

Description

Technical Field

[0001] This invention relates to the field of enzymes, and more specifically to a laccase mutant and its preparation and application. Background Technology

[0002] Laccase (EC1.10.3.2) is a polyphenol oxidase containing four copper ions, belonging to the multi-copper oxidase (MCOs) family. Laccases from different sources exhibit significant differences in catalytic properties, and are classified according to their origin into animal / plant laccases and microbial laccases. Microbial laccases can be further divided into fungal laccases and bacterial laccases. Laccases possess characteristics such as high single-electron redox potential, strong catalytic activity, and a broad substrate range. They can oxidatively degrade many harmful substances and have been applied in the food industry, dyes, decolorization, pulping, bleaching, and papermaking wastewater treatment. Current research has found that laccases have great application potential in the degradation of mycotoxins such as zearalenone (ZEN) and aflatoxin B1 (AFB1). Therefore, how to provide a laccase with high degradation activity and stability is a technical problem faced by researchers in this field. Summary of the Invention

[0003] The technical problem solved by this invention is how to provide a laccase with high degradation activity and stability.

[0004] To address the aforementioned technical problems, the present invention provides the following applications.

[0005] Proteins can be used in any of the following applications:

[0006] A1) Applications in the enzymatic hydrolysis of zearalenone or the preparation of enzymatically hydrolyzed zearalenone products;

[0007] A2) Applications in the enzymatic hydrolysis of aflatoxin B1 or in the preparation of enzymatically hydrolyzed aflatoxin B1 products;

[0008] A3) Applications in the enzymatic hydrolysis of ABTS or the preparation of enzymatically hydrolyzed ABTS products;

[0009] The protein is any one of the following:

[0010] B1) The amino acid sequence is that of the protein shown in sequence 3;

[0011] B2) A protein having more than 80% identity and the same function as the protein shown in B1) obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in B1).

[0012] B3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in B1) or B2).

[0013] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0014] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0015] In the aforementioned proteins, the 80% or more identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0016] In this application, the amino acid sequence of the protein shown in Sequence 1 (SEQ ID No. 1) consists of 513 amino acids. It is named Wt-CotA protein or protein Wt-CotA. Its encoding gene is the Wt-CotA gene. The nucleotide sequence of the Wt-CotA gene is shown in Sequence 2.

[0017] In this application, the amino acid sequence of the protein shown in Sequence 3 (SEQ ID No. 3) consists of 513 amino acids. It is named T480A-CotA protein or protein T480A-CotA. Its encoding gene is the T480A-CotA gene. The nucleotide sequence of the T480A-CotA gene is shown in Sequence 4.

[0018] The coding gene for Wt-CotA protein (nucleotide sequence of Sequence 2 (SEQ ID No. 2)) was modified by mutating C (deoxycytosine nucleotide) at position 1440 to G (deoxyguanine nucleotide) and A (deoxyadenine nucleotide) at position 1438 to G (deoxyguanine nucleotide), thus obtaining the coding gene for T480A-CotA protein (nucleotide sequence of Sequence 4 (SEQ ID No. 4)). This resulted in a mutation of T (threonine) at position 480 of the Wt-CotA protein (amino acid sequence of Sequence 1) to A (alanine), yielding the T480A-CotA protein (amino acid sequence of Sequence 3).

[0019] In the above applications, the protein is derived from Bacillus subtilis.

[0020] The Bacillus subtilis mentioned above may be Bacillus subtilis ZJ-2019-1-1.

[0021] Of the proteins described above, the protein is used in any of the following applications:

[0022] A1) Applications in the enzymatic hydrolysis of zearalenone or the preparation of enzymatically hydrolyzed zearalenone products;

[0023] A2) Applications in the enzymatic hydrolysis of aflatoxin B1 or in the preparation of enzymatically hydrolyzed aflatoxin B1 products;

[0024] A3) Applications in the enzymatic hydrolysis of ABTS or the preparation of enzymatically hydrolyzed ABTS products.

[0025] In this application, the CAS number of the zearalenone is 17924-92-4.

[0026] In this application, the CAS number of aflatoxin B1 is 1162-65-8.

[0027] To address the aforementioned technical problems, the present invention also provides the following applications.

[0028] Applications of biomaterials in any of the following:

[0029] A1) Applications in the enzymatic hydrolysis of zearalenone or the preparation of enzymatically hydrolyzed zearalenone products;

[0030] A2) Applications in the enzymatic hydrolysis of aflatoxin B1 or in the preparation of enzymatically hydrolyzed aflatoxin B1 products;

[0031] A3) Applications in the enzymatic hydrolysis of ABTS or the preparation of enzymatically hydrolyzed ABTS products;

[0032] The biomaterial is any one of the following:

[0033] D1) The nucleic acid molecule that encodes the above-mentioned protein;

[0034] D2) expresses the gene encoding the nucleic acid molecule described in D1);

[0035] D3) contains an expression cassette containing the gene described in D2);

[0036] D4) A recombinant vector containing the gene described in D2), or a recombinant vector containing the expression cassette described in D3);

[0037] D5) Recombinant microorganisms containing the gene described in D2), or recombinant microorganisms containing the expression cassette described in D3), or recombinant microorganisms containing the recombinant vector described in D4);

[0038] D6) A transgenic plant cell line containing the gene described in D2), or a transgenic plant cell line containing the expression cassette described in D3), or a transgenic plant cell line containing the recombinant vector described in D4;

[0039] D7) Transgenic plant tissue containing the gene described in D2), or transgenic plant tissue containing the expression cassette described in D3), or transgenic plant tissue containing the recombinant vector described in D4;

[0040] D8) A transgenic plant organ containing the gene described in D2), or a transgenic plant organ containing the expression cassette described in D3), or a transgenic plant organ containing the recombinant vector described in D4).

[0041] In the nucleic acid molecule described in D1), those skilled in the art can easily mutate the nucleic acid molecule encoding the protein of the present invention using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides that have 80% or more identity with the nucleotide sequence of the nucleic acid molecule encoding the protein obtained by the present invention, and that have the function of expressing the coding gene of the nucleic acid molecule encoding the protein, are all derived from and equivalent to the nucleotide sequence of the present invention.

[0042] The aforementioned 80% or higher identity can be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0043] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of amino acid sequences, then the identity value (%) can be obtained.

[0044] The nucleic acid molecule described in D1) can be sequence 4.

[0045] To address the aforementioned technical problems, the present invention also provides a composition.

[0046] The composition comprises the aforementioned protein, acetylsuccione, syringaldehyde, and / or luteolin.

[0047] In the above description, the composition comprises the aforementioned protein and acetylsuccinone. The molar ratio of the protein to acetylsuccinone is 20 μg: 1 mmol.

[0048] The composition described above comprises the aforementioned protein and syringaldehyde. The molar ratio of the protein to syringaldehyde is 20 μg: 1 mmol.

[0049] The composition described above comprises the aforementioned protein and luteolin. The molar ratio of the protein to luteolin is 20 μg: 1 mmol.

[0050] To address the aforementioned technical problems, the present invention also provides the following applications.

[0051] The composition may be used in any of the following applications:

[0052] A1) Applications in the enzymatic hydrolysis of zearalenone or the preparation of enzymatically hydrolyzed zearalenone products;

[0053] A2) Applications in the enzymatic hydrolysis of aflatoxin B1 or in the preparation of enzymatically hydrolyzed aflatoxin B1 products;

[0054] A3) Applications in the enzymatic hydrolysis of ABTS or the preparation of enzymatically hydrolyzed ABTS products;

[0055] The composition includes the protein described above, and also includes at least one of acetylsuccinone, syringaldehyde, and luteolin.

[0056] To address the aforementioned technical problems, the present invention also provides a protein-microbial complex.

[0057] The complex comprises bacterial cells and the aforementioned protein, wherein the protein is adsorbed onto the surface of Bacillus subtilis cells.

[0058] In the above text, the adsorption can be electrostatic adsorption.

[0059] To address the aforementioned technical problems, this invention also provides a method for preparing protein-microbe complexes.

[0060] The method includes the step of mixing and incubating Bacillus subtilis spores with the aforementioned protein.

[0061] In the above text, the incubation condition is a pI value of 5. The buffer solution with a pI value of 5 can be a citrate buffer. The citrate buffer is a 10 mM citrate solution.

[0062] In the above text, the temperature for the mixed incubation is 25°C.

[0063] In the above text, the rotation speed for the mixed incubation is 250 rpm / min.

[0064] In the above text, the incubation time is 2 hours.

[0065] In the above text, the mass ratio of the protein in Bacillus subtilis spores is 0.01g:10μg.

[0066] The method described above also includes a supernatant removal step. The supernatant removal can be performed at a rate of 10,000 g, 4°C, for 5 minutes.

[0067] The above-described composite or the composite prepared by the above method may be used in any of the following applications:

[0068] A1) Applications in the enzymatic hydrolysis of zearalenone or the preparation of enzymatically hydrolyzed zearalenone products;

[0069] A2) Applications in the enzymatic hydrolysis of aflatoxin B1 or in the preparation of enzymatically hydrolyzed aflatoxin B1 products;

[0070] A3) Applications in the enzymatic hydrolysis of ABTS or the preparation of enzymatically hydrolyzed ABTS products.

[0071] To address the aforementioned technical problems, the present invention also provides an enzymatic hydrolysis method.

[0072] The method includes any of the following:

[0073] E1) includes the step of mixing and reacting the above-mentioned protein with zearalenone, aflatoxin B1 and / or ABTS;

[0074] E2) includes the step of mixing and reacting the above composition with zearalenone, aflatoxin B1 and / or ABTS;

[0075] E3) includes the step of mixing and reacting the above-mentioned protein-microbe complex or the complex prepared by the above method with zearalenone, aflatoxin B1 and / or ABTS.

[0076] In the above text, E1), the zearalenone, aflatoxin B1 and / or ABTS are any one of zearalenone, aflatoxin B1 and / or ABTS.

[0077] In the above text, the mass ratio of the protein to zearalenone is 20 μg: 2 μg.

[0078] In the above text, the mass ratio of the protein to aflatoxin B1 is 20 μg: 2 μg.

[0079] In the above text, E2), the zearalenone, aflatoxin B1 and / or ABTS are any one of zearalenone, aflatoxin B1 and / or ABTS.

[0080] In the above text, E3), the zearalenone, aflatoxin B1 and / or ABTS are any one of zearalenone, aflatoxin B1 and / or ABTS.

[0081] In the above text, the mass ratio of the protein-bacterial complex to zearalenone is 0.01 g: 2 μg.

[0082] In the above text, the mass ratio of the protein-bacterial complex to aflatoxin B1 is 0.01 g: 2 μg.

[0083] Beneficial effects

[0084] This invention discloses a laccase mutant, its preparation, and its application. The technical problem it solves is to provide an enzyme mutant with higher enzyme activity, and also to provide an enzyme-Bacillus subtilis complex that can make the enzyme activity more stable. Specifically, the protein disclosed is any one of the following: B1) a protein with the amino acid sequence shown in Sequence 3; B2) a protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in B1, possessing more than 80% identity and the same function as the protein shown in B1); B3) a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in B1) or B2). Using the above proteins alone can degrade zearalenone and aflatoxin B1. Alternatively, mixing them with Bacillus subtilis to prepare a complex can improve enzyme stability and can be used for industrial production. Attached Figure Description

[0085] Figure 1 Elution optimization of laccase mutant T480A-CotA under different concentrations of imidazole.

[0086] Figure 2 The specific activities of wild-type laccase Wt-CotA and its mutant T480A-CotA in degrading ABTS were measured at 37℃. Wt-CotA is the wild-type laccase, and T480A-CotA is the laccase mutant.

[0087] Figure 3 The degradation rate of 2 μg / mL ZEN and AFB1 by the laccase mutant T480A-CotA at different pH and 60℃ over 8 h was calculated.

[0088] Figure 4 The degradation rates of wild-type laccase Wt-CotA (at a concentration of 20 μg / mL) and its mutant T480A-CotA on 2 μg / mL ZEN and AFB1 were measured at 30 °C for 24 h.

[0089] Figure 5 The degradation rates of 10 μg / mL ZEN and AFB1 by the laccase mutant T480A-CotA at a concentration of 20 μg / mL and the natural mediator at 37℃ for 3 h are given.

[0090] Figure 6 The degradation activity of wild-type laccase Wt-CotA and its mutant T480A-CotA against ABTS was measured after standing at room temperature for 7 and 14 days at a concentration of 20 μg / mL. Detailed Implementation

[0091] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0092] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0093] The following examples used SPSS 11.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used. p < 0.05 (*) indicates a significant difference, p < 0.01 (**) indicates a highly significant difference, and p < 0.001 (***) indicates a highly significant difference.

[0094] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0095] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0096] The terms “include,” “including,” “have,” and “contain” are all open-ended terms, meaning that they include but are not limited to.

[0097] The term “gene” refers to a segment of DNA involved in the production of a polypeptide chain; it includes regions before and after the coding region (leader and tail regions) involved in the transcription / translation of the gene product and the regulation of said transcription / translation, as well as insertion sequences (introns) between individual coding regions (exons).

[0098] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. This term can be applied to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers.

[0099] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as subsequently modified amino acids such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. In this document, amino acids are represented using the commonly used three-letter or single-letter codes recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature. Similarly, nucleotides are represented using their generally accepted single-letter codes.

[0100] The term "Bacillus subtilis" refers to bacteria belonging to the kingdom Bacteria, family Bacillaceae, and genus Bacillus. Its CAS number is 68038-70-0.

[0101] The term "identity" is used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical...). Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including: Needleman et al. (1970) J. Mol. Biol. 48: 443, a homology alignment algorithm; Smith et al. (1981) Adv. Appl. Math. 2: 482, a local homology algorithm; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444, a similarity search method; and the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (19...). 97); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266: 460-480 (1996)); or GAP, BESTFIT, BLASTAltschul et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, MountainView, California.

[0102] The term "protein tag" refers to a specific amino acid sequence attached to the N-terminus or C-terminus of a target protein using gene recombination technology in genetic engineering. These tags are used to assist in the purification, detection, and functional studies of the target protein. Different protein tags have different functions and characteristics, and selecting an appropriate protein tag can significantly improve the efficiency of protein research. These include, but are not limited to, His tags, GST tags, MBP tags, HA tags, Myc tags, eGFP / eCFP / eYFP / mCherry tags, and SUMO tags.

[0103] The term "nucleic acid" or "nucleic acid molecule" refers to a polymer containing at least two deoxynucleotides or nucleotides in single or double strands. Unless specifically limited, the term covers nucleic acids containing known analogs of natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced by a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsukae et al., J. Biol. Chem. 260: 2605-2608 (1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)). A “nucleotide” contains a sugar, deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together by phosphate groups. "Bases" include purines and pyrimidines, further including natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that replace new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates (esters), and alkyl halides. DNA can exist as antisense, plasmid DNA, portions of plasmid DNA, pre-compressed DNA, polymerase chain reaction (PCR) products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups.

[0104] The term "coding gene" refers to a DNA sequence that can be transcribed into mRNA, or further translated into protein.

[0105] The term "reaction" refers to the environmental conditions or optimal environmental conditions provided by two or more substances to maintain the reaction state, allowing the reaction to proceed normally.

[0106] The term "enzymatic hydrolysis" refers to the process of breaking down large molecular compounds into smaller molecular compounds through enzyme-catalyzed decomposition. In enzymatic hydrolysis, enzymes act as biological catalysts, accelerating the rate of chemical reactions and breaking down large molecules into smaller ones. The enzyme in this application may be the protein T480A-CotA. The amino acid sequence of protein T480A-CotA is shown in Sequence 3.

[0107] The term "complex" refers to an aggregate of elements that combine to form a single entity and possess a specific function or characteristic. In this application, the complex is a complex composed of Bacillus subtilis and laccase. The laccase includes, but is not limited to, the laccase mutant T480A-CotA and laccase Wt-CotA. The amino acid sequence of the laccase mutant T480A-CotA includes, but is not limited to, sequence 3. The amino acid sequence of the laccase Wt-CotA includes, but is not limited to, sequence 1 (SEQ ID No. 1). The Bacillus subtilis includes, but is not limited to, Bacillus subtilis ZJ-2019-1. The accession number of Bacillus subtilis ZJ-2019-1 is CGMCC No. 23636. The Bacillus subtilis may be Bacillus subtilis spores.

[0108] The term "composition" refers to a formulation of multiple preparations.

[0109] The term "amplification product" refers to nucleic acid products produced through nucleic acid amplification technology.

[0110] The term "zearalenone" refers to the substance with CAS number 17924-92-4.

[0111] The substance referred to as “aflatoxin B1” has the CAS number 1162-65-8.

[0112] Bacillus subtilis ZJ-2019-1 is described in the patent application publication number CN113913340A (patent title: A strain of Bacillus subtilis and its application in the degradation of zearalenone, application number 202111316138.9), in which the name of Bacillus subtilis (BJ-2019-1-1) is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 23636.

[0113] Preservation Instructions

[0114] Strain name: Bacillus subtilis;

[0115] Latin name: Bacillus subtilis;

[0116] Strain number: ZJ-2019-1;

[0117] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0118] The abbreviation for the depository institution is CGMCC.

[0119] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0120] Date of deposit: October 21, 2021;

[0121] Registration number at the Preservation Center: CGMCC No. 23636.

[0122] Example 1: Construction of genetically engineered bacteria containing laccase mutants and those containing wild-type laccase

[0123] Construction of genetically engineered bacteria containing laccase mutants

[0124] 1. Obtaining the laccase mutant gene

[0125] This invention is based on CotA, an enzyme extracted from Bacillus subtilis ZJ-2019-1-1 (disclosed in CN113913340A, named Bacillus subtilis ZJ-2019-1 in the patent, with accession number CGMCC No. 23636), which was isolated, identified and preserved in our laboratory. The enzyme was subjected to molecular docking and molecular dynamics simulation with zearalenone and aflatoxin. After selecting random mutations and combining with literature, the 480th site, which is most likely to enhance activity, was selected for T-to-A mutation.

[0126] 2. Construction of engineered laccase mutant bacteria

[0127] The mutated laccase sequence was used, with pET-28a as the plasmid vector, NcoI and XhoI as endonucleases, and *E. coli* BL21 as the host cell. Synthesis was performed by a third-party company to obtain engineered *E. coli* bacteria containing the mutant enzyme gene. The specific method is as follows:

[0128] 1. Total RNA was extracted from Bacillus subtilis ZJ-2019-1 and reverse transcribed to obtain cDNA.

[0129] 2. Using the cDNA obtained in step 1 as a template, PCR amplification was performed using a primer pair composed of T480A-CotA-F and T480A-CotA-R under the action of the high-fidelity amplification enzyme PrimeSTAR. The PCR amplification product was recovered to obtain the PCR amplification product.

[0130] T480A-CotA-F: 5'-CATGCCATGGCATGATGACC-3' (Sequence 5; SEQ ID No. 5);

[0131] T480A-CotA-R: 5'-GGTCATCATGCCATGGCATG-3' (Sequence 6; SEQ ID No. 6).

[0132] PCR reaction program: 95℃ pre-denaturation for 5 min; 98℃ for 10 s, 55℃ for 30 s, 75℃ for 1.5 min, 35 cycles; 72℃ for 10 min.

[0133] 3. Take the PCR amplification product obtained in step 2, and perform double digestion with restriction endonucleases NcoI and XhoI. Recover the digested products to obtain the PCR amplification product digested product.

[0134] 4. Take the vector pET-28a, and perform double digestion with restriction endonucleases NcoI and XhoI. Recover the vector backbone to obtain the pET-28a digestion product.

[0135] 5. Ligate the PCR amplification product obtained in step 3 with the enzyme digestion product of the vector pET-28a obtained in step 4 to obtain the recombinant plasmid pET-28a-T480A-CotA.

[0136] The recombinant plasmid pET-28a-T480A-CotA is obtained by replacing the small fragment between the NcoI and XhoI recognition sites of the restriction endonuclease in the vector pET-28a(+) (Sangon Biotech (Shanghai), catalog number B540183) with the nucleotides shown in sequence 4 (SEQ ID No. 4) in the sequence listing, while keeping the other sequences of the vector pET-28a unchanged. The resulting recombinant vector is named recombinant plasmid pET-28a-T480A-CotA.

[0137] Sequence 4 (SEQ ID No. 4) is as follows:

[0138]

[0139] The nucleotide sequence of sequence 4 (SEQ ID No. 4) encodes a protein with the amino acid sequence of sequence 3 (SEQ ID No. 3), which is as follows:

[0140] MTLEKFVDALPIPDTLKPVQQSKEKTYYEVTMEECTHQLHRDLPPTRLWGYNGLFPGPTIEVKRNENVYVKWMNNLPSTHFLPIDHTIHHSDSQHEEPEVKTVVHLHGGVTPDDSDGYPEAWFSKDFE QTGPYFKREVYHYPNQQRGAILWYHDHAMALTRLNVYAGLVGAYIIHDPKEKRLKLPSDEYDVPLLITDRTINEDGSLFYPSAPENPSPSLPNPSIVPAFCGETILVNGKVWPYLEVEPRKYRFRVINA SNTRTYNLSLDNGGDFIQIGSDGGLLPRSVKLNSFSLAPAERYDIIIDFTAYEGESIILANSAGCGGDVNPETDANIMQFRVTKPLAQKDESRKPKYLASYPSVQHERIQNIRTLKLAGTQDEYGRPV LLLNNKRWHDPVTETPKVGTTEIWSIINPTRGTHPIHLHLVSFRVLDRRPFDIARYQESGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAAAFGPYSGRYVWHCHILEHEDYDMMRPMDITDPHK.

[0141] 6. Transform the recombinant plasmid pET-28a-T480A-CotA into E. coli BL21 to obtain BL21 / recombinant plasmid pET-28a-T480A-CotA. BL21 / recombinant plasmid pET-28a-T480A-CotA contains the recombinant plasmid.

[0142] pET-28a-T480A-CotA Escherichia coli BL21.

[0143] Construction of genetically engineered bacteria containing wild-type laccase

[0144] 1. Obtaining the wild-type laccase gene

[0145] This invention uses wild-type Bacillus subtilis preserved in this experiment.

[0146] 2. Construction of wild-type mutant engineered bacteria

[0147] The wild-type laccase sequence was used as a plasmid vector, pET-28a(+), and Escherichia coli BL21 was used as the host cell. The synthesis was commissioned to a third-party company to obtain engineered E. coli containing the wild-type enzyme gene. The specific method is as follows:

[0148] 1. Genomic DNA of Bacillus subtilis ZJ-2019-1 was extracted according to the instructions of the bacterial genomic DNA extraction kit from Tiangen (Beijing) Co., Ltd. A forward primer 5'-ATGACACTTGAAAAATTTGTGGATGC-3' (sequence 7; SEQ ID No. 7) and a reverse primer 5'-TTATTTATGGGGATCTGTTATATC-3' (sequence 8; SEQ ID No. 8) were designed.

[0149] 2. The target gene (Bacillus subtilis ZJ-2019-1 genomic DNA) was amplified by PCR. The PCR products were subjected to 1% agarose gel electrophoresis and recovered by a full-length gold agarose gel DNA recovery kit (PCR reaction program: 95℃ pre-denaturation for 5 min; 98℃ for 10 s, 55℃ for 30 s, 75℃ for 1.5 min, 35 cycles; 72℃ for 10 min).

[0150] 3. The original plasmid was digested with enzymes, and pET-28a(+) (Sangon Biotech (Shanghai)) was added to prepare the cloning reaction system. The reaction was carried out at room temperature for 5 min, and then the reaction system was placed in an ice bath to construct the recombinant plasmid.

[0151] 4. Transform the recombinant plasmid into *E. coli* DH5α for amplification. Transfer 10 μL of the resulting recombinant plasmid into 100 μL of competent *E. coli* BL21 cells, mix well, and incubate on ice for 30 min. Then, incubate at 42°C for 45 s, followed by an ice bath for 2 min, keeping the sample as still as possible during this process. Add the competent cells to antibiotic-free LB broth and incubate at 37°C for 1-2 h at 200 rpm / min. Then, spread 150 μL of the bacterial culture evenly onto LB agar plates containing 50 μg / mL ampicillin and incubate overnight at 37°C for positive transformant selection.

[0152] 5. Single colonies were picked from LB agar plates and cultured overnight in LB liquid medium containing 50 μg / mL ampicillin (Beyotime, CASST008). 1 μL of the bacterial culture was used for colony PCR (primers as above). Positive transformant plasmids verified by colony PCR were extracted. The extracted plasmids were divided into two parts. One part was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The results showed that the recombinant plasmid pET-28a-Wt-CotA was obtained. The recombinant plasmid pET-28a-Wt-CotA is obtained by replacing the small fragment between the NcoI and XhoI recognition sites of the vector pET-28a with the nucleotides shown in sequence 2 (SEQ ID No. 2) in the sequence listing, while keeping other sequences of the vector pET-28a unchanged. This recombinant vector was named recombinant plasmid pET-28a-Wt-CotA.

[0153] After sequencing verification confirmed the correct construction of the recombinant plasmid pET-28a-Wt-CotA, another portion of the plasmid was transformed into *E. coli* competent cells BL21 to express the His-tagged laccase Wt-CotA. This yielded *BL21* / recombinant plasmid pET-28a-Wt-CotA, which is *E. coli* BL21 containing the recombinant plasmid pET-28a-Wt-CotA.

[0154] Sequence 2 (SEQ ID No. 2) is as follows:

[0155]

[0156] The nucleotide sequence of sequence 2 encodes a protein with the amino acid sequence of sequence 1 (SEQ ID No. 1), which is as follows:

[0157] MTLEKFVDALPIPDTLKPVQQSKEKTYYEVTMEECTHQLHRDLPPTRLWGYNGLFPGPTIEVKRNENVYVKWMNNLPSTHFLPIDHTIHHSDSQHEEPEVKTVVHLHGGVTPDDSDGYPEAWFSKDFE QTGPYFKREVYHYPNQQRGAILWYHDHAMALTRLNVYAGLVGAYIIHDPKEKRLKLPSDEYDVPLLITDRTINEDGSLFYPSAPENPSPSLPNPSIVPAFCGETILVNGKVWPYLEVEPRKYRFRVINA SNTRTYNLSLDNGGDFIQIGSDGGLLPRSVKLNSFSLAPAERYDIIIDFTAYEGESIILANSAGCGGDVNPETDANIMQFRVTKPLAQKDESRKPKYLASYPSVQHERIQNIRTLKLAGTQDEYGRPV LLLNNKRWHDPVTETPKVGTTEIWSIINPTRGTHPIHLHLVSFRVLDRRPFDIARYQESGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAATFGPYSGRYVWHCHILEHEDYDMMRPMDITDPHK.

[0158] 6. Transform the recombinant plasmid pET-28a-Wt-CotA into Escherichia coli BL21 to obtain BL21 / recombinant plasmid pET-28a-Wt-CotA. BL21 / recombinant plasmid pET-28a-Wt-CotA is Escherichia coli BL2 containing the recombinant plasmid pET-28a-Wt-CotA.

[0159] The BL21 / recombinant plasmid pET-28a-Wt-CotA can express the Wt-CotA protein. The BL21 / recombinant plasmid pET-28a-T480A-CotA can express the T480A-CotA protein.

[0160] The coding gene for Wt-CotA protein (nucleotide sequence of Sequence 2 (SEQ ID No. 2)) was modified by mutating C (deoxycytosine nucleotide) at position 1440 to G (deoxyguanine nucleotide) and A (deoxyadenine nucleotide) at position 1438 to G (deoxyguanine nucleotide), thus obtaining the coding gene for T480A-CotA protein (nucleotide sequence of Sequence 4 (SEQ ID No. 4)). This resulted in a mutation of T (threonine) at position 480 of the Wt-CotA protein (amino acid sequence of Sequence 1) to A (alanine), yielding the T480A-CotA protein (amino acid sequence of Sequence 3).

[0161] Example 2: Expression of laccase-containing mutant genetically engineered bacteria and purification of mutant proteins

[0162] Successfully transformed BL21 / recombinant plasmid pET-28a-T480A-CotA and BL21 / recombinant plasmid pET-28a-Wt-CotA were picked and cultured overnight in 10 mL of liquid LB medium containing kanamycin (50 μg / mL). The samples were then transferred at a 1% inoculum to 300 mL of LB liquid medium containing 50 μg / mL kanamycin. The medium was then incubated on a 37°C shaker at 220 rpm. The absorbance value (OD) was measured... 600 When the concentration of α-carboxylic acid (α-carboxylic acid) was 0.6, IPTG (Solepro, CAS: 367-93-1) and CuSO4 (Sinopharm Reagent, CAS No. 7758-99-8) were added to a final concentration of 0.2 mmol / L, respectively. The temperature was lowered to 18℃ and the shaking speed was reduced to 150 rpm / min, and expression was induced for 16 h. The bacterial cell pellets of BL21 / recombinant plasmid pET-28a-T480A-CotA and BL21 / recombinant plasmid pET-28a-Wt-CotA were harvested by centrifugation (8,000×g, 4℃) for 25 min.

[0163] Purification of mutant proteins:

[0164] Six g of the above-mentioned bacterial cell pellets (BL21 / recombinant plasmid pET-28a-T480A-CotA and BL21 / recombinant plasmid pET-28a-Wt-CotA bacterial cell pellets) were resuspended in 100 mL of lysis buffer (25 mmol / L Tris, 500 mmol / L NaCl, pH 6.9), placed on ice for sonication to lyse the cells, and centrifuged (10,000×g, 4℃, 20 min) to remove cell debris. The supernatants were collected to obtain T480A-CotA and Wt-CotA protein supernatants. The N-terminus of the recombinant laccase mutant T480A-CotA and the N-terminus of the recombinant wild-type laccase Wt-CotA both contain a histidine tag (6×His). Ni 2+ -NTA pre-packed chromatographic columns were used to purify recombinant proteins using a fully automated protein purification system. Elution was performed using purification buffers (25 mmol / L Tris, 500 mmol / L NaCl, pH 6.9) containing different imidazole concentrations (15%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%). The target protein was eluted with imidazole concentrations ranging from 40% to 100%, as shown in the SDS-PAGE electrophoresis bands. Figure 1 ( Figure 1 The markers in the figure represent protein markers with sizes of 40, 60, and 80 kDa, where 15%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% represent different imidazole concentrations. T480A-CotA protein purification supernatant (eluted with 225 mM imidazole) and Wt-CotA protein purification supernatant (eluted with 225 mM imidazole) were obtained for subsequent experiments. The concentration of T480A-CotA protein in the T480A-CotA protein purification supernatant was 0.323 mg / mL, and the concentration of Wt-CotA protein in the Wt-CotA protein purification supernatant was 0.296 mg / mL.

[0165] Example 3: Enzymatic Properties Detection of Mutant T480A-CotA

[0166] Method for determining laccase activity using ABTS as a substrate:

[0167] Accurately weigh 274.3 mg of ABTS (Aladdin reagent, CAS No. A109612), add 10 mL of ultrapure water, vortex to mix, and then take 0.2 mL and add it to 7.8 mL of buffer solution containing the same solute as the laccase to be tested. Vortex to mix again to obtain the working solution. Take 1.6 mL of the working solution and add it to 0.4 mL of the test samples (T480A-CotA protein purification supernatant and Wt-CotA protein purification supernatant), mix quickly, and then measure the absorbance at 420 nm using a quartz cuvette (Shanghai Meipuda Instrument Co., Ltd., 10 mm). Calculate the absorbance difference between 0 min and 1 min and record it as the absorbance difference value.

[0168] Enzyme activity calculation formula = (enzyme dilution factor × 1000 × absorbance difference × total volume of enzyme activity assay system) / (36,000 mol) -1 ·L·cm -1 ×Reaction system enzyme liquid volume × Cuvette diameter (The cuvette is a quartz cuvette purchased from Shanghai Meipuda Instrument Co., Ltd., specification 10mm)). Enzyme specific activity = enzyme activity / enzyme concentration.

[0169] The results are as follows Figure 2 (The vertical axis represents specific activity (U / mg), T480A-CotA is the exogenous expressed protein of BL21 / recombinant plasmid pET-28a-T480A-CotA (T480A-CotA protein purification supernatant), and Wt-CotA is the exogenous expressed protein of BL21 / recombinant plasmid pET-28a-Wt-CotA (Wt-CotA protein purification supernatant)). As shown, the specific activity of T480A-CotA is 1.9, which is significantly higher than the specific activity of wild-type Wt-CotA (1.1).

[0170] Detection of AFB1 degradation rate and ZEN degradation rate:

[0171] The CAS number for ZEN (zearalenone) is 17924-92-4; the CAS number for AFB1 (aflatoxin B1) is 1162-65-8.

[0172] The high-performance liquid chromatography (HPLC) method for the detection of ZEN (zearalenone) and AFB1 (aflatoxin B1) is as follows:

[0173] Take 200 μL of the sample to be tested, add 600 μL of methanol, mix with a vortex mixer for 1 min, centrifuge the mixture at room temperature for 10 min (12,000 × g), extract the supernatant after centrifugation with a syringe, filter it through a 0.22 μm filter membrane, and then inject it into an HPLC sample vial for sample detection.

[0174] Specific HPLC detection conditions for AFB1:

[0175] 1. The chromatographic column was an Agilent SB-C18 (150mm × 4.6mm, 5μm);

[0176] 2. The mobile phase was methanol:acetonitrile:water = 22:22:56, the column temperature was 40℃, the injection volume was 10μL, and the flow rate was 1.0mL / min;

[0177] 3. Fluorescence detector: excitation wavelength (Ex) = 365nm, emission wavelength (Em) = 430nm.

[0178] AFB1 degradation rate (%) = (AFB1 peak area in control group - AFB1 peak area in experimental group) / AFB1 peak area in control group × 100%

[0179] ZEN HPLC Specific Detection Conditions

[0180] 1. The chromatographic column was an Agilent SB-C18 (150mm × 4.6mm, 5μm);

[0181] 2. The mobile phase was methanol:acetonitrile:water = 8:46:46, the column temperature was 30℃, the injection volume was 10μL, and the flow rate was 1.0mL / min;

[0182] 3. Fluorescence detector: Excitation wavelength (Ex) = 235 nm, emission wavelength (Em) = 460 nm

[0183] ZEN degradation rate (%) = (ZEN peak area of ​​control group - ZEN peak area of ​​experimental group) / ZEN peak area of ​​control group × 100%;

[0184] AFB1 degradation rate (%) = (AFB1 peak area in control group - AFB1 peak area in experimental group) / AFB1 peak area in control group × 100%.

[0185] A certain amount of AFB1 and ZEN were weighed and dissolved in methanol. AFB1 solution and ZEN solution were obtained separately. The concentration of AFB1 in the AFB1 solution was 30.4 mmol / L. The concentration of ZEN in the ZEN solution was 31.8 mmol / L.

[0186] AFB1 degradation:

[0187] Buffer preparation:

[0188] Citrate buffer: Prepare 0.01 mol / L citric acid and 0.01 mol / L sodium citrate solutions separately using deionized water, according to the relative molecular masses of citric acid and sodium citrate. Mix the 0.01 mol / L citric acid and sodium citrate solutions in an appropriate ratio to achieve final pH values ​​of 4.0 and 5.0, respectively. Autoclave and set aside for later use, obtaining pH3, pH4, and pH5 buffers. Phosphate buffer (PBS): Prepare 0.01 mol / L sodium dihydrogen phosphate and 0.01 mol / L disodium hydrogen phosphate solutions separately using deionized water, according to the relative molecular masses of sodium dihydrogen phosphate and disodium hydrogen phosphate. Mix the 0.01 mol / L sodium dihydrogen phosphate and disodium hydrogen phosphate solutions in an appropriate ratio to achieve final pH values ​​of 6.0, 7.0, and 8.0, respectively. Autoclave and set aside for later use, obtaining pH6, pH7, and pH8 buffers. Glycine-Sodium Hydroxide Buffer: Prepare 0.01 mol / L glycine solutions and 0.01 mol / L sodium hydroxide solutions separately using deionized water according to the relative molecular masses of glycine and sodium hydroxide. Mix the 0.01 mol / L glycine solutions and 0.01 mol / L sodium hydroxide solutions in an appropriate ratio to make the final pH values ​​9.0 and 10.0. Autoclave and set aside for later use to obtain buffer solutions with pH values ​​of 9.0 and 10.

[0189] All the above reagents were purchased from Sinopharm Reagent Center. Citric acid solution CAS number: 77-92-9; sodium citrate solution CAS number: 68-04-2; sodium dihydrogen phosphate CAS number: 7558-80-7; disodium hydrogen phosphate CAS number: 7558-79-4; glycine CAS number: 56-40-6; sodium hydroxide CAS number: 1310-73-2.

[0190] The experiment was divided into 7 groups: pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, and pH 10. The following procedures were performed on pH 4:

[0191] After adding pH 4 buffer to the AFB1 solution, T480A-CotA protein purification supernatant was added to make the final concentration of AFB1 0.00607 mmol / L, the final concentration of T480A-CotA 20 μg / mL, and the pH 4. The degradation rate (%) of AFB1 was detected after 3 h at 37℃ (the detection method is as shown above). The experiment was repeated three times.

[0192] pH5 group:

[0193] The only difference from the pH4 group is that the pH4 buffer is replaced with a pH5 buffer to make the pH 5. The rest of the operation is the same as the pH4 group.

[0194] pH6 group:

[0195] The only difference from the pH4 group is that the pH4 buffer is replaced with a pH6 buffer to make the pH 6. The rest of the operation is the same as the pH4 group.

[0196] pH7 group:

[0197] The only difference from the pH4 group is that the pH7 buffer is used instead of the pH4 buffer to make the pH 7. The rest of the operation is the same as the pH4 group.

[0198] pH8 group:

[0199] The only difference from the pH4 group is that the pH4 buffer is replaced with a pH8 buffer to make the pH 8. The rest of the operation is the same as the pH4 group.

[0200] pH9 group:

[0201] The only difference from the pH4 group is that the pH4 buffer is replaced with a pH9 buffer to make the pH 9. The rest of the operation is the same as the pH4 group.

[0202] pH10 group:

[0203] The only difference from the pH4 group is that the pH4 buffer is replaced with a pH10 buffer to make the pH 10. All other operations are the same as the pH4 group.

[0204] ZEN degradation:

[0205] The experiment was divided into 7 groups: pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, and pH 10. The following procedures were performed on pH 4:

[0206] After adding pH 4 buffer to the ZEN solution, T480A-CotA protein purification supernatant was added to make the final ZEN concentration 0.00636 mmol / L, the final T480A-CotA concentration 20 μg / mL, and the pH 4. The ZEN degradation rate (%) was detected after 3 h at 37℃ (the detection method is as shown above). The experiment was repeated three times.

[0207] pH5 group:

[0208] The only difference from the pH4 group is that the pH4 buffer is replaced with a pH5 buffer to make the pH 5. The rest of the operation is the same as the pH4 group.

[0209] pH6 group:

[0210] The only difference from the pH4 group is that the pH4 buffer is replaced with a pH6 buffer to make the pH 6. The rest of the operation is the same as the pH4 group.

[0211] pH7 group:

[0212] The only difference from the pH4 group is that the pH7 buffer is used instead of the pH4 buffer to make the pH 7. The rest of the operation is the same as the pH4 group.

[0213] pH8 group:

[0214] The only difference from the pH4 group is that the pH4 buffer is replaced with a pH8 buffer to make the pH 8. The rest of the operation is the same as the pH4 group.

[0215] pH9 group:

[0216] The only difference from the pH4 group is that the pH4 buffer is replaced with a pH9 buffer to make the pH 9. The rest of the operation is the same as the pH4 group.

[0217] pH10 group:

[0218] The only difference from the pH4 group is that the pH4 buffer is replaced with a pH10 buffer to make the pH 10. All other operations are the same as the pH4 group.

[0219] The results are as follows Figure 3 ( Figure 3 The left bar chart represents the AFB1 degradation rate (%), with the horizontal axis 4-10 corresponding to pH groups 4, 5, 6, 7, 8, 9, and 10, respectively; the right bar chart represents the ZEN degradation rate (%), with the horizontal axis 4-10 corresponding to pH groups 4, 5, 6, 7, 8, 9, and 10, respectively. The results indicate that T480A-CotA can effectively degrade ZEN and AFB1 between pH 7 and 10.

[0220] Comparative experiment on AFB1 degradation rate:

[0221] The experiment was divided into the Wt-CotA group and the T480A-CotA group. The T480A-CotA group underwent the following procedures:

[0222] After adding T480A-CotA protein purification supernatant and phosphate buffer at pH 7 (preparation method see Example 3) to the AFB1 solution, the final concentration of AFB1 was 0.00607 mmol / L, and the final concentration of Wt-CotA protein was 20 μg / mL. After 3 h at 37℃, the degradation rate (%) of AFB1 was detected (detection method as shown above). The experiment was repeated three times.

[0223] Wt-CotA group:

[0224] The only difference from the T480A-CotA group is that the T480A-CotA protein purification supernatant is replaced with the Wt-CotA protein purification supernatant; all other operations are the same as the T480A-CotA group.

[0225] Comparative experiment on ZEN degradation rate:

[0226] The experiment was divided into the Wt-CotA group and the T480A-CotA group. The T480A-CotA group underwent the following procedures:

[0227] After adding T480A-CotA protein purification supernatant and phosphate buffer at pH 7 (preparation method see Example 3) to the ZEN solution, the final ZEN concentration was 0.00636 mmol / L and the final Wt-CotA protein concentration was 20 μg / mL. After 3 h at 37℃, the ZEN degradation rate (%) was detected (detection method as shown above). The experiment was repeated three times.

[0228] Wt-CotA group:

[0229] The only difference from the T480A-CotA group is that the T480A-CotA protein purification supernatant is replaced with the Wt-CotA protein purification supernatant; all other operations are the same as the T480A-CotA group.

[0230] The results are as follows Figure 4 ( Figure 4 In the comparison experiment (AFB1 is the AFB1 degradation rate, ZEN is the ZEN degradation rate, Wt-CotA is the Wt-CotA group, and T480A-CotA is the T480A-CotA group), under the same conditions, T480A-CotA has a higher ability to degrade ZEN and AFB1 than wild-type Wt-CotA.

[0231] Example 4: Method for combining mutant T480A-CotA with natural mediators and bacterial spores

[0232] Instructions for using natural mediators to enhance the activity of the laccase mutant T480A-CotA:

[0233] A certain amount of acetylsuccine, syringaldehyde, or luteolin was weighed and dissolved in 50% methanol. Acetylsyringone solution, syringaldehyde solution, or luteolin solution were obtained respectively. The concentration of acetylsuccine in the acetylsuccine solution was 10 mmol / L. The concentration of syringaldehyde in the syringaldehyde solution was 10 mmol / L. The concentration of luteolin in the luteolin solution was 10 mmol / L.

[0234] AFB1 degradation:

[0235] The experiment was divided into four groups: acetylsuccione solution group, syringaldehyde solution group, luteolin solution group, and CK group. The acetylsuccione solution group underwent the following procedures:

[0236] After adding Wt-CotA protein purification supernatant and phosphate buffer at pH 7 (preparation method see Example 3) to the AFB1 solution, a certain amount of acetylsuccinone solution was added to bring the final concentration of acetylsuccinone to 1 mM, the final concentration of Wt-CotA protein to 20 μg / mL, and the final concentration of AFB1 to 20 mg / L. The degradation rate (%) of AFB1 was detected after 3 hours at 37℃ (detection method as shown above), and the experiment was repeated three times.

[0237] The eugenol solution group was operated on as follows:

[0238] The only difference from the acetyl eugenol solution group is that the acetyl eugenol solution is replaced with eugenol solution; the rest of the operation is the same as the acetyl eugenol solution group.

[0239] The luteolin solution group was operated on as follows:

[0240] The only difference from the acetylsyringone solution group is that the acetylsyringone solution is replaced with luteolin solution; the rest of the operation is the same as the acetylsyringone solution group.

[0241] CK group:

[0242] The only difference from the acetylsylgenone solution group was that the acetylsylgenone solution was replaced with distilled water; all other procedures were the same as the acetylsylgenone solution group. Results were as follows: Figure 5 As shown.

[0243] ZEN degradation:

[0244] The experiment was divided into four groups: acetylsuccione solution group, syringaldehyde solution group, luteolin solution group, and CK group. The acetylsuccione solution group underwent the following procedures:

[0245] After adding Wt-CotA protein purification supernatant and phosphate buffer (pH 7, preparation method as shown in Example 3) to the ZEN solution, a certain amount of acetylsuccione solution was added to bring the final concentration of acetylsuccione to 1 mM, the final concentration of Wt-CotA protein to 20 μg / mL, and the final concentration of ZEN to 20 mg / L. The ZEN degradation rate (%) was detected after 3 hours at 37°C (detection method as shown above), and the experiment was repeated three times.

[0246] The eugenol solution group was operated on as follows:

[0247] The only difference from the acetyl eugenol solution group is that the acetyl eugenol solution is replaced with eugenol solution; the rest of the operation is the same as the acetyl eugenol solution group.

[0248] The luteolin solution group was operated on as follows:

[0249] The only difference from the acetylsyringone solution group is that the acetylsyringone solution is replaced with luteolin solution; the rest of the operation is the same as the acetylsyringone solution group.

[0250] CK group:

[0251] The only difference from the acetylsylgenone solution group was that the acetylsylgenone solution was replaced with distilled water; all other procedures were the same as the acetylsylgenone solution group. Results were as follows: Figure 5 ( Figure 5 In the diagram, CK represents the CK group, acetylsuccione represents the acetylsuccione solution group, luteolin represents the luteolin solution group, and syringaldehyde represents the syringaldehyde solution group. Figure 5 The left bar chart shows the AFB1 degradation rate (%), and the right bar chart shows the ZEN degradation rate (%).

[0252] The results showed that all three mediators could degrade more than 70% of 10 μg / mL ZEN and AFB1 within 3 hours at 37℃ using 20 μg / mL T480A-CotA protein. Figure 5 As shown in the figure. Compared with the control group, the addition of acetylsuccinone, luteolin, and syringaldehyde increased the degradation rate of AFB1 by 76.5%, 67.8%, and 77.6%, respectively, and the degradation rate of ZEN by 60.4%, 64.7%, and 60.4%, respectively. Figure 5 ).

[0253] Example 5: Enhancing the stress resistance of the laccase mutant T480A-CotA using Bacillus subtilis ZJ-2019-1 spores.

[0254] A method for enhancing the stress resistance of the laccase mutant T480A-CotA using Bacillus subtilis ZJ-2019-1 spores:

[0255] Bacillus subtilis ZJ-2019-1 was inoculated into ordinary LB medium and cultured with shaking at 37℃ and 250 rpm / min for 12 h. After removing the supernatant by centrifugation (10,000×g, 4℃, 5 min), the cell pellet was dried at 60℃ for 2 h to obtain Bacillus subtilis spores.

[0256] A 10 mM citric acid solution (Sinopharm Reagent, CAS No. 77-92-9) and a sodium citrate solution (Sinopharm Reagent, CAS No. 68-04-2) were prepared. The pH of the citric acid solution was adjusted to 4-5 using the sodium citrate solution to prepare the citric acid buffer. 0.5 mL of the citric acid buffer was used to resuspend Bacillus subtilis spores, and 10 μg of the laccase mutant T480A-CotA (added as the supernatant from T480A-CotA protein purification) was added. The mixture was incubated at 25°C with shaking at 250 rpm / min for 2 h, followed by centrifugation (10,000 × g, 4°C, 5 min). After removing the supernatant, the laccase mutant T480A-CotA adsorbed by Bacillus subtilis spores was obtained. The experiment was repeated three times.

[0257] The only difference between this method and the one used to prepare the laccase mutant T480A-CotA by adsorbing Bacillus subtilis spores is that Bacillus subtilis spores are not added, thus obtaining the laccase mutant T480A-CotA for later use.

[0258] The laccase mutants T480A-CotA and T480A-CotA, adsorbed with Bacillus subtilis spores, were placed at room temperature for 7 and 14 days, respectively, and then the laccase activity was determined using ABTS as a substrate.

[0259] Accurately weigh 274.3 mg of ABTS (Aladdin Reagent, CAS No. A109612), add 10 mL of ultrapure water, vortex to mix, and then take 0.2 mL and add it to 7.8 mL of buffer solution containing the same solute as the laccase to be tested. Vortex to mix again to obtain the working solution. Take 1.6 mL of the working solution and add it to 0.4 mL of the test sample (laccase mutant T480A-CotA and laccase mutant T480A-CotA adsorbed by Bacillus subtilis spores). Mix quickly, and then measure the absorbance at 420 nm using a quartz cuvette (Shanghai Meipuda Instrument Co., Ltd., 10 mm). Calculate the absorbance difference between 0 min and 1 min and record it as the absorbance difference value.

[0260] Enzyme activity calculation formula = (enzyme dilution factor × 1,000 × absorbance difference × total volume of enzyme activity assay system) / (36,000 mol) -1 ·L·cm -1 ×Reaction system enzyme liquid volume × cuvette diameter). Enzyme specific activity = enzyme activity / enzyme concentration.

[0261] The results showed that after being placed at room temperature for 7 and 14 days, the laccase mutant T480A-CotA exhibited 0% activity in degrading ABTS, while the activity of the laccase mutant T480A-CotA after spore adsorption remained at 68.42% and 61.37%, respectively. Figure 6In this study, the enzyme was a laccase mutant T480A-CotA, and the immobilized enzyme was a laccase mutant T480A-CotA adsorbed onto Bacillus subtilis spores.

[0262] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The use of the composition in any of the following: A1) Applications in the enzymatic hydrolysis of zearalenone or the preparation of enzymatically hydrolyzed zearalenone products; A2) Applications in the enzymatic hydrolysis of aflatoxin B1 or in the preparation of enzymatically hydrolyzed aflatoxin B1 products; The composition includes protein and luteolin; The protein is any one of the following: B1) The amino acid sequence is that of the protein shown in sequence 3; B2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in B1).

2. An enzymatic hydrolysis method, characterized in that, The method includes the steps of mixing and reacting the composition of claim 1 with zearalenone and / or aflatoxin B1.

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