Laccase mutant as well as preparation and application thereof

Abstract

The invention discloses a laccase mutant as well as preparation and application thereof. The technical problem to be solved is to provide an enzyme mutant with higher enzyme activity and a complex formed by an enzyme with more stable enzyme activity and bacillus subtilis. The protein is any one of the following proteins: B1) a protein with an amino acid sequence shown as a sequence 3; b2) a protein which is obtained by substitution and / or deletion and / or addition of amino acid residues of the protein B1), has 80% or more of identity with the protein B1) and has the same function as the protein B1); and B3) a fusion protein obtained by connecting the N terminal or / and the C terminal of the protein B1) or B2) with a protein tag. When the protein is independently used, zearalenone and aflatoxin B1 can be degraded, or when the protein is mixed with bacillus subtilis to prepare a complex, the stability of enzyme can be improved, and the protein can be used for industrial production.

Description

Technical Field

[0001] The invention relates to the field of enzymes, and in particular to a laccase mutant and a preparation and application thereof. Background Art

[0002] Laccase (EC1.10.3.2) is a polyphenol oxidase containing four copper ions and belongs to the multi-copper oxidase (MCOs) family. The catalytic properties of laccases from different sources vary greatly. According to their sources, they are divided into animal and plant laccases and microbial laccases. Microbial laccases can be divided into fungal laccases and bacterial laccases. Laccases have the characteristics of high single-electron redox potential, strong catalytic activity, and a wide range of catalytic substrates. It can oxidatively degrade many harmful substances and has been used in food industry, dyes, decolorization, pulping, bleaching and papermaking wastewater treatment. Existing studies have found that laccase has great application potential in the degradation of mycotoxins such as zearalenone (abbreviated as ZEN) and aflatoxin B1 (abbreviated as AFB1). Therefore, how to provide a laccase with high degradation activity and stability is a technical problem faced by people in this field. Summary of the invention

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

[0004] In order to solve the above technical problems, the present invention provides the following applications.

[0005] The application of protein in any of the following:

[0006] A1) Application in enzymatic hydrolysis of zearalenone or preparation of enzymatic hydrolysis of zearalenone products;

[0007] A2) Application in enzymatic hydrolysis of aflatoxin B1 or preparation of enzymatic hydrolysis of aflatoxin B1 products;

[0008] A3) Application in enzymatic hydrolysis of ABTS or preparation of enzymatic hydrolysis ABTS products;

[0009] The protein is any of the following:

[0010] B1) The amino acid sequence is the protein shown in SEQ ID NO: 3;

[0011] B2) a protein having more than 80% identity with the protein shown in B1) and having the same function as the protein shown in B1) obtained by substitution and / or deletion and / or addition of amino acid residues of the protein shown in B1);

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

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

[0014] In the above-mentioned proteins, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

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

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

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

[0018] The C (deoxycytosine nucleotide) at position 1440 of the coding gene of the Wt-CotA protein (nucleotide sequence is sequence 2 (SEQ ID No. 2)) was mutated to G (deoxyguanine nucleotide), and the A (deoxyadenine nucleotide) at position 1438 was mutated to G (deoxyguanine nucleotide), and the coding gene of the T480A-CotA protein (nucleotide sequence is sequence 4 (SEQ ID No. 4)) was obtained. Thus, the T (threonine) at position 480 of the Wt-CotA protein (amino acid sequence is sequence 1) was mutated to A (alanine), and the T480A-CotA protein (amino acid sequence is sequence 3) was obtained.

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

[0020] In the above, the Bacillus subtilis may be Bacillus subtilis ZJ-2019-1-1.

[0021] In the above-mentioned protein, the protein is used in any of the following applications:

[0022] A1) Application in enzymatic hydrolysis of zearalenone or preparation of enzymatic hydrolysis of zearalenone products;

[0023] A2) Application in enzymatic hydrolysis of aflatoxin B1 or preparation of enzymatic hydrolysis of aflatoxin B1 products;

[0024] A3) Application in enzymatic hydrolysis of ABTS or preparation of enzymatic hydrolysis ABTS products.

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

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

[0027] In order to solve the above technical problems, the present invention also provides the following applications.

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

[0029] A1) Application in enzymatic hydrolysis of zearalenone or preparation of enzymatic hydrolysis of zearalenone products;

[0030] A2) Application in enzymatic hydrolysis of aflatoxin B1 or preparation of enzymatic hydrolysis of aflatoxin B1 products;

[0031] A3) Application in enzymatic hydrolysis of ABTS or preparation of enzymatic hydrolysis ABTS products;

[0032] The biological material is any of the following:

[0033] D1) a nucleic acid molecule encoding the above protein;

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

[0035] D3) 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) a recombinant microorganism containing the gene described in D2), or a recombinant microorganism containing the expression cassette described in D3), or a recombinant microorganism 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] D1) In the nucleic acid molecule, a person skilled in the art can easily mutate the nucleic acid molecule encoding the protein of the present invention by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 80% or more identity with the nucleotide sequence of the nucleic acid molecule encoding the protein isolated by the present invention and having the function of expressing the coding gene of the nucleic acid molecule encoding the protein are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

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

[0043] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching to calculate the identity of an amino acid sequence, the value (%) of identity can then be obtained.

[0044] D1) The nucleic acid molecule may be sequence 4.

[0045] In order to solve the above technical problems, the present invention also provides a composition.

[0046] The composition comprises the above protein, acetosyringone, syringaldehyde and / or luteolin.

[0047] In the above, the composition comprises the above protein and acetosyringone, wherein the amount ratio of the protein to acetosyringone is 20 μg: 1 mMol.

[0048] In the above, the composition comprises the above protein and syringaldehyde, wherein the mass ratio of the protein to syringaldehyde is 20 μg: 1 mMol.

[0049] In the above, the composition comprises the above protein and luteolin, wherein the amount ratio of the protein to luteolin is 20 μg: 1 mMol.

[0050] In order to solve the above technical problems, the present invention also provides the following applications.

[0051] The composition is used in any of the following:

[0052] A1) Application in enzymatic hydrolysis of zearalenone or preparation of enzymatic hydrolysis of zearalenone products;

[0053] A2) Application in enzymatic hydrolysis of aflatoxin B1 or preparation of enzymatic hydrolysis of aflatoxin B1 products;

[0054] A3) Application in enzymatic hydrolysis of ABTS or preparation of enzymatic hydrolysis ABTS products;

[0055] The composition comprises the above protein and at least one of acetosyringone, syringaldehyde and luteolin.

[0056] In order to solve the above technical problems, the present invention also provides a protein bacterial complex.

[0057] The complex comprises bacteria and the above protein, and the protein is adsorbed on the surface of Bacillus subtilis.

[0058] In the above, the adsorption may be electrostatic adsorption.

[0059] In order to solve the above technical problems, the present invention also provides a method for preparing the protein bacterial complex.

[0060] The method comprises the steps of mixing and incubating the Bacillus subtilis spores with the above-mentioned protein.

[0061] In the above, the mixed incubation condition is a PI value of 5. The buffer with a PI value of 5 can be a citric acid buffer. The citric acid buffer is a 10 mM citric acid solution.

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

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

[0064] In the above, the mixed incubation time is 2h.

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

[0066] In the above, the method further comprises a step of removing the supernatant, which can be performed at 10,000×g, 4° C., and 5 min.

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

[0068] A1) Application in enzymatic hydrolysis of zearalenone or preparation of enzymatic hydrolysis of zearalenone products;

[0069] A2) Application in enzymatic hydrolysis of aflatoxin B1 or preparation of enzymatic hydrolysis of aflatoxin B1 products;

[0070] A3) Application in enzymatic hydrolysis of ABTS or preparation of enzymatic hydrolysis ABTS products.

[0071] In order to solve the above technical problems, the present invention also provides an enzymatic hydrolysis method.

[0072] The method includes any of the following:

[0073] E1) comprising the steps of mixing and reacting the above protein with zearalenone, aflatoxin B1 and / or ABTS;

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

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

[0076] In the above, in E1), the zearalenone, aflatoxin B1 and / or ABTS is 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, in E2), the zearalenone, aflatoxin B1 and / or ABTS is any one of zearalenone, aflatoxin B1 and / or ABTS.

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

[0081] In the above, 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 0.01g:2μg.

[0083] Beneficial Effects

[0084] The present invention discloses a laccase mutant and its preparation and application, and solves the technical problem of providing an enzyme mutant with higher enzyme activity, and also provides an enzyme and Bacillus subtilis complex that can make the enzyme activity more stable. Specifically disclosed is a protein, and the protein is any of the following: B1) the amino acid sequence is the protein shown in sequence 3; B2) the protein described in B1 is obtained by replacing and / or deleting and / or adding amino acid residues, and has more than 80% identity with the protein shown in B1) and has the same function; B3) the N-terminus or / and C-terminus of the protein described in B1) or B2) is connected to a protein tag to obtain a fusion protein. The above protein can be used alone to degrade zearalenone and aflatoxin B1. Or it can be mixed with Bacillus subtilis to form a complex, which can improve the stability of the enzyme and can be used for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 To optimize the elution of laccase mutant T480A-CotA under different imidazole concentrations.

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

[0087] Figure 3 The degradation rate of 2 μg / mL ZEN and AFB1 by laccase mutant T480A-CotA at a concentration of 20 μg / mL at different pH and 60°C within 8 h.

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

[0089] Figure 5 The degradation rate of 10 μg / mL ZEN and AFB1 by 20 μg / mL laccase mutant T480A-CotA and natural mediator at 37°C for 3 h.

[0090] Figure 6 The degradation activity of wild-type laccase Wt-CotA and its mutant T480A-CotA on ABTS at a concentration of 20 μg / mL after being left at room temperature for 7 and 14 days. DETAILED DESCRIPTION

[0091] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0092] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0093] The following examples were processed using SPSS11.5 statistical software, and the experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used, and p < 0.05 (*) indicated a significant difference, p < 0.01 (**) indicated a very significant difference, and p < 0.001 (***) indicated a very significant difference.

[0094] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

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

[0096] The terms "include", "including", "have", "contain", etc. are all open terms, meaning including but not limited to.

[0097] The term "gene" refers to a segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer regions) that are involved in the transcription / translation of the gene product and the regulation of said transcription / translation, as well as intervening sequences (introns) between individual coding regions (exons).

[0098] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms can be applied to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers 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 mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are amino acids encoded by the genetic code, as well as subsequently modified amino acids, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. In this article, amino acids may be represented by the common three-letter codes or single-letter codes recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be represented by their generally accepted single-letter codes.

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

[0101] When the term "identity" is used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is 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 program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Science Institute, New York); Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (19 97); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, and include, but are 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., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

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

[0103] The term "nucleic acid" or "nucleic acid molecule" refers to a polymer that is present in a single or double-stranded form and comprises at least two deoxynucleotides or nucleotides. Unless specifically limited, the term encompasses nucleic acids comprising known analogs of natural nucleotides that have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise noted, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences as well as sequences that are explicitly noted. Specifically, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka 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" comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. The nucleotides are linked by the phosphate group. "Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that substitute new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and halogenated alkyls. The DNA can be present as antisense, plasmid DNA, a portion of plasmid DNA, pre-compressed DNA, a product of polymerase chain reaction (PCR), a vector (P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, chromosomal DNA, or a derivative of these groups.

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

[0105] The term "reaction" refers to providing environmental conditions or optimal environmental conditions for two or more substances to maintain a reaction state so that the reaction can proceed normally.

[0106] The term "enzymatic hydrolysis" refers to the process of decomposing macromolecular compounds into small molecular compounds by enzyme-catalyzed decomposition. In the enzymatic hydrolysis process, the enzyme acts as a biological catalyst to accelerate the speed of chemical reactions and decompose macromolecular substances into small molecular substances. The enzyme of the present application may be protein T480A-CotA. The amino acid sequence of protein T480A-CotA is shown in Sequence 3.

[0107] The term "complex" refers to a collection of elements that are combined with each other and have certain functions or obvious characteristics. In the present application, the complex is a complex composed of Bacillus subtilis and laccase combined with each other. The laccase includes but is not limited to 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 that shown in sequence 1 (SEQ ID No.1). The Bacillus subtilis includes but is not limited to Bacillus subtilis ZJ-2019-1. The deposit number of the Bacillus subtilis ZJ-2019-1 is CGMCCNo.23636. The Bacillus subtilis may be a Bacillus subtilis spore.

[0108] The term "composition" refers to a preparation of various substances.

[0109] The term "amplification product" refers to a nucleic acid product produced by a nucleic acid amplification technique.

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

[0111] The term "aflatoxin B1" refers to the substance with the CAS number 1162-65-8.

[0112] Bacillus subtilis ZJ-2019-1 is recorded in a patent with application publication number CN113913340A (patent name: a strain of Bacillus subtilis and its application in degrading zearalenone, application number: 202111316138.9). The name in the patent is Bacillus subtilis ZJ-2019-1-1, and it is deposited in the General Microbiology Center of the China National Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 23636.

[0113] Collection Instructions

[0114] Bacteria species name: Bacillus subtilis;

[0115] Latin name: Bacillus subtilis;

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

[0117] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0118] Abbreviation of depository institution: CGMCC;

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

[0120] Deposit date: October 21, 2021;

[0121] The registration number of the Collection Center is: CGMCC No.23636.

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

[0123] Construction of genetically engineered bacteria containing laccase mutations

[0124] 1. Obtaining laccase mutant genes

[0125] The present invention is based on CotA obtained by heterologously expressing an enzyme extracted from Bacillus subtilis ZJ-2019-1-1 (Bacillus subtilis disclosed in CN113913340A, the name in the patent is Bacillus subtilis ZJ-2019-1, and its preservation number is CGMCC No. 23636) isolated, identified and preserved in this laboratory. The enzyme is molecularly docked and molecular dynamics simulated with zearalenone and aflatoxin, and random mutations are selected and combined with literature to select the 480th site that is most likely to improve activity for mutation from T to A.

[0126] 2. Construction of laccase mutant engineered bacteria

[0127] The mutant laccase sequence was synthesized by entrusting a third party company with pET-28a as a plasmid vector, NcoI and XhoI as endonucleases, and Escherichia coli BL21 as a host cell to obtain an engineered Escherichia coli containing the mutant enzyme gene. The specific method is as follows:

[0128] 1. Extract total RNA from Bacillus subtilis ZJ-2019-1 and reverse transcribe it to obtain cDNA.

[0129] 2. Using the cDNA obtained in step 1 as a template, using a primer pair consisting of T480A-CotA-F and T480A-CotA-R, perform PCR amplification under the action of high-fidelity amplification enzyme PrimeSTAR, recover the PCR amplification product, and 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: pre-denaturation at 95°C for 5 min; 98°C for 10 s, 55°C for 30 s, 75°C for 1.5 min, 35 cycles; 72°C for 10 min.

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

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

[0135] 5. The PCR amplification product obtained in step 3 and the vector pET-28a digestion product obtained in step 4 were connected 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 restriction endonuclease NcoI recognition site and the XhoI recognition site of the vector pET-28a(+) (Shanghai Bioengineering, catalog number B540183) with the nucleotides shown in sequence 4 (SEQ ID No.4) in the sequence table, while keeping the other sequences of the vector pET-28a unchanged, and the obtained 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) can encode a protein having an amino acid sequence of sequence 3 (SEQ ID No.3), and sequence 3 is specifically as follows:

[0140] MTLEKFVDALPIPDTLKPVQQSKEKTYYEVTMEECTHQLHRDLPPTRLWGYNGLFPGPTIEVKRNENVYVKWMNNLPSTHFLPIDHTIHHSDSQHEEPEVKTVVHLHGGVTPDDSDGYPEAWFSKDFE QTGPYFKREVYHYPNQQRGAILWYHDHAMALTRLNVYAGLVGAYIIHDPKEKRLKLPSDEYDVPLLITDRTINEDGSLFYPSAPENPSPSLPNPSIVPAFCGETILVNGKVWPYLEVEPRKYRFRVINA SNTRTYNLSLDNGGDFIQIGSDGGLLPRSVKLNSFSLAPAERYDIIIDFTAYEGESIILANSAGCGGDVNPETDANIMQFRVTKPLAQKDESRKPKYLASYPSVQHERIQNIRTLKLAGTQDEYGRPV LLLNNKRWHDPVTETPKVGTTEIWSIINPTRGTHPIHLHLVSFRVLDRRPFDIARYQESGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAAAFGPYSGRYVWHCHILEHEDYDMMRPMDITDPHK.

[0141] 6. The recombinant plasmid pET-28a-T480A-CotA was transferred into Escherichia coli BL21 to obtain BL21 / recombinant plasmid pET-28a-T480A-CotA. BL21 / recombinant plasmid pET-28a-T480A-CotA is a recombinant plasmid containing

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

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

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

[0145] The present invention is based on the wild-type Bacillus subtilis preserved in this experiment.

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

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

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

[0149] 2. The target gene (Bacillus subtilis ZJ-2019-1 genomic DNA) was amplified by PCR amplification technology. The PCR amplification product was subjected to 1% agarose gel electrophoresis, and the PCR product was recovered by full-form gold agarose gel DNA recovery kit (PCR reaction procedure: 95°C pre-denaturation for 5 min; 98°C for 10 s, 55°C for 30 s, 75°C for 1.5 min, 35 cycles; 72°C for 10 min).

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

[0151] 4. Transfer the recombinant plasmid into E. coli DH5α for amplification. Transfer 10 μL of the obtained recombinant plasmid into 100 μL of E. coli competent BL21, mix well and place in ice bath for 30 minutes, then place in 42°C water bath for 45 seconds, and then place in ice bath for 2 minutes. Try to keep the sample still during this period. Add the competent cells to LB liquid culture medium without antibiotics, incubate at 200 rpm / min, 37°C for 1-2 hours, then take 150 μL of bacterial solution and evenly spread it on LB solid culture medium plate containing 50 μg / mL ampicillin, invert at 37°C overnight, and screen positive transformants.

[0152] 5. Pick a single colony on the LB plate and culture it overnight in LB liquid medium containing 50 μg / mL ampicillin (Biyuntian, CASST008), take 1 μL of bacterial solution for colony PCR (primers as above), extract the positive transformant plasmid verified by colony PCR, and divide the extracted plasmid into two parts, one part is sent to Shanghai Shenggong Biological Company for sequencing, and the test results show that the recombinant plasmid pET-28a-Wt-CotA is obtained. The recombinant plasmid pET-28a-Wt-CotA is a small fragment between the restriction endonuclease NcoI recognition site and the XhoI recognition site of the vector pET-28a replaced with the nucleotide shown in sequence 2 (SEQ ID No. 2) in the sequence table, and the other sequences of the vector pET-28a are kept unchanged, and the obtained recombinant vector is named recombinant plasmid pET-28a-Wt-CotA.

[0153] After sequencing verified that the recombinant plasmid pET-28a-Wt-CotA was constructed correctly, the other part of the plasmid was transformed into the E. coli competent cell BL21 to express the His-tagged laccase Wt-CotA. BL21 / recombinant plasmid pET-28a-Wt-CotA was obtained, and BL21 / recombinant plasmid pET-28a-Wt-CotA was the 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 can encode a protein having an amino acid sequence of sequence 1 (SEQ ID No. 1), and sequence 1 is specifically as follows:

[0157] MTLEKFVDALPIPDTLKPVQQSKEKTYYEVTMEECTHQLHRDLPPTRLWGYNGLFPGPTIEVKRNENVYVKWMNNLPSTHFLPIDHTIHHSDSQHEEPEVKTVVHLHGGVTPDDSDGYPEAWFSKDFE QTGPYFKREVYHYPNQQRGAILWYHDHAMALTRLNVYAGLVGAYIIHDPKEKRLKLPSDEYDVPLLITDRTINEDGSLFYPSAPENPSPSLPNPSIVPAFCGETILVNGKVWPYLEVEPRKYRFRVINA SNTRTYNLSLDNGGDFIQIGSDGGLLPRSVKLNSFSLAPAERYDIIIDFTAYEGESIILANSAGCGGDVNPETDANIMQFRVTKPLAQKDESRKPKYLASYPSVQHERIQNIRTLKLAGTQDEYGRPV LLLNNKRWHDPVTETPKVGTTEIWSIINPTRGTHPIHLHLVSFRVLDRRPFDIARYQESGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAATFGPYSGRYVWHCHILEHEDYDMMRPMDITDPHK.

[0158] 6. The recombinant plasmid pET-28a-Wt-CotA was transferred into Escherichia coli BL21 to obtain BL21 / recombinant plasmid pET-28a-Wt-CotA. BL21 / recombinant plasmid pET-28a-Wt-CotA is Escherichia coli BL 2 containing the recombinant plasmid pET-28a-Wt-CotA.

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

[0160] The C (deoxycytosine nucleotide) at position 1440 of the coding gene of the Wt-CotA protein (nucleotide sequence is sequence 2 (SEQ ID No. 2)) was mutated to G (deoxyguanine nucleotide), and the A (deoxyadenine nucleotide) at position 1438 was mutated to G (deoxyguanine nucleotide), and the coding gene of the T480A-CotA protein (nucleotide sequence is sequence 4 (SEQ ID No. 4)) was obtained. Thus, the T (threonine) at position 480 of the Wt-CotA protein (amino acid sequence is sequence 1) was mutated to A (alanine), and the T480A-CotA protein (amino acid sequence is sequence 3) was obtained.

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

[0162] The successfully transformed BL21 / recombinant plasmid pET-28a-T480A-CotA and BL21 / recombinant plasmid pET-28a-Wt-CotA were picked up, cultured overnight in 10 mL of liquid LB medium containing kanamycin (50 μg / mL), and the samples were transferred to 300 mL of LB liquid medium containing 50 μg / mL kanamycin at a 1% inoculum. Then, they were placed on a constant temperature shaker at 37°C and shaken at a speed of 220 rpm / min. When the absorbance value (OD 600 ) was 0.6, IPTG (Solaibao, CAS: 367-93-1) and CuSO4 (Sinopharm, CAS No. 7758-99-8) were added at a final concentration of 0.2 mmol / L, and the temperature was lowered to 18°C, the shaking speed was reduced to 150 rpm / min, and the expression was induced for 16 hours. The BL21 / recombinant plasmid pET-28a-T480A-CotA and BL21 / recombinant plasmid pET-28a-Wt-CotA bacterial precipitates were harvested by centrifugation (8,000×g, 4°C) for 25 minutes.

[0163] Purification of mutant proteins:

[0164] 6 g of the above bacterial precipitates (BL21 / recombinant plasmid pET-28a-T480A-CotA and BL21 / recombinant plasmid pET-28a-Wt-CotA bacterial precipitates) were resuspended in 100 mL lysis buffer (25 mmol / L Tris, 500 mmol / L NaCl, pH 6.9), placed on ice for ultrasonic treatment, cell disruption, and centrifugation (10,000 × g, 4 ° C, 20 min) to remove cell debris, and the supernatant was taken to obtain T480A-CotA protein supernatant and Wt-CotA protein supernatant. The N-terminus of the recombinant laccase mutant T480A-CotA carries a histidine tag (6×His) and the N-terminus of the recombinant laccase wild-type Wt-CotA carries a histidine tag (6×His), and Ni was used to 2+ -NTA pre-packed chromatographic column was combined and the recombinant protein was purified in a fully automatic protein purification system. The target protein was eluted with purification buffer (25mmol / LTris, 500mmol / L NaCl, pH 6.9) containing different imidazole concentrations (15%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%). 40%-100% imidazole can elute the target protein. The SDS-PAGE electrophoresis band is as follows Figure 1 ( Figure 1 The marker is a protein maker, and its size is 40, 60 and 80 kDa, respectively, wherein 15%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% represent different imidazole concentrations, respectively). The purified supernatant of T480A-CotA protein (eluted with 225 mM imidazole) and the purified supernatant of Wt-CotA protein (eluted with 225 mM imidazole) were obtained for subsequent experiments. In the purified supernatant of T480A-CotA protein, the concentration of T480A-CotA protein was 0.323 mg / mL, and in the purified supernatant of Wt-CotA protein, the concentration of Wt-CotA protein was 0.296 mg / mL.

[0165] Example 3 Detection of enzymatic properties of mutant T480A-CotA

[0166] Laccase activity assay using ABTS as substrate:

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

[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 solution volume × cuvette diameter (the cuvette is a quartz cuvette purchased from Shanghai Meipuda Instrument Co., Ltd., with a specification of 10 mm)). 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 an exogenously expressed protein from BL21 / recombinant plasmid pET-28a-T480A-CotA (purified supernatant of T480A-CotA protein); and Wt-CotA is an exogenously expressed protein from BL21 / recombinant plasmid pET-28a-Wt-CotA (purified supernatant of Wt-CotA protein)). As shown in FIG. 1 , the specific activity of T480A-CotA is 1.9, which is significantly higher than the specific activity of the wild-type Wt-CotA of 1.1).

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

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

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

[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 through a 0.22 μm filter membrane, and then inject into an HPLC sample bottle for sample detection.

[0174] Specific HPLC detection conditions of AFB1:

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

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

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

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

[0179] Specific HPLC detection conditions of ZEN

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

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

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

[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 of ​​control group - AFB1 peak area of ​​experimental group) / AFB1 peak area of ​​control group × 100%.

[0185] A certain amount of AFB1 and ZEN were weighed and dissolved in methanol to obtain AFB1 solution and ZEN solution, respectively. In the AFB1 solution, the concentration of AFB1 was 30.4 mMol / L. In the ZEN solution, the concentration of ZEN was 31.8 mMol / L.

[0186] Degradation of AFB1:

[0187] Buffer configuration:

[0188] Citric acid buffer: According to the relative molecular weights of citric acid and sodium citrate, 0.01mol / L citric acid solution and 0.01mol / L sodium citrate solution were prepared with deionized water, and 0.01mol / L citric acid solution and 0.01mol / L sodium citrate solution were mixed in appropriate proportions to make the final pH values ​​4.0 and 5.0, and then sterilized by high pressure for use, to obtain pH3 buffer, pH4 buffer and pH5 buffer, respectively. Phosphate buffer (PBS): According to the relative molecular weights of sodium dihydrogen phosphate and disodium hydrogen phosphate, 0.01mol / L sodium dihydrogen phosphate solution and 0.01mol / L disodium hydrogen phosphate solution were prepared with deionized water, and 0.01mol / L sodium dihydrogen phosphate solution and 0.01mol / L disodium hydrogen phosphate solution were mixed in appropriate proportions to make the final pH values ​​6.0, 7.0 and 8.0, and then sterilized by high pressure for use, to obtain pH6 buffer, pH7 buffer and pH8 buffer, respectively. Glycine-sodium hydroxide buffer: According to the relative molecular masses of glycine and sodium hydroxide, use deionized water to prepare 0.01 mol / L glycine solution and 0.01 mol / L sodium hydroxide solution respectively, mix 0.01 mol / L glycine solution and 0.01 mol / L sodium hydroxide solution in appropriate proportions to make the final pH values ​​​​9.0 and 10.0, sterilize by high pressure, and set aside to obtain buffer solutions of pH 9.0 and 10.

[0189] The above reagents were purchased from Sinopharm Reagents, citric acid solution CAS No. 77-92-9, sodium citrate solution CAS No. 68-04-2, sodium dihydrogen phosphate CAS No. 7558-80-7, disodium hydrogen phosphate CAS No. 7558-79-4, glycine CAS No. 56-40-6, sodium hydroxide CAS No. 1310-73-2)

[0190] The experiment was divided into 7 groups, pH4 group, pH5 group, pH6 group, pH7 group, pH8 group, pH9 group and pH10 group. The pH4 group was subjected to the following operations:

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

[0192] pH5 group:

[0193] The only difference from the pH 4 group was that the pH 5 buffer was used to replace the pH 4 buffer to make the pH 5, and the rest of the operations were the same as the pH 4 group.

[0194] pH6 group:

[0195] The only difference from the pH 4 group was that the pH 4 buffer was replaced with a pH 6 buffer to make the pH 6, and the rest of the operations were the same as the pH 4 group.

[0196] pH7 group:

[0197] The only difference from the pH 4 group was that the pH 7 buffer was used to replace the pH 4 buffer to make the pH 7, and the rest of the operations were the same as the pH 4 group.

[0198] pH8 group:

[0199] The only difference from the pH 4 group was that the pH 8 buffer was used to replace the pH 4 buffer to make the pH 8, and the rest of the operations were the same as the pH 4 group.

[0200] pH9 group:

[0201] The only difference from the pH 4 group was that the pH 9 buffer was used to replace the pH 4 buffer to make the pH 9, and the rest of the operations were the same as the pH 4 group.

[0202] pH10 group:

[0203] The only difference from the pH 4 group was that the pH 10 buffer was used to replace the pH 4 buffer to make the pH 10, and the rest of the operations were the same as the pH 4 group.

[0204] Degradation of ZEN:

[0205] The experiment was divided into 7 groups, pH4 group, pH5 group, pH6 group, pH7 group, pH8 group, pH9 group and pH10 group. The pH4 group was subjected to the following operations:

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

[0207] pH5 group:

[0208] The only difference from the pH 4 group was that the pH 5 buffer was used to replace the pH 4 buffer to make the pH 5, and the rest of the operations were the same as the pH 4 group.

[0209] pH6 group:

[0210] The only difference from the pH 4 group was that the pH 4 buffer was replaced with a pH 6 buffer to make the pH 6, and the rest of the operations were the same as the pH 4 group.

[0211] pH7 group:

[0212] The only difference from the pH 4 group was that the pH 7 buffer was used to replace the pH 4 buffer to make the pH 7, and the rest of the operations were the same as the pH 4 group.

[0213] pH8 group:

[0214] The only difference from the pH 4 group was that the pH 8 buffer was used to replace the pH 4 buffer to make the pH 8, and the rest of the operations were the same as the pH 4 group.

[0215] pH9 group:

[0216] The only difference from the pH 4 group was that the pH 9 buffer was used to replace the pH 4 buffer to make the pH 9, and the rest of the operations were the same as the pH 4 group.

[0217] pH10 group:

[0218] The only difference from the pH 4 group was that the pH 10 buffer was used to replace the pH 4 buffer to make the pH 10, and the rest of the operations were the same as the pH 4 group.

[0219] The results are as follows Figure 3 ( Figure 3 The left bar graph shows the degradation rate of AFB1 (%), and the horizontal axes 4-10 correspond to pH4 group, pH5 group, pH6 group, pH7 group, pH8 group, pH9 group and pH10 group, respectively; the right bar graph shows the degradation rate of ZEN (%), and the horizontal axes 4-10 correspond to pH4 group, pH5 group, pH6 group, pH7 group, pH8 group, pH9 group and pH10 group, respectively. The results show that T480A-CotA can effectively degrade ZEN and AFB1 between pH 7-10.

[0220] Comparative experiment of AFB1 degradation rate:

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

[0222] After adding the purified supernatant of T480A-CotA protein 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° C., the degradation rate (%) of AFB1 was detected (detection method as shown above), and the experiment was repeated three times.

[0223] Wt-CotA group:

[0224] The only difference from the T480A-CotA group was that the T480A-CotA protein purification supernatant was replaced with the Wt-CotA protein purification supernatant, and the rest of the operations were the same as the T480A-CotA group.

[0225] Comparative experiment of ZEN degradation rate:

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

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

[0228] Wt-CotA group:

[0229] The only difference from the T480A-CotA group was that the T480A-CotA protein purification supernatant was replaced with the Wt-CotA protein purification supernatant, and the rest of the operations were the same as the T480A-CotA group.

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

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

[0232] Method for enhancing the activity of laccase mutant T480A-CotA using natural mediators:

[0233] A certain amount of acetosyringone, syringaldehyde or luteolin was weighed and dissolved in 50% methanol. An acetosyringone solution, a syringaldehyde solution or a luteolin solution was obtained. In the acetosyringone solution, the concentration of acetosyringone was 10 mMol / L. In the syringaldehyde solution, the concentration of syringaldehyde was 10 mMol / L. In the luteolin solution, the concentration of luteolin was 10 mMol / L.

[0234] Degradation of AFB1:

[0235] The experiment was divided into 4 groups: acetosyringone solution group, syringaldehyde solution group, luteolin solution group and CK group. The acetosyringone solution group underwent the following operations:

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

[0237] The syringaldehyde solution was used to perform the following operations:

[0238] The only difference from the acetosyringone solution group is that the acetosyringone solution is replaced by syringaldehyde solution, and the rest of the operations are the same as those of the acetosyringone solution group.

[0239] The luteolin solution group was subjected to the following operations:

[0240] The only difference from the acetosyringone solution group was that the acetosyringone solution was replaced with luteolin solution, and the rest of the operations were the same as those of the acetosyringone solution group.

[0241] CK group:

[0242] The only difference from the acetosyringone solution group is that the acetosyringone solution was replaced with distilled water, and the rest of the operations were the same as the acetosyringone solution group. Figure 5 shown.

[0243] Degradation of ZEN:

[0244] The experiment was divided into 4 groups: acetosyringone solution group, syringaldehyde solution group, luteolin solution group and CK group. The acetosyringone solution group underwent the following operations:

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

[0246] The syringaldehyde solution was used to perform the following operations:

[0247] The only difference from the acetosyringone solution group is that the acetosyringone solution is replaced by syringaldehyde solution, and the rest of the operations are the same as those of the acetosyringone solution group.

[0248] The luteolin solution group was subjected to the following operations:

[0249] The only difference from the acetosyringone solution group was that the acetosyringone solution was replaced with luteolin solution, and the rest of the operations were the same as those of the acetosyringone solution group.

[0250] CK group:

[0251] The only difference from the acetosyringone solution group is that the acetosyringone solution was replaced with distilled water, and the rest of the operations were the same as the acetosyringone solution group. Figure 5 ( Figure 5 In the experiment, CK was the CK group, acetosyringone was the acetosyringone solution group, luteolin was the luteolin solution group, and syringaldehyde was the syringaldehyde solution group. Figure 5 The left bar graph shows the degradation rate of AFB1 (%), and the right bar graph shows the degradation rate of ZEN (%).

[0252] The results showed that all three mediators could degrade 10 μg / mL ZEN and AFB1 by more than 70% within 3 h at 37°C with 20 μg / mL T480A-CotA protein. Figure 5 Compared with the control group, the degradation rates of AFB1 were increased by 76.5%, 67.8% and 77.6% after adding acetosyringone, luteolin and syringaldehyde, respectively, and the degradation rates of ZEN were increased by 60.4%, 64.7% and 60.4%, respectively ( Figure 5 ).

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

[0254] Method for improving stress resistance of 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 at 37°C and 250 rpm / min for 12 hours. After removing the supernatant by centrifugation (10,000×g, 4°C, 5 min), the cell pellet was dried at 60°C for 2 hours to obtain Bacillus subtilis spores.

[0256] Prepare 10mM citric acid solution (Sinopharm Reagent, CAS No. 77-92-9) and sodium citrate solution (Sinopharm Reagent CAS No. 68-04-2), use sodium citrate solution to adjust the citric acid solution to pH 4-5 and use it as citric acid buffer for standby use, take 0.5mL citric acid buffer, resuspend the spores of Bacillus subtilis, add 10μg of laccase mutant T480A-CotA (added in the form of T480A-CotA protein purified supernatant), shake culture at 250rpm / min at 25℃ for 2h, and centrifuge (10,000×g, 4℃, 5min). After removing the supernatant, the laccase mutant T480A-CotA adsorbed by Bacillus subtilis spores was obtained, and the experiment was repeated three times.

[0257] The method for preparing the laccase mutant T480A-CotA adsorbed by Bacillus subtilis spores is different from the method for preparing the laccase mutant T480A-CotA adsorbed by Bacillus subtilis spores in that the laccase mutant T480A-CotA is obtained without adding Bacillus subtilis spores for standby use.

[0258] The laccase activity of the mutant T480A-CotA and the mutant T480A-CotA adsorbed by Bacillus subtilis spores was determined at room temperature for 7 and 14 days using ABTS as substrate:

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

[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 solution volume × colorimetric cup 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 ABTS degradation activity of the laccase mutant T480A-CotA was 0%, while the activity of the laccase mutant T480A-CotA after spore adsorption was still maintained at 68.42% and 61.37% ( Figure 6In the present invention, the enzyme is a laccase mutant T480A-CotA, and the immobilized enzyme is a laccase mutant T480A-CotA adsorbed by Bacillus subtilis spores).

[0262] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. The application of protein in any of the following: A1) Application in enzymatic hydrolysis of zearalenone or preparation of enzymatic hydrolysis of zearalenone products; A2) Application in enzymatic hydrolysis of aflatoxin B1 or preparation of enzymatic hydrolysis of aflatoxin B1 products; A3) Application in enzymatic hydrolysis of ABTS or preparation of enzymatic hydrolysis ABTS products; The protein is any of the following: B1) The amino acid sequence is the protein shown in SEQ ID NO: 3; B2) a protein having more than 80% identity with the protein shown in B1) and having the same function as the protein shown in B1) obtained by substitution and / or deletion and / or addition of amino acid residues of the protein shown in B1); B3) A fusion protein obtained by connecting the N-terminus and / or C-terminus of the protein described in B1) or B2) to a protein tag.

2. The use according to claim 1, characterized in that: The protein is derived from Bacillus subtilis.

3. Application of biomaterials in any of the following: A1) Application in enzymatic hydrolysis of zearalenone or preparation of enzymatic hydrolysis of zearalenone products; A2) Application in enzymatic hydrolysis of aflatoxin B1 or preparation of enzymatic hydrolysis of aflatoxin B1 products; A3) Application in enzymatic hydrolysis of ABTS or preparation of enzymatic hydrolysis ABTS products; The biological material is any of the following: D1) A nucleic acid molecule encoding the protein according to claim 1; D2) expressing the gene encoding the nucleic acid molecule described in D1); D3) an expression cassette containing the gene described in D2); D4) a recombinant vector containing the gene described in D2) or a recombinant vector containing the expression cassette described in D3); D5) A recombinant microorganism containing the gene described in D2), or a recombinant microorganism containing the expression cassette described in D3), or a recombinant microorganism containing the recombinant vector described in D4).

4. Application of the composition in any of the following: A1) Application in enzymatic hydrolysis of zearalenone or preparation of enzymatic hydrolysis of zearalenone products; A2) Application in enzymatic hydrolysis of aflatoxin B1 or preparation of enzymatic hydrolysis of aflatoxin B1 products; A3) Application in enzymatic hydrolysis of ABTS or preparation of enzymatic hydrolysis ABTS products; The composition comprises the protein of claim 1, and further comprises at least one of acetosyringone, syringaldehyde and luteolin.

5. A protein bacterial complex, characterized in that The complex comprises bacteria and the protein according to claim 1, and the protein is adsorbed on the surface of Bacillus subtilis.

6. A method for preparing a protein bacterial complex, characterized in that: The method comprises the steps of mixing and incubating Bacillus subtilis spores with the protein of claim 1.

7. Use of the protein bacteria according to claim 5 or the protein bacteria complex prepared by the method according to claim 6 in any of the following: A1) Application in enzymatic hydrolysis of zearalenone or preparation of enzymatic hydrolysis of zearalenone products; A2) Application in enzymatic hydrolysis of aflatoxin B1 or preparation of enzymatic hydrolysis of aflatoxin B1 products; A3) Application in enzymatic hydrolysis of ABTS or preparation of enzymatic hydrolysis ABTS products.

8. An enzymatic hydrolysis method, characterized in that: The method includes any of the following: E1) comprising the step of mixing and reacting the protein according to claim 1 or 2 with zearalenone, aflatoxin B1 and / or ABTS; E2) comprising the step of mixing and reacting the composition of claim 4 with zearalenone, aflatoxin B1 and / or ABTS; E3) comprises the steps of mixing and reacting the complex described in claim 5 or the complex prepared by the method described in claim 6 with zearalenone, aflatoxin B1 and / or ABTS.

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