Laccase mutant and its application

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to laccase mutants, encoding genes and applications. The laccase mutant is obtained by subjecting the original laccase to W141C, T198P, R341S, I389T and S476R mutations, and has the amino acid sequence shown in the sequence table SEQ ID NO.2. The optimal reaction pH of the mutated laccase is 4.5. After being treated at pH 3.0-9.0 for 24 hours, the relative enzyme activity is still maintained at more than 90%. The optimal reaction temperature is 85°C, and it can retain more than 95% of the enzyme activity under the conditions of 40-80°C for 15 hours. The laccase mutant provided by the present invention is expressed in Pichia pastoris X33 via the expression plasmid pPICZαA and is grown at 60m 3 The average fermentation enzyme activity in the fermenter was 106673 U / L, which could further reduce the production cost of laccase and promote the widespread application of laccase in the degradation of fungal toxins.

Description

Technical field:

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a laccase mutant and a coding gene and application thereof. Background technology:

[0002] Laccase is a polyphenol oxidase containing four copper ions, which belongs to the copper blue oxidase and exists in the form of monomeric glycoprotein.

[0003] At present, a large number of bacterial laccase genes have been cloned, such as CueO of Escherichia coli, CotA of Bacillus subtilis, CgL1 of Corynebacterium glutamicum, CopA of Pseudomonas syringae, etc. Compared with laccases derived from eukaryotic organisms, bacterial laccases have the advantages of convenient gene cloning, good thermal stability, wide range of enzyme activity pH values, and no glycosylation modification.

[0004] The optimum pH value and suitable temperature of laccase vary depending on the species, and also vary depending on the substrate, but they are all within the acidic pH range and are not suitable for use in the alkaline pH range. The amino acids in the active part of laccase contain acidic and alkaline groups. Due to different dissociation states at different pH values, there are three main ways in which pH affects enzyme activity: acid or alkali changes the spatial structure of the enzyme, causing the enzyme to lose activity; acid or alkali affects the dissociation state of the active part of the enzyme, making the substrate unable to be decomposed; acid or alkali destroys the dissociation state of the substrate, making it impossible for the substrate to bind to the enzyme.

[0005] Domestic research on the application of laccase mainly focuses on pulp bleaching, dye wastewater decolorization, food quality improvement, and degradation of aflatoxin in feed. Its industrial application is generally in high temperature and acid-base change environment, such as the high temperature and alkaline environment requirements in the scouring and bleaching process in the papermaking industry; in the production of juice and other beverages, the phenolic substances in the acidic environment are degraded to clarify the beverage to ensure the stability of the beverage quality; adding laccase during high-temperature baking of food can make the food more flavorful and chewy; in the deep processing of feed, laccase is added in different pH and high-temperature cooking stages to remove aflatoxin.

[0006] The enzyme has poor stability in acid, alkali and high temperature environments, resulting in unsatisfactory enzymolysis effects and low fermentation enzyme activity, which leads to high application costs and difficulty in industrial promotion and application. Therefore, the problems that need to be solved in the industrial application of laccase at this stage include but are not limited to the following: 1. High temperature resistance; 2. Acid and alkali resistance; 3. High fermentation enzyme activity. Summary of the invention:

[0007] The main purpose of the present invention is to provide a laccase mutant and its gene, a recombinant expression vector and a recombinant strain containing the gene, and a method for preparing laccase by the recombinant bacteria, aiming to solve the problems of poor acid, alkali and heat resistance and low fermentation enzyme activity level of laccase in the prior art.

[0008] To achieve the above-mentioned purpose, one of the technical solutions provided by the present invention is a laccase mutant, which is obtained by mutating W, T, R, I, S at positions 141, 198, 341, 389 and 476 in the amino acid sequence of the wild-type laccase shown in SEQ ID NO.1 to C, P, S, T, R, respectively. The amino acid sequence of the mutant is shown in the sequence table SEQ ID NO.2.

[0009] The present invention also provides a gene encoding the mutant;

[0010] Furthermore, the nucleotide sequence of the coding gene is shown in the sequence listing SEQ ID NO.4.

[0011] The second technical solution provided by the present invention is the application of the above-mentioned laccase mutant, especially the application in the degradation of fungal toxins, especially the degradation of aflatoxin; more particularly the application in the degradation of aflatoxin in the feed industry.

[0012] The third technical solution provided by the present invention is a recombinant vector or a recombinant strain comprising the above-mentioned laccase mutation encoding gene;

[0013] Preferably, the expression plasmid used in the recombinant vector is pPICZαA;

[0014] Preferably, the host cell used by the recombinant strain is Pichia pastoris X33.

[0015] A fourth technical solution provided by the present invention is the application of the above-mentioned recombinant expression vector or recombinant strain, especially in the preparation of the laccase mutant described in the first technical solution;

[0016] Furthermore, the method for preparing a laccase mutant using the above recombinant strain is as follows:

[0017] Culture in fermenter: inoculate 4-6% of the volume of the fermenter medium, culture temperature 28-30°C, tank pressure 0.04-0.08MPa, initial speed 100rpm, initial air volume 200m 3 / h, maximum speed 600rpm, maximum ventilation volume 4000m 3 / h;

[0018] Feed control: When the wet weight of the bacteria reaches 60-70g / L, start to feed 30-40% glycerol at a rate of 600-700kg / h. When the wet weight of the bacteria reaches 150-160g / L, stop feeding glycerol. Starve for 1-1.5h, then start to feed methanol. In the first 1-2h, the flow rate of methanol is 100-150kg / h. After 1-2h, the flow rate of methanol increases by 30-50kg per hour until the maximum feed rate is 300-350kg / h. Maintain this feed rate until the end of fermentation.

[0019] Control of dissolved oxygen: Before starting to add methanol, the dissolved oxygen is controlled to 30-40% by adjusting the rotation speed and air volume. After starting to add methanol, the dissolved oxygen is controlled to 20%-30%. After reaching the maximum rotation speed and ventilation volume, the dissolved oxygen is no longer controlled.

[0020] Ammonia supplementation controls pH at 4.5-5.5;

[0021] When the autolysis of the bacteria is serious, the fermentation is terminated, and the culture cycle is 180-200h.

[0022] Furthermore, the culture conditions in the seed tank are: 5-7% inoculation amount, temperature 28-30°C, tank pressure 0.04-0.08Mpa, initial speed 100rpm, initial air volume 20m 3 / h, the dissolved oxygen is controlled to be greater than 30% by increasing the speed and air volume, the maximum speed is 600rpm, and the maximum ventilation volume is 200m 3 / h, add ammonia to control pH at 4.5-5.5, and culture for 48-50h;

[0023] Further, the seed culture medium is composed of (w / v): 2-3% glycerol, 4-6% monoammonium phosphate, 0.2-0.5% disodium hydrogen phosphate, 2-2.5% magnesium sulfate, 1-1.3% potassium sulfate, 0.1-0.3% calcium sulfate, 0.1-0.2% potassium hydroxide, 0.02-0.08% phosphoric acid, and the rest is water, with a pH of 4.5-5.5;

[0024] Furthermore, the fermentation medium consists of (w / v): 5-6% glycerol, 6-7% monoammonium phosphate, 0.3-0.5% disodium hydrogen phosphate, 2.5-3.5% magnesium sulfate, 1-1.6% potassium sulfate, 0.1-0.4% calcium sulfate, 0.1-0.2% potassium hydroxide, 0.03-0.1% phosphoric acid, and the rest is water, with a pH of 4.5-5.5.

[0025] Beneficial effects:

[0026] The laccase mutant provided by the present invention has an optimum reaction pH of 4.5. After being treated at pH 3.0-9.0 for 24 hours, the relative enzyme activity is still maintained at more than 90%. The optimum reaction temperature is 85°C. After being kept at 40-80°C for 15 hours, more than 95% of the enzyme activity can still be retained.

[0027] The laccase mutant provided by the present invention is expressed in Pichia pastoris X33 via expression plasmid pPICZαA. 3 The average fermentation enzyme activity in the fermenter was 106673U / L, which can further reduce the production cost of laccase and promote the widespread application of laccase in the degradation of fungal toxins.

[0028] In addition, the present invention confirms 5 amino acid sites in laccase that are related to acid-base resistance and heat resistance, and proves the importance of these sites to the acid-base resistance and heat resistance of the enzyme, which provides important clues for studying the acid-base resistance and heat resistance of laccase, and provides a reliable reference basis for the research of other laccases. Description of the drawings:

[0029] Figure 1 The optimal reaction pH curve of laccase before and after mutation;

[0030] Figure 2 pH stability curves of laccase before and after mutation;

[0031] Figure 3 The optimal reaction temperature curve of laccase before and after mutation;

[0032] Figure 4 Temperature stability curves of laccase before and after mutation;

[0033] Figure 5 The curve of aflatoxin B1 degradation rate before and after laccase mutation at the optimal reaction temperature. Specific implementation method:

[0034] The present invention is described in detail below through specific implementation cases. The specific implementation cases are only for illustration and are not intended to limit the scope of implementation of the present invention.

[0035] The laccase mutant of the present invention is obtained by subjecting the amino acid sequence of the wild-type laccase to point mutations of W141C, T198P, R341S, I389T and S476R. The amino acid sequence of the laccase mutant is shown in SEQ ID NO.2.

[0036] Identification of the laccase mutant used in the present invention:

[0037] The mutated amino acid in the mutant is represented by "original amino acid + position + replaced amino acid". For example, W141C means that the amino acid at position 141 is replaced by W to C. The position number corresponds to the amino acid sequence number of the wild-type laccase in SEQ ID NO.1. The information is shown in the following table.

[0038]

[0039] The specific sequence information is as follows:

[0040] The amino acid sequence of wild-type laccase is shown in SEQ ID NO.1:

[0041] MALEKFADELPIIETLQPQKVTMKECFHKLHSAVIGTPDGITWHYEQEGTGATPPDSDGYLH

[0042] RDLPPTRLWGYNGLFPGPTIWSGDSMCLHARQACGSRRASIPDKTVVHLDVKTVVHLHGG

[0043] KTVVHLHGGNQDENVYIKWMNDLPDKHDDSDGYPEVTPDDSDGYPEHHSESYPNKQRG

[0044] ALLWYHDHAMAITHGGVTPGHQEPDVRLKLPSDAWFTKDEYDVPLLITDRTIYNKFGDIV

[0045] YTGPAVPPPPSEFKETGPYFEREVYHKPLAQNGGKFIQIGSDGKDESRKPKYLASYPSGCGG

[0046] DADPDTDANVMQFRVTGEHIILTNGTPYMEVEPRTYRFRILNASNTRMLKLTNTQDKWSIIN

[0047] PMGGTHPIHLHLVSFRVLDRRPFDIARIHLHLVSFQILDRRPFDLERYGRPVLLLNNKRWHDP

[0048] VTEACGNTILVNGKAWFGPYTGRYVWHCHILEHEDYGLLPRSVKTQSISLAPAERYFLPIDH

[0049] TIHHSDSQHEEPEVRYVWHCHILEHEDYQFGLPWVQIAIFRASRGFYTTRKYLDMMRPMDVTDKQ*

[0050] The amino acid sequence of the mutant laccase is shown in SEQ ID NO.2:

[0051] MALEKFADELPIIETLQPQKVTMKECFHKLHSAVIGTPDGITWHYEQEGTGATPPDSDGYLH

[0052] RDLPPTRLWGYNGLFPGPTIWSGDSMCLHARQACGSRRASIPDKTVVHLDVKTVVHLHGG

[0053] KTVVHLHGGNQDENVYIKCMNDLPDKHDDSDGYPEVTPDDSDGYPEHHSESYPNKQRGA

[0054] LLWYHDHAMAITHGGVPPGHQEPDVRLKLPSDAWFTKDEYDVPLLITDRTIYNKFGDIVYT

[0055] GPAVPPPPSEFKETGPYFEREVYHKPLAQNGGKFIQIGSDGKDESRKPKYLASYPSGCGGDA

[0056] DPDTDANVMQFRVTGEHIILTNGTPYMEVEPRTYRFSILNASNTRMLKLTNTQDKWSIINPM

[0057] GGTHPIHLHLVSFRVLDRRPFDTARIHLHLVSFQILDRRPFDLERYGRPVLLLNNKRWHDPV

[0058] TEACGNTILVNGKAWFGPYTGRYVWHCHILEHEDYGLLPRSVKTQSIRLAPAERYFLPIDHT

[0059] IHHSDSQHEEPEVRYVWHCHILEHEDYQFGLPWVQIAIFRASRGFYTTRKYLDMMRPMDVTDKQ*

[0060] The nucleotide sequence of the wild-type laccase is shown in SEQ ID NO.3:

[0061] ATGGCACTAGAAAAATTTGCAGATGAACTGCCGATTATCGAAACACTCCAGCCGCAGAA

[0062] GGTCACCATGAAGGAATGCTTTCACAAGCTGCACTCCGCTGTCATTGGTACCCCGGACG

[0063] GCATTACTTGGCACTATGAGCAAGAAGGCACCGGCGCAACGCCGCCGGACAGCGACGG

[0064] CTACCTTCACCGCGATCTCCCGCCGACCCGGCTGTGGGGGTATAACGGTTTATTTCCCGG

[0065] CCCGACGATCTGGTCTGGGGATTCCATGTGCTTGCACGCTCGACAAGCGTGTGGGAGCC

[0066] GTCGAGCCAGCATCCCTGATAAGACTGTCGTTCATTTAGACGTGAAAACCGTCGTTCATT

[0067] TACACGGCGGCAAAACCGTCGTCCATTTACACGGAGGAAACCAAGATGAAAACGTCTAT

[0068] ATTAAATGGATGAATGACCTTCCGGATAAGCATGATGATAGTGACGGGTATCCAGAGGTC

[0069] ACTCCAGATGACAGCGACGGTTATCCTGAGCACCATTCAGAAAGCTATCCAAACAAACA

[0070] GCGCGGGGCTCTGTTATGGTATCACGATCACGCCATGGCGATTACGCACGGAGGCGTCA

[0071] CGCCAGGCCATCAGGAACCCGATGTCCGCTTAAAACTGCCTTCAGACGCATGGTTCACA

[0072] AAGGATGAATACGATGTGCCGCTTCTTATCACAGACCGCACGATCTATAACAAATTCGGC

[0073] GACATTGTGTATACAGGCCCCGCCGTCCCGCCTCCTCCAAGTGAATTTAAGGAAACGGG

[0074] TCCTTATTTTGAGAGAGAGGTATACCATAAACCGTTGGCACAAAACGGCGGGAAGTTTAT

[0075] CCAAATCGGTTCTGACGGCAAAGATGAAAGCAGAAAGCCAAAGTACCTTGCCTCATACC

[0076] CTTCCGGCTGCGGGGGCGACGCCGATCCGGATACAGACGCCAATGTGATGCAATTCCGC

[0077] GTCACAGGAGAACATATCATTTTAACGAACGGCACCCCGTATATGGAAGTTGAACCGCG

[0078] GACATATCGTTTCCGCATCCTTAATGCCTCCAATACGAGGATGCTTAAACTCACAAATAC

[0079] GCAAGACAAATGGTCGATTATCAATCCGATGGGGGGAACCCATCCGATTCACCTGCATTT

[0080] GGTCTCCTTCCGTGTGTTGGACCGGCGTCCGTTTGATATCGCGCGTATACACCTGCACCT

[0081] TGTTTCCTTCCAAATCCTTGACCGGCGCCCTTTTGACTTAGAGCGTTACGGCAGACCCGT

[0082] CCTTCTTCTTAACAACAAACGCTGGCACGATCCTGTCACTGAAGCATGCGGAAATACCA

[0083] TTCTCGTCAATGGTAAAGCGTGGTTCGGGCCATACACTGGGCGGTACGTATGGCATTGCC

[0084] ACATTCTTGAGCATGAAGACTATGGACTGCTTCCCCGCTCTGTCAAAACACAATCTATCA

[0085] GCTTAGCCCCGGCAGAGCGGTATTTCCTTCCGATTGATCACACCATTCATCACAGTGACA

[0086] GCCAGCATGAAGAGCCCGAGGTACGCTACGTATGGCACTGTCATATTTTAGAACATGAA

[0087] GATTACCAATTCGGTCTGCCGTGGGTGCAAATCGCTATTTTCCGCGCATCCCGAGGTTTTT

[0088] ATACGACGAGAAAGTATCTGGACATGATGAGACCGATGGACGTCACGGATAAGCAGTAA

[0089] Nucleotide sequence of the mutant laccase, as shown in SEQ ID NO.4:

[0090] ATGGCACTAGAAAAATTTGCAGATGAACTGCCGATTATCGAAACACTCCAGCCGCAGAA

[0091] GGTCACCATGAAGGAATGCTTTCACAAGCTGCACTCCGCTGTCATTGGTACCCCGGACG

[0092] GCATTACTTGGCACTATGAGCAAGAAGGCACCGGCGCAACGCCGCCGGACAGCGACGG

[0093] CTACCTTCACCGCGATCTCCCGCCGACCCGGCTGTGGGGGTATAACGGTTTATTTCCCGG

[0094] CCCGACGATCTGGTCTGGGGATTCCATGTGCTTGCACGCTCGACAAGCGTGTGGGAGCC

[0095] GTCGAGCCAGCATCCCTGATAAGACTGTCGTTCATTTAGACGTGAAAACCGTCGTTCATT

[0096] TACACGGCGGCAAAACCGTCGTCCATTTACACGGAGGAAACCAAGATGAAAACGTCTAT

[0097] ATTAAATGCATGAATGACCTTCCGGATAAGCATGATGATAGTGACGGGTATCCAGAGGTC

[0098] ACTCCAGATGACAGCGACGGTTATCCTGAGCACCATTCAGAAAGCTATCCAAACAAACA

[0099] GCGCGGGGCTCTGTTATGGTATCACGATCACGCCATGGCGATTACGCACGGAGGCGTCC

[0100] CGCCAGGCCATCAGGAACCCGATGTCCGCTTAAAACTGCCTTCAGACGCATGGTTCACA

[0101] AAGGATGAATACGATGTGCCGCTTCTTATCACAGACCGCACGATCTATAACAAATTCGGC

[0102] GACATTGTGTATACAGGCCCCGCCGTCCCGCCTCCTCCAAGTGAATTTAAGGAAACGGG

[0103] TCCTTATTTTGAGAGAGAGGTATACCATAAACCGTTGGCACAAAACGGCGGGAAGTTTAT

[0104] CCAAATCGGTTCTGACGGCAAAGATGAAAGCAGAAAGCCAAAGTACCTTGCCTCATACC

[0105] CTTCCGGCTGCGGGGGCGACGCCGATCCGGATACAGACGCCAATGTGATGCAATTCCGC

[0106] GTCACAGGAGAACATATCATTTTAACGAACGGCACCCCGTATATGGAAGTTGAACCGCG

[0107] GACATATCGTTTCAGCATCCTTAATGCCTCCAATACGAGGATGCTTAAACTCACAAATAC

[0108] GCAAGACAAATGGTCGATTATCAATCCGATGGGGGGAACCCATCCGATTCACCTGCATTT

[0109] GGTCTCCTTCCGTGTGTTGGACCGGCGTCCGTTTGATACCGCGCGTATACACCTGCACCT

[0110] TGTTCCTTCCAAATCCTTGACCGGCGCCCTTTTGACTTAGAGCGTTACGGCAGACCCGT

[0111] CCTTCTTCTTAACAACAAACGCTGGCACGATCCTGTCACTGAAGCATGCGGAAATACCA

[0112] TTCTCGTCAATGGTAAAGCGTGGTTTCGGGCCATACACTGGGCGGTACGTATGGCATTGCC

[0113] ACATTCTTGAGCATGAAGACTATGGACTGCTTCCCCGCTCTGTCAAAACACAATCTATCC

[0114] GCTTAGCCCCGGCAGAGCGGTATTTCCTTCCGATTGATCACACCATTCATCACAGTGACA

[0115] GCCAGCATGAAGAGCCCGAGGTACGCTACGTATGGCACTGTCATATTTTAGAACATGAA

[0116] GATTACCAATTCGGTCTGCCGTGGGTGCAAATCGCTATTTTCCGCGCATCCCGAGGTTTTT

[0117] ATACGACGAGAAAGTATCTGGACATGATGAGACCGATGGACGTCACGGATAAGCAGTAA

[0118] The present invention will be further explained below in conjunction with specific implementation modes.

[0119] Example 1 Obtaining laccase mutant gene, expression vector and recombinant strain

[0120] 1.1 Obtaining laccase mutant genes

[0121] With the help of FoldX, Rosetta and YASARA software, an electronic library of acid-base resistant and heat-stable mutants of laccase was constructed. At the same time, a semi-rational design method was used to analyze the catalytic mechanism, and it was finally determined that the laccase mutant of the present invention was obtained by mutating the tryptophan, threonine, arginine, isoleucine and serine at the 141st, 198th, 341st, 389th and 476th positions in the amino acid sequence of the wild-type laccase from Bacillus pumilus NGJ763 to cysteine, proline, serine, threonine and arginine, respectively.

[0122] 300 mL of the logarithmic phase seed solution of Bacillus pumilus NGJ763 was taken into 6 50 mL centrifuge tubes, centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the precipitated bacteria were washed twice with 600 mL of physiological saline, and then sent to Shanghai Biotech for genome sequencing to obtain the coding gene sequence and upstream and downstream sequence information of the wild-type laccase. According to the obtained genome, primers Y1 (containing EcoRⅠ restriction site) and Y2 (containing XbaⅠ restriction site) were used to amplify the laccase gene CotA (SEQ ID NO.3), and connected to the T vector for storage, and the recombinant vector was named T-CotA. The site-directed mutagenesis kit used the kit of Shanghai Biotech, model B639281, and the specific operation steps were as follows:

[0123] (1) Primers for amplifying the wild-type laccase gene and various point mutations.

[0124] Table 1 The primers used for the above point mutation are as follows

[0125] Primer name Sequence (5'-3') Primer length (bp) Y1 CCGGAATTCATGGCACTAGAAAAATT 26 Y2 CTAGTCTAGATTACTGCTTATCCGTG 26 W141C-1 AACGTCTATTAAATGCATGAATGACCTTCCG 33 W141C-2 CGGAAGGTCATTCATGCATTTAATATAGACGTT 33 T198P-1 TTACGCACGGAGGCGTCCCGCCAGGCCATCAGGA 34 T198P-2 TCCTGATGGCCTGGCGGGACGCCTCCGTGCGTAA 34 R341S-1 ACATATCGTTTCAGCATCCTTAATGCCTCCAA 32 R341S-2 TTGGAGGCATTAAGGATGCTGAAACGATATGT 32 I389T-1 CGGCGTCCGTTTGATACCGCGCGTATACACCTGC 34 I389T-2 GCAGGTGTATACGCGCGGTATCAAACGGACGCCG 34 S476R-1 ACACAATCTATCCGCTTAGCCCCGGCAGAGCGGTA 35 S476R-2 TACCGCTCTGCCGGGGCTAAGCGGATAGATTGTGT 35

[0126] (2) Point mutation: Taking the point mutation of W141C as an example, use the two primers W141C-1 and W141C-2, and the recombinant T-vector T-CotA containing the wild-type laccase gene CotA as a template. According to the system in the point mutation kit, add other reagents and perform PCR amplification. After the amplification, take 10 μL of the above mutation PCR amplification product and perform 0.8% agarose gel electrophoresis detection. After the correct band is found, add 1 μL of digestion enzyme to the mutation product, mix well, and incubate at 37°C for 60 minutes.

[0127] (3) Transformation: Add 5 μL of the mutated product to 50 μL of DH5α competent cells, mix well, and place on ice for 30 min; heat shock at 42°C for 45 s and immediately cool on ice for 10 min; add 500 μL of LB medium, culture at 180 r / min, and 37°C for 1 h; then centrifuge the culture at 10,000 r / min for 5 min, discard part of the supernatant, retain 100-150 μL of the supernatant, flick to suspend the bacteria, take all the bacterial solution to plate, and culture at 37°C overnight.

[0128] (4) Verification of positive clones: Pick a single colony in 500 μL LB medium containing ampicillin resistance, culture at 200 r / min for 2-3 h, and then identify by PCR. Send the screened positive clones for sequencing. Compare the sequencing results and select the positive clones that have the mutation of G to C at position 423 in the sequence shown in nucleotide sequence SEQ ID NO.3. The positive clone is the mutant containing the recombinant plasmid with the correct mutation.

[0129] (5) Mutation of other sites: Using the mutant screened correctly as above as a template, the primer pairs corresponding to each mutation site in Table 1 were used one by one, and the same method as above was used to mutate each site, and finally the laccase mutant gene shown in SEQ ID NO.4 was screened out.

[0130] The laccase gene BCotA (SEQ ID NO. 4) obtained after the above point mutations was connected to a T vector for storage and named as T-BCotA vector.

[0131] The present invention connects the mutant laccase gene to the expression vector pPICZαA to obtain pPICZαA-BCotA, and then expresses the laccase mutant in Pichia pastoris X33 through the expression vector pPICZαA-BCotA, as follows: 1.2 Obtaining the recombinant expression vector

[0132] 1.2.1 Enzyme digestion

[0133] The recombinant T-vector T-BCotA containing the laccase mutant gene and the expression vector pPICZαA were digested with restriction endonucleases EcoRⅠ and XbaⅠ, respectively. The digestion system is as follows:

[0134] Component name volume EcoR I 1μL XB 1μL 10×M Buffer 2μL Vector T-BCotA (or pPICZαA) ≤1 μg Sterile water up to 20μL Overall system 20μL

[0135] The above reaction system was digested overnight in a 37°C water bath to obtain the linear fragment of the laccase mutant gene BCotA and the linearized vector pPICZαA.

[0136] 1.2.2 Connection

[0137] The above linear fragments were identified by 1% agarose gel electrophoresis, purified and recovered using a gel recovery kit, and then ligated using T4 DNA ligase. The ligation system is as follows:

[0138] Component name volume Mutated BCotA gene 13μL pPICZαA linear vector 4μL 10×T4 Buffer 2μL T4 DNA Ligase 1μL Overall system 20μL

[0139] The above reaction was carried out at 16°C for 16 h.

[0140] The ligation product was used to transform Escherichia coli DH5α: 100 μL of competent E. coli was added with 20 μL of the above ligation product, mixed thoroughly and placed in an ice bath for 30 minutes, then in a 42°C water bath for 90 seconds, followed by an ice bath for 5 minutes, followed by addition of 500 μL of resistance-free LB medium, incubated at 37°C, 150 rpm on a shaker for 1 hour, then spread on an LB plate containing bleomycin (final concentration 25 μg / ml), and incubated at 37°C for 12 hours.

[0141] Screening of the recombinant expression vector pPICZαA-BCotA: Select a single colony on the plate and culture it in LB liquid culture medium (containing bleomycin). After 12 hours, use primers Y1 and Y2 to screen the recombinant expression vector pPICZαA-BCotA by colony PCR.

[0142] Extraction of recombinant expression vector pPICZαA-BCotA: For the positive clones with correct sequencing, inoculate them into a test tube containing LB liquid culture medium and culture them at 37°C and 150 rpm for 10 h until OD 600 About 1.0, and then use a plasmid extraction kit to extract the recombinant expression vector pPICZαA-BCotA.

[0143] 1.3 Obtaining recombinant strains

[0144] The recombinant expression vector pPICZαA-BCotA was linearized with restriction endonuclease SacI and digested at 37°C overnight. The linearization digestion reaction system was:

[0145]

[0146] The above reaction system was reacted at 37°C for 2h. The plasmid after enzyme digestion was purified with a DNA purification kit and transferred into Pichia pastoris X33 competent cells by electroporation (voltage 2.0k V, capacitance 25μF, resistance 200Ω, and electroporation time 5ms). 200μL was spread on YPDS solid medium (10g / L yeast extract, 20g / L peptone, 20g / L glucose, 1mol / L sorbitol, 20g / L agar, autoclaved at 115°C for 20min) containing a final concentration of Zeocin of 100ug / ml, and inverted cultured at 30°C. The resulting strain was the X33 / pPICZαA-BCotA Pichia pastoris engineered bacteria.

[0147] 1.4 Obtaining wild-type laccase gene expression strain

[0148] Using the same technical means as in Examples 1.2 and 1.3 above, the recombinant T vector T-CotA containing the original laccase gene and the expression vector pPICZαA were digested with restriction endonucleases EcoRⅠ and XbaⅠ, respectively, and then ligated to obtain the expression vector pPICZαA-CotA expressing the wild-type laccase gene, which was then linearized with restriction endonuclease SacI and transferred into Pichia pastoris X33 competent cells to obtain the expression engineering strain X33 / pPICZαA-CotA of the original laccase gene.

[0149] Example 2 Preparation of laccase

[0150] 2.1 Preparation of mutant laccase

[0151] The recombinant strain X33 / pPICZαA-BCotA constructed in Example 1 was used for fermentation preparation, and the method was as follows:

[0152] 3m 3 Seed culture in seed tank: 6% inoculation, temperature 30°C, tank pressure 0.04-0.08Mpa, initial speed 100rpm, initial air volume 20m 3 / h, the dissolved oxygen is controlled to be greater than 30% by increasing the speed and air volume, the maximum speed is 600rpm, and the maximum ventilation volume is 200m 3 / h, ammonia was added to control the pH at 5.5, and the culture was carried out for 49h;

[0153] Seed culture medium composition: 3% glycerol, 5% monoammonium phosphate, 0.3% disodium hydrogen phosphate, 2.5% magnesium sulfate, 1% potassium sulfate, 0.3% calcium sulfate, 0.2% potassium hydroxide, 0.06% phosphoric acid, pH 5.5;

[0154] 60m 3 Fermentation in the fermenter: inoculate 6% of the volume of the fermenter medium, culture temperature 30°C, tank pressure 0.04-0.08MPa, initial speed 100rpm, initial air volume 200m 3 / h, maximum speed 600rpm, maximum ventilation volume 4000m 3 / h; Feed control: When the wet weight of the bacteria reaches 65g / L, start to feed 35% glycerol at a flow rate of 650kg / h. When the wet weight of the bacteria reaches 150g / L, stop feeding glycerol, starve for 1.3h, and then start to feed methanol. In the first 2h, the flow rate of methanol is 130kg / h. After 2h, the flow acceleration of methanol increases by 30kg per hour until the maximum feed rate is 300kg / h. Maintain this feed rate until the end of fermentation. Control of dissolved oxygen: Before starting to feed methanol, control the dissolved oxygen to 30% by adjusting the speed and air volume. After starting to feed methanol, control the dissolved oxygen to 20%. After reaching the maximum speed and ventilation volume, the dissolved oxygen is no longer controlled. Ammonia is added to control the pH at 5.5. When the bacteria are seriously autolyzed, the fermentation is terminated. The culture cycle is 200h.

[0155] The fermentation medium composition: glycerol 5%, monoammonium phosphate 7%, disodium hydrogen phosphate 0.5%, magnesium sulfate 2.5%, potassium sulfate 1%, calcium sulfate 0.4%, potassium hydroxide 0.2%, phosphoric acid 0.03%, pH 5.5.

[0156] The fermentation results of three batches are as follows, and the average fermentation enzyme activity is: 106673U / L.

[0157] batch Fermentation cycle (h) Fermentation enzyme activity (U / L) 1 200 106561 2 200 107032 3 200 106426

[0158] 2.2 Preparation of wild-type laccase

[0159] The same fermentation method as in Example 2.1 was used to carry out three batches of fermentation using the X33 / pPICZαA-CotA recombinant bacteria. The results were as follows: the average fermentation enzyme activity was 30334 U / L.

[0160] batch Fermentation cycle (h) Fermentation enzyme activity (U / L) 1 200 29793 2 200 30182 3 200 31027

[0161] Example 3 Determination of laccase activity

[0162] 3.1 Definition of laccase activity involved in the present invention

[0163] When the ABTS method is used to determine the activity of laccase, the amount of enzyme required to convert 1 μmol of substrate per minute is defined as an activity unit (unless otherwise specified, the reaction conditions are pH 5.5 and temperature 38° C.).

[0164] 3.2 Enzyme activity determination steps

[0165] Preheating: Take 2.6 mL of phosphate-citrate buffer solution with a pH value of 5.5 in a test tube, add 0.5 mL of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) solution (the final concentration of ABTS is 0.5 mM) in the test tube and preheat in a 38°C water bath for 3 min;

[0166] Reaction: Add 0.1 mL of laccase solution and shake evenly;

[0167] Measurement: The shaken sample was subjected to kinetic measurement using a spectrophotometer. The change in OD value per second within 30 seconds was measured at a wavelength of 420 nm (the reaction rate was a uniform reaction) and the enzyme activity was calculated.

[0168] Enzyme activity formula:

[0169]

[0170] Where: △OD: change in absorbance OD; V 总 : total reaction volume; n: enzyme dilution multiple; △t: reaction time; V0: enzyme volume; ε: molar absorption coefficient of substrate 3.6×10 4 (mol / L) - 1 cm - 1.

[0171] Example 4 Enzymatic properties of laccase mutants

[0172] The laccase mutant provided by the present invention has an optimum reaction pH of 4.5. After being treated at pH 3.0-9.0 for 24 hours, the relative enzyme activity is still maintained at more than 90%. The optimum reaction temperature is 85° C., and after being kept at 40-80° C. for 15 hours, the enzyme activity can still be retained at more than 95%, as follows:

[0173] (1) Optimal reaction pH

[0174] The supernatant obtained by centrifuging the fermentation broth of batch 1 in Example 2.1 and batch 1 in Example 2.2 was taken as a crude enzyme solution, and the laccase activity was measured at pH values ​​of 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0 at a temperature of 38°C, and the relative enzyme activity was calculated, with the highest enzyme activity being 100%. The measurement results are as follows: Figure 1 As shown, the activity of the mutant laccase was highest at pH 4.5.

[0175] (2) Alkali resistance

[0176] The supernatant obtained by centrifugation of the fermentation broth of batch 1 of Example 2.1 and batch 1 of Example 2.2 was taken as a crude enzyme solution, and the pH of the enzyme solution was adjusted to 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 respectively with 0.1M NaOH or 0.1M HCl, and then placed at room temperature for 24 hours, and then the enzyme activity was measured at 38°C and pH 4.5, and the relative enzyme activity was calculated, with the original enzyme activity as 100%. The measurement results are as follows: Figure 2 As shown, after being treated at pH 3.0-9.0 for 24 h, the relative enzyme activity of the mutant laccase still remained above 90%.

[0177] (3) Optimum reaction temperature

[0178] The supernatant obtained by centrifugation of the fermentation broth of batch 1 in Example 2.1 and batch 1 in Example 2.2 was taken as the crude enzyme solution, and the laccase activity was measured at 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95° C. under pH 5.5, and the relative enzyme activity was calculated, with the highest enzyme activity being 100%. The results are shown in FIG. Figure 3 As shown, the optimal reaction temperature of the mutant laccase is 85℃.

[0179] (4) Thermal stability

[0180] The supernatant obtained by centrifuging the fermentation broth of batch 1 of Example 2.1 and batch 1 of Example 2.2 was taken as the crude enzyme solution, and the enzyme solution was incubated at 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95°C for 15 hours. After the incubation, the enzyme activity was measured at 38°C and pH 5.5, and the relative enzyme activity was calculated, with the original enzyme activity as 100%. The experimental results are as follows Figure 4 As shown in the figure, the mutant laccase can still maintain more than 95% enzyme activity when kept at 40-80℃ for 15h. The thermal stability is significantly improved compared with the wild-type laccase.

[0181] Example 5 Degradation experiment of aflatoxin by mutated laccase

[0182] In this example, the degradation ability of wild-type laccase and mutant laccase on aflatoxin was analyzed.

[0183] 5.1 Enzymatic hydrolysis method

[0184] Enzymes with the same protein concentration (final concentration in the degradation system: 0.003 ng / mL) were used for degradation, and the concentration of aflatoxin B1 was 50 μg / mL. In order to achieve the best enzymatic hydrolysis effect and comparability, the wild-type laccase and the mutant laccase were placed at the optimal reaction temperatures of 55°C and 85°C, respectively, and in phosphate-citrate buffer solutions with their respective optimal pH values ​​of 6.0 and 4.5, respectively, to degrade aflatoxin, and the content of aflatoxin B1 was determined every 20 minutes (the content of aflatoxin B1 was determined according to the method of national standard GB / T 17480-2008).

[0185] 5.2 Results Analysis

[0186] According to the above method, the present invention measured the degradation efficiency of wild-type laccase and mutant laccase on aflatoxin B1. Figure 5 As shown in the figure (the horizontal axis is time min, the vertical axis is degradation efficiency), the modified laccase is significantly better than the wild-type laccase in degrading aflatoxin B1. The wild-type laccase takes 180 minutes to completely degrade aflatoxin B1 in the system, while the modified laccase only takes 60 minutes to completely degrade aflatoxin B1 in the system.

[0187] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A laccase mutant, characterized in that: The amino acid sequence of the laccase mutant is shown in SEQ ID NO.

2.

2. A gene encoding the laccase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

4.

4. A recombinant vector or recombinant strain comprising the coding gene according to claim 2.

5. The recombinant vector according to claim 4, characterized in that The expression plasmid used was pPICZαA.

6. The recombinant strain according to claim 4, characterized in that The host bacteria used was Pichia pastoris X33.

7. Use of the recombinant vector or recombinant strain according to claim 4 in producing the laccase mutant according to claim 1.

8. Use of the laccase mutant according to claim 1, characterized in that: It is used in the degradation of aflatoxin.

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