High-temperature resistant laccase, preparation and application thereof

Abstract

The application belongs to the technical field of genetic engineering of enzymes, and particularly relates to a high-temperature-resistant laccase, preparation and application. Priestia aryabhattai The application obtains S281E mutant and S281E / N387D mutant by rationally designing and molecularly modifying laccase genes from a source. The enzyme activity of the mutants S281E and S281E / N387D is respectively increased by 72.7% and 40.4% relative to that of the wild type; under the condition of 70 DEG C and 60 min of incubation, the residual enzyme activity of WT is 25.2%, the residual enzyme activity of S281E is 45.1%, and the residual enzyme activity of S281E / N387D is 80.2%.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme genetic engineering, and in particular relates to a high-temperature resistant laccase, and its preparation and application. Background Art

[0002] Agro-industrial wastes, such as rice bran, wheat bran, bagasse, corn stover, and peanut shells, are rich in lignocellulose. Currently, most of these wastes are dumped or incinerated, resulting in ecological pollution and slowed decomposition. Therefore, converting lignocellulose into a viable commodity is crucial. Lignocellulose, a renewable substrate widely used in the production of reducing sugars and biofuels, primarily consists of hemicellulose, cellulose, and lignin.

[0003] To effectively release cellulose and hemicellulose, lignin removal pretreatment is essential. Currently, commonly used industrial lignin removal processes include strong-alkali, high-temperature pulping and strong-acid, high-temperature xylose (or furfural) removal. However, these processes face environmental restrictions and development prohibitions. Therefore, bioenzymatic methods, as an alternative to traditional chemical lignin degradation methods, can effectively reduce pollution and improve economic efficiency.

[0004] In lignin degradation, laccase (EC1.10.3.2) has attracted considerable attention due to its unique catalytic properties. First discovered in the sap of the rhubarb tree, laccase is a metallo-oxidase capable of catalyzing the oxidation of over 200 substrates. Laccase oxidizes phenolic compounds, opening the phenolic rings in lignin molecules and breaking the lignin chains, thereby increasing their biodegradability and bioavailability.

[0005] Laccases come from a wide range of sources, including plants, insects, fungi, and bacteria. Fungal laccases play an important role in lignin degradation, pigmentation, and plant-pathogen interactions. Although fungal laccases perform well under mild conditions (optimum temperature 30-60°C, optimum pH 3.0-6.0), their application is limited in high-temperature and extreme pH environments. In contrast, bacterial laccases, particularly those from Bacillus, possess superior thermal and pH stability, making them suitable for applications such as lignocellulose degradation, pulp bleaching, and wastewater treatment.

[0006] Bacillus expression systems offer numerous advantages for industrial applications, including efficient protein secretion, non-pathogenicity, rapid growth, and low nutrient requirements. These characteristics make Bacillus an ideal choice for enzyme production, further promoting its potential in lignocellulose degradation and other industrial applications. Pichia pastoris also offers numerous advantages for protein production, including a strong and tightly regulated alcohol oxidase (AOX1) promoter, enabling inducible expression, and a secretory pathway similar to that of higher eukaryotes, enabling folding and post-translational modification of proteins entering the secretory pathway.

[0007] When it comes to enzyme engineering, there are two main approaches: irrational and rational protein molecular design. Irrational design, in the absence of information about a protein's three-dimensional structure, involves generating mutants with desired properties through gene mutation and high-throughput screening. Rational design, on the other hand, relies on known protein structures to target specific amino acid residues for targeted mutagenesis to construct novel protein molecules. Compared to irrational design, rational design requires less work and is more likely to yield effective mutants.

[0008] In summary, laccase can degrade lignin in agricultural and industrial wastes into lignocellulose. When used synergistically with cellulases and hemicellulases, it can improve the conversion efficiency of reducing sugars. Bioenzymatic methods, particularly laccase degradation, can effectively transform these wastes, reduce environmental pollution, and achieve sustainable resource utilization. Future research should focus on improving the enzyme's stability and catalytic efficiency to promote its widespread industrial application.

[0009] Therefore, in the present invention, Priestia aryabhattai The laccase gene from the original source was rationally designed, and mutants with improved heat resistance were obtained through mutation screening. The mutants were then expressed and prepared using Bacillus and Pichia pastoris expression systems. Summary of the Invention

[0010] The purpose of the present invention is to modify the existing laccase based on the problem of high temperature application scenarios to obtain a heat-resistant laccase mutant. Priestia aryabhattai Laccase gene from lac ) was expressed in Escherichia coli BL21, and then mutated by overlapping PCR technology. The mutants were screened using ABTS to identify mutants with improved heat resistance, and their efficient expression and preparation were achieved in Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Pichia pastoris.

[0011] The technical route for achieving the purpose of the present invention is summarized as follows:

[0012] right Priestia aryabhattaiThe laccase gene from the original strain was rationally mutated and screened using the Escherichia coli BL21 expression system to generate two thermostable mutants, S281E and S281E / N387D. After incubation at 70°C for 60 minutes, the residual enzyme activity of the WT, S281E, and S281E / N387D was 25.2%, 45.1%, and 80.2%, respectively. Furthermore, the mutant laccases were efficiently produced using Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, and Pichia pastoris expression systems.

[0013] One of the technical solutions provided by the present invention is a laccase mutant, which is obtained by causing at least one of the mutations S281E and N387D to occur on the wild-type laccase shown in SEQ ID NO.1;

[0014] Furthermore, the laccase mutant is an S281E mutant, and the amino acid sequence is shown in SEQ ID NO.3;

[0015] Furthermore, the coding gene of the S281E mutant lacm1 , the nucleotide sequence is shown in SEQ ID NO.4;

[0016] Furthermore, the laccase mutant is an S281E / N387D mutant, and the amino acid sequence is shown in SEQ ID NO.5;

[0017] Furthermore, the coding gene of the S281E / N387D mutant lacm2 , the nucleotide sequence is shown in SEQ ID NO.6.

[0018] The second technical solution provided by the present invention is a recombinant plasmid or recombinant strain containing the above mutant encoding gene;

[0019] Furthermore, the host cell used by the recombinant strain is Escherichia coli, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis or Pichia pastoris;

[0020] Furthermore, the host cell used by the recombinant strain is Escherichia coli BL21, Bacillus subtilis WB600, Bacillus amyloliquefaciens CGMCC NO.11218, Bacillus licheniformis 2709 or Pichia pastoris GS115;

[0021] Furthermore, the expression vector used in the recombinant plasmid is: pET-28a (+), pBSA43 or pPIC9K;

[0022] Preferably, the recombinant strain is obtained by connecting the mutant encoding gene to the expression vector pET-28a(+) and expressing it in the host Escherichia coli.

[0023] The third technical solution provided by the present invention is the use of the recombinant plasmid or recombinant strain described in the second technical solution in producing the laccase mutant described in the first technical solution.

[0024] The fourth technical solution provided by the present invention is the application of the laccase mutant described in the first technical solution, especially in the degradation of lignin.

[0025] Beneficial effects:

[0026] The invention uses overlapping PCR technology to perform site-directed mutagenesis on wild-type laccase, and the enzyme activities of mutants S281E and S281E / N387D are increased by 72.7% and 40.4% respectively compared with the wild type; under the conditions of 70°C and insulation for 60 minutes, the residual enzyme activity of WT is 25.2%, the residual enzyme activity of S281E is 45.1%, and the residual enzyme activity of S281E / N387D is 80.2%. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The electrophoresis diagram of the PCR amplification of the wild-type laccase gene of the present invention is

[0028] Among them: M is DNA Marker, 1 is laccase gene.

[0029] Figure 2 The recombinant plasmid pET- lac Enzyme digestion verification diagram

[0030] Among them: M is DNA Marker, 1 is the recombinant plasmid pET- lac through Nco I and Xho I double enzyme digestion electrophoresis;

[0031] Figure 3 The SDS-PAGE images of the mutants S281E and S281E / N387D after purification are shown in FIG.

[0032] Wherein: M is protein marker, 1 is S281E purified sample, and 2 is S281E / N387D purified sample. DETAILED DESCRIPTION

[0033] The technical content of the present invention will be further described below in conjunction with the embodiments, but the present invention is not limited to these embodiments, and the protection scope of the present invention cannot be limited by the following embodiments.

[0034] 1. Some of the culture media and solutions used in the examples of the present invention are as follows:

[0035] LB medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, and the rest is water.

[0036] LBS medium: 91.1 g sorbitol, 10 g NaCl, 5 g yeast extract, and 10 g tryptone dissolved in 1 L water.

[0037] The solid medium of the above medium was supplemented with 2% agar.

[0038] 20 mM phosphate buffer: 38 mL 0.2 M NaH2PO4, 62 mL 0.2 M Na2HPO4, pH 7.0.

[0039] SP salt solution (1 L): K2HPO4 18.34 g, KH2PO4 6.0 g, (NH4)2SO4 2.0 g, sodium citrate 1.0 g, MgSO4·7H2O 0.2 g, add 800 mL of water to dissolve, and after complete dissolution, continue to add water to make up to 1 L.

[0040] SP I medium (200 mL): 195.2 mL of SP salt solution, 0.8 mL of 5% casein hydrolysate, 2 mL of 10% yeast extract, and 2 mL of 5% glucose solution. Mix well and dispense 5 mL of each aliquot into sterilized empty test tubes and store at 4°C.

[0041] SP II medium: 292.8 mL of SP salt solution, 1.2 mL of 5% casein hydrolysate, 3 mL of 10% yeast extract, 3 mL of 5% glucose solution, 1.5 mL of 100 mM calcium chloride, and 1.5 mL of 50 mM magnesium chloride. Mix well, and dispense 2 mL of each into sterilized empty test tubes. Store at 4°C.

[0042] Wash Buffer (mM): Weigh 29.25 g of NaCl, 2.42 g of Tris, and 3.5 g of imidazole and dissolve them in ultrapure water to a volume of 1 L. Filter through a 0.22 µm microporous membrane to remove impurities and store in a refrigerator at 4 °C away from light.

[0043] Elution Buffer (mM): Weigh 29.25 g of NaCl, 2.42 g of Tris, and 13.6 g of imidazole and dissolve them in ultrapure water to a volume of 1 L. Filter through a 0.22 µm microporous membrane to remove impurities and store in a refrigerator at 4 °C away from light.

[0044] Lysis Buffer (mM): Weigh 29.25 g NaCl, 2.42 g Tris, and 1.4 g imidazole and fully dissolve them in ultrapure water to a volume of 1 L. Filter through a 0.22 µm microporous membrane to remove impurities and store in a refrigerator at 4 °C away from light.

[0045] 2. The following definitions are adopted in the present invention:

[0046] (1) Nomenclature of amino acid and DNA sequences

[0047] The generally accepted IUPAC nomenclature for amino acid residues is used, using the three-letter / one-letter code format. The generally accepted IUPAC nomenclature for DNA nucleic acid sequences is used.

[0048] (2) Identification of laccase mutants

[0049] The mutated amino acid in a mutant is represented by "original amino acid + position + substituted amino acid." For example, S281E indicates that the amino acid at position 281 is substituted from Ser in the wild-type to Glu; N387D indicates that the amino acid at position 387 is substituted from Asn in the wild-type to Asp. The position numbers correspond to the amino acid sequence numbering of the wild-type laccase in SEQ ID NO. 1.

[0050] In this invention, lowercase italics lac Indicates the gene encoding wild-type laccase, lowercase italic lacm1 Indicates the gene encoding the mutant S281E, in lowercase italics lacm2 The gene encoding the mutant S281E / N387D is shown in the following table.

[0051]

[0052] In the present invention, the wild-type laccase amino acid sequence is shown in SEQ ID NO.1:

[0053] MELKKFVDILPIPSIIRPKGMYKGRPFYDVCMIETLHKFHRDLPKTKVWGYNGLVPGPTFNVEKNQPIYVRWANNLPEKHFLPVDKTIHGASHNPEVRTVVHLHGSPSEPASDGHPEAWFTRGFRQTGPHFMKEIYHYTNGERATALWYHDHALGVTRLNVYAGLAGLYFIRDEQERSLSLPKGKYEVPLIIQDKTFNPDGSLFYPAQPDNPSPNLPYPSIVPSFLGDTITVNGKVWPFLKVEPRKYRFRLLNASNTRTYQFQLSNLRPFALIGTDGGLLSRPIKVKSLDVSPAERIDIVIDFSGLEGKNVILQDGFESENPTGEIMEFQVTKFLSCPDQSRLPPLLSHIDRIPLNKVKKIRRLTLNDSQDEFGRLMLLFDDKEWMNPATETPLLNSVEIWELVNLTPGIHPIHVHLVNFRVLDRYDQNGNLVAPLPADFGLKDTVLVGPGETVRIIMKFQPFSGDYVWHCHRLEHEDHDMMRPLKIIPSNRNKYKSN

[0054] In the present invention, the amino acid sequence of laccase mutant S281E is shown as SEQ ID NO.3:

[0055] MELKKFVDILPIPSIIRPKGMYKGRPFYDVCMIETLHKFHRDLPKTKVWGYNGLVPGPTFNVEKNQPIYVRWANNLPEKHFLPVDKTIHGASHNPEVRTVVHLHGSPSEPASDGHPEAWFTRGFRQTGPHFMKEIYHYTNGERATALWYHDHALGVTRLNVYAGLAGLYFIRDEQERSLSLPKGKYEVPLIIQDKTFNPDGSLFYPAQPDNPSPNLPYPSIVPSFLGDTITVNGKVWPFLKVEPRKYRFRLLNASNTRTYQFQLSNLRPFALIGTDGGLLERPIKVKSLDVSPAERIDIVIDFSGLEGKNVILQDGFESENPTGEIMEFQVTKFLSCPDQSRLPPLLSHIDRIPLNKVKKIRRLTLNDSQDEFGRLMLLFDDKEWMNPATETPLLNSVEIWELVNLTPGIHPIHVHLVNFRVLDRYDQNGNLVAPLPADFGLKDTVLVGPGETVRIIMKFQPFSGDYVWHCHRLEHEDHDMMRPLKIIPSNRNKYKSN

[0056] In the present invention, the amino acid sequence of the laccase mutant S281E / N387D is shown in SEQ ID NO.5:

[0057] MELKKFVDILPIPSIIRPKGMYKGRPFYDVCMIETLHKFHRDLPKTKVWGYNGLVPGPTFNVEKNQNQVRWANNLPEKHFLPVDKTIHGASHNPEVRTVVHLHGSPSEPASDGHPEAWFTRGF RQTGPHFMKEIYHYTNGERATALWYHDHALGVTRLNVYAGLAGLYFIRDEQERSLSLPKGKYEVPLIIQDKTFNPDGSLFYPAQPDNPSPNLPYPSIVPSFLGDTITVNGKVWPFLKVEPRKYRF RLLNASNTRTYQFQLSNLRPFALIGTDGGLLERPIKVKSLDVSPAERIDIVIDFSGLEGKNVILQDGFESENPTGEIMEFQVTKFLSCPDQSRLPPLLSHIDRIPLNKVKKIRRLTLNDSQDEF GRLMLLFDDKEWMDPATETPLLNSVEIWELVNLTPGIHPIHVHLVNFRVLDRYDQNGNLVAPLPADFGLKDTVLVGPGETVRIIMKFQPFSGDYVWHCHRLEHEDHDMMRPLKIIPSNRNKYKSN

[0058] The present invention will be further explained below through specific examples.

[0059] Example 1: Obtaining wild-type laccase gene

[0060] 1. The wild-type laccase gene was obtained from a laboratory-stored Priestia aryabhattai The genome of TCCC 11368 strain was extracted using Bacterial DNA Kit D3350-02 from OMEGA, USA, according to the manufacturer's instructions.

[0061] (1) Strain activation: Use an inoculating loop to dip the bacterial solution from the glycerol tube, inoculate it onto a solid culture medium plate, draw three lines, and incubate at 37°C for 12 hours;

[0062] (2) Transfer: Pick a single colony from the culture plate and inoculate it into 5 mL of liquid LB medium. Incubate at 220 rpm and 37 °C for 12 h.

[0063] (3) Collecting bacteria: Take an appropriate amount of culture medium and dispense it into a 1.5 mL EP tube. Centrifuge at 12000 rpm for 2 min and discard the supernatant.

[0064] (4) Add 250 μL of ddH2O to resuspend the cells, and add 50 μL of 50 mg / mL lysozyme, and incubate at 37°C for 20 min.

[0065] (5) Add 100 µL BTL Buffer and 20 µL Proteinase K and vortex;

[0066] (6) Incubate in a 55°C water bath for 40-50 min, shaking and mixing every 20-30 min;

[0067] (7) Add 5 μL of RNase, invert and mix several times, and let stand at room temperature for 5 min;

[0068] (8) Centrifuge at 12,000 rpm for 2 min, remove the undigested portion, and transfer the supernatant to a new 1.5 mL EP tube;

[0069] (9) Add 220 μL of BDL Buffer, shake to mix, and incubate in a 65°C water bath for 10 min;

[0070] (10) Add 220 μL of anhydrous ethanol and mix thoroughly by pipetting;

[0071] (11) Transfer the liquid in the EP tube to the adsorption column, let it stand for 1 min, centrifuge it at 12000 rpm for 1 min, and pour the filtrate back into the recovery column. Repeat twice and discard the waste liquid.

[0072] (12) Add 500 μL HBC Buffer, centrifuge at 12,000 rpm for 1 min, and discard the filtrate;

[0073] (13) Add 700 μL DNA Wash Buffer, centrifuge at 12,000 rpm for 1 min, and discard the filtrate;

[0074] (14) Add 500 μL DNA Wash Buffer, centrifuge at 12,000 rpm for 1 min, and discard the filtrate;

[0075] (15) Centrifuge at 12,000 rpm for 2 min, discard the waste liquid tube, place the recovery column in a new EP tube, place in a 55°C metal bath for 10 min, and air dry;

[0076] (16) Add 40 μL of 55 ℃ ddH2O for elution, discard the recovery column, and store the genome at -20 ℃.

[0077] 2. Amplification of the wild-type laccase gene

[0078] Using the genome obtained in step 1 as a template, amplify the coding gene of wild-type laccase lac.

[0079] A pair of primers were designed upstream and downstream of the ORF frame to introduce restriction enzyme sites. Nco I. Xho I. Wild-type laccase encoding gene lac The primer sequences (upstream primer LAC-F, downstream primer LAC-R) are as follows:

[0080] LAC-F: ATA CCATGG GCGAGTTAAAAAAATTTGTTGATATC (the underlined part is Nco I restriction enzyme cutting site)

[0081] LAC-R: GTG CTCGAG GTTTGATTTGTATTTATTACGATTGGA (the underlined part is Xho I restriction enzyme site);

[0082] Using the genome obtained in step 1 as a template, perform PCR amplification of the sequence. The total system is 50 μL, and its composition is:

[0083]

[0084] The amplification program was set as follows: pre-denaturation: 98°C for 30 s; denaturation: 98°C for 10 s; annealing: 54°C for 20 s; extension: 72°C for 8 s; 30 cycles of the above reaction; and extension: 72°C for 10 min.

[0085] The PCR amplification product was electrophoresed on 0.8% agarose gel, and a 1494 bp band was obtained ( Figure 1 ), the PCR product was recovered using a small amount of DNA recovery kit to obtain the wild-type laccase gene of the present invention. lac( SEQ ID NO.2). lac and pET-28a(+) plasmids were cleaved with restriction enzymes Nco I and Xho I was double-enzyme digested and the gel was recovered. lac Connected with pET vector to obtain recombinant plasmid pET- lac , double enzyme digestion verification Figure 2 The recombinant plasmid was then transformed into E. coli BL21 to obtain a recombinant strain named BL21 / pET- lac .

[0086] Example 2: Construction of laccase mutants

[0087] According to the results of protein molecular dynamics prediction, sites with a greater impact on protein thermal stability were selected for mutation, including S281E and S281E / N387D. The mutation primers were designed as follows:

[0088] f-S281E:TGGCCTTCTTGAACGTCC

[0089] r-S281E: GGACGTTCAA GAAGGCCA

[0090] f-N387D:GGAGTGGATGGATCCAGC

[0091] r-N387D:GCTGGATCCA TCCACTCC

[0092] The mutant S281E / N387D can be obtained by performing N387D mutation on the mutant S281E.

[0093] 1. In the first step of overlapping PCR, the upper and lower arms are performed separately. Taking S281E as an example, the upstream reaction system is:

[0094]

[0095] The downstream reaction system is:

[0096]

[0097] The amplification program was as follows: pre-denaturation at 98°C for 30 min; denaturation at 98°C for 10 s, annealing at 54°C for 20 s, and extension at 72°C for 5 s, for 30 cycles; and extension at 72°C for 10 min.

[0098] 2. After gel excision and recovery of upstream and downstream fragments, PCR was performed. The reaction system was:

[0099]

[0100] The amplification program was as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 54°C for 20 s, and extension at 72°C for 8 s, for 5 cycles; and extension at 72°C for 10 min.

[0101] 3. After PCR, 2 μL of primers LAC-F and LAC-R were added to the system and PCR amplification was performed as follows: 98°C pre-denaturation for 30 seconds; 98°C denaturation for 10 seconds, 54°C annealing for 20 seconds, 72°C extension for 10 seconds, 30 cycles; 72°C extension for 10 minutes. The PCR amplification product was subjected to 0.8% agarose gel electrophoresis and recovered using a small DNA recovery kit to obtain the gene encoding the laccase site-directed mutant S281E. lacm1 .

[0102] 4. Laccase site-directed mutant gene lacm1 After connecting with the expression vector pET, it was transformed into JM109 and its plasmid was extracted to obtain the recombinant plasmid pET- lacm1 , and then the recombinant plasmid pET- lacm1 Transform into E. coli BL21. Activate the BL21 transformed cells onto a newly divided Kan plate and incubate them upside down at 37°C for 12 hours. Then, under sterile conditions, pick a single mutant colony and inoculate it into liquid LB medium containing Kan resistance. The plasmid was extracted and sent for sequencing to confirm the S281E mutant gene. lacm1 The positive transformants were named BL21 / pET- lacm1 .

[0103] 5. Using recombinant plasmid pET- lacm1 As a template, the overlapping PCR and transfection verification steps were repeated using LAC-F, r-N387D, LAC-R, and f-N387D as upstream and downstream primers to obtain the coding gene of the mutant S281E / N387D. lacm2 , and the recombinant plasmid pET- lacm2 , and recombinant bacteria BL21 / pET- lacm2 .

[0104] 6. The wild-type laccase recombinant bacteria BL21 / pET- lac .

[0105] Example 3 Expression and purification of laccase

[0106] 1. Induced expression of recombinant strains

[0107] (1) On the LB plate, pick the recombinant bacteria BL21 / pET- lac or BL21 / pET- lacm1 or BL21 / pET- lacm2 A single colony was inoculated into a 5 mL LB tube (containing 50 μg / mL Kan) and cultured in a shaker at 37 °C for 12 h.

[0108] (2) Transfer the recombinant bacterial suspension into 250 mL of LB medium (final concentration of 50 μg / mL Kan) and culture in a shaking incubator at 37°C until the OD600 reaches approximately 0.6.

[0109] (3) Add 125 μL of IPTG (final concentration 0.5 mmol / L) and induce the culture in a shaking incubator at 16°C for 16-20 h;

[0110] (4) The fermentation broth was purified to prepare wild-type laccase and various laccase mutants.

[0111] 2. Ni column purification of recombinant protein

[0112] (1) Disruption of bacterial cells

[0113] The fermentation broth was collected using a centrifuge cup and centrifuged at 10,000 rpm for 15 min. The supernatant was discarded and 20 mL of Lysis buffer was added to aspirate the bacteria. Ultrasonic waves were used to disrupt the bacteria, destroy the cell walls, and release the proteins inside the bacteria.

[0114] After the disruption was completed, the bacterial solution was poured into a 50 mL centrifuge tube and centrifuged at 12000 rpm for 30 min at 4 °C to collect the supernatant.

[0115] (2) Binding of WT, S281E, and S281E / N387D to nickel columns

[0116] a. Before nickel column purification, add an appropriate amount of ddH2O to the purification column and add two column volumes of Lysis buffer to equilibrate the resin.

[0117] b. Combine the equilibrated resin and bacterial supernatant, place on a magnetic stirrer, and stir at 80-100 rpm for 1 hour while maintaining a low temperature (4°C).

[0118] (3) Protein purification

[0119] a. Add the binding solution to the purification column in 2-3 times in the chromatography cabinet;

[0120] b. After the binding solution is completely filtered out, add 10 mL of wash buffer to elute the impurities bound to the resin;

[0121] c. Finally, add 10 mL of pre-chilled Elution Buffer to the purification column to elute the target protein bound to the resin and collect the filtrate.

[0122] d. Transfer the entire eluate to an ultrafiltration centrifuge tube and centrifuge until 1 mL of the solution remains. Add pre-chilled 50 mM Tris-HCl buffer, pH 7.0, and repeat the replacement twice. This yields purified WT, S281E, and S281E / N387D proteins. The purified mutants S281E and S281E / N387D were analyzed by SDS-PAGE. Figure 3 shown.

[0123] Example 4 Laccase activity and temperature stability determination

[0124] 1. ABTS laccase activity determination method:

[0125] (1) Take 200 μL of pH 5.0 citric acid-sodium hydrogen phosphate buffer (50 mM, containing 5 mM Cu 2+ ) in a 96-well ELISA plate and incubate in a water bath at 80 °C for 1 min;

[0126] (2) Add 10 μL of purified laccase protein diluted to an appropriate concentration, mix well, and place in an 80 °C water bath for 1 min.

[0127] (3) Add 30 μL of ABTS (50 mM) and mix thoroughly by pipetting. Incubate the mixture in a water bath at 80 °C for 10 min. Record the initial and final OD values ​​at 420 nm.

[0128]

[0129] Where: △OD = OD end - OD start;

[0130] V1 represents the total volume of the reaction system (µL);

[0131] △t represents the reaction time (min);

[0132] V2: volume of enzyme solution added (µL);

[0133] ε represents the molar absorption coefficient of the product at 420 nm, 36 mM -1 cm -1 ;

[0134] d represents the inner diameter of the 96-well ELISA plate / the thickness of the optical path (cm).

[0135] Enzyme specific activity (U / mg) = enzyme activity / protein concentration.

[0136] The specific activities of the three purified laccases, WT, S281E, and S281E / N387D, obtained in Example 2 were measured using the above enzyme activity assay method. The values ​​are shown in the following table:

[0137]

[0138] 2. Determination of laccase thermal stability

[0139] The purified laccase protein in Example 2 was appropriately diluted with pH 7 phosphate buffer and incubated at 70°C for 60 minutes. The residual activity after incubation was measured using the laccase activity assay method in step 1 of Example 4. The data are shown in the following table:

[0140]

[0141] Example 5 Expression and Preparation of Laccase Mutants in Bacillus subtilis

[0142] The genes encoding the laccase mutants S281E and S281E / N387D lacm1 and lacm2 and wild-type laccase encoding gene lac The new recombinant plasmid pBSA43- lacm1 、pBSA43- lacm2 and pBSA43- lac ;

[0143] The recombinant plasmids were transformed into Bacillus subtilis WB600, and the mutant recombinant bacteria WB600 / pBSA43- lacm1 、WB600 / pBSA43- lacm2 and wild-type laccase recombinant strain WB600 / pBSA43- lac .

[0144] The recombinant strain WB600 / pBSA43- lacm1 、WB600 / pBSA43- lacm2 and WB600 / pBSA43- lac Each cell was inoculated into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL), cultured overnight at 37°C, 220 rpm, and then transferred to 50 mL of fresh fermentation medium (containing kanamycin, 50 µg / mL) at a 2% inoculum size. The culture was continued at 37°C, 220 rpm for 48 h (fermentation medium (g / L): corn flour 64, soybean meal 40, 2.7% amylase, Na2HPO4 4, KH2PO4 0.3, and the rest water; kept at 90°C for 30 min and sterilized at 121°C for 20 min).

[0145] Laccase activity in Bacillus subtilis fermentations was determined using the ATBS method described in Example 4 (the fermentation broth was centrifuged and the supernatant was used as the crude enzyme solution for activity measurement). The wild-type enzyme activity in Bacillus subtilis was 943.25 U / mL, the fermentation broth of S281E was 1620.2 U / mL, and the fermentation broth of S281E / N387D was 1325.5 U / mL.

[0146] Example 6 Expression and Preparation of Laccase Mutants in Recombinant Bacillus amyloliquefaciens Strains

[0147] The genes encoding the laccase mutants S281E and S281E / N387D lacm1 and lacm2and wild-type laccase encoding gene lac The new recombinant plasmid pBSA43- lacm1 、pBSA43- lacm2 and pBSA43- lac ;

[0148] The recombinant plasmids were transformed into Bacillus amyloliquefaciens CGMCC No.11218, and the mutant recombinant strain CGMCC No.11218 / pBSA43- lacm1 、CGMCC No.11218 / pBSA43- lacm2 and wild-type laccase recombinant strain CGMCC No.11218 / pBSA43- lac .

[0149] The recombinant strain CGMCC No.11218 / pBSA43- lacm1 、CGMCC No.11218 / pBSA43- lacm2 and CGMCC No.11218 / pBSA43- lac Each cell was inoculated into 5 mL of fermentation medium (containing kanamycin, 50 μg / mL), cultured at 37°C, 220 rpm overnight, and then transferred to 50 mL of fresh fermentation medium (containing kanamycin, 50 μg / mL) at a 2% inoculum size. The culture was continued at 37°C, 220 rpm for 48 h (fermentation medium (g / L): corn flour 64, soybean meal 40, 2.7 amylase, Na2HPO4 4, KH2PO4 0.3, and the rest water; kept at 90°C for 30 min and sterilized at 121°C for 20 min).

[0150] The ATBS method described in Example 4 was used to determine the activity of laccases obtained from fermentation with Bacillus amyloliquefaciens (the fermentation broth was centrifuged and the supernatant was used as the crude enzyme solution for enzyme activity determination). In Bacillus amyloliquefaciens, the wild-type enzyme had an activity of 1559.25 U / mL, the fermentation broth of S281E had an activity of 2638.6 U / mL, and the fermentation broth of S281E / N387D had an activity of 2114.8 U / mL.

[0151] Example 7 Expression and Preparation of Laccase Mutants in Recombinant Bacillus licheniformis Strains

[0152] The genes encoding the laccase mutants S281E and S281E / N387D lacm1 and lacm2 and wild-type laccase encoding gene lac The new recombinant plasmid pBSA43- lacm1 、pBSA43- lacm2 and pBSA43- lac ;

[0153] The recombinant plasmids were transformed into Bacillus licheniformis 2709, and the mutant recombinant bacteria 2709 / pBSA43- lacm1 、2709 / pBSA43- lacm2 and wild-type laccase recombinant strain 2709 / pBSA43- lac .

[0154] The recombinant strain 2709 / pBSA43- lacm1 、2709 / pBSA43- lacm2 and 2709 / pBSA43- lac Each cell was inoculated into 5 mL of fermentation medium (containing kanamycin, 50 μg / mL), cultured at 37°C, 220 rpm overnight, and then transferred to 50 mL of fresh fermentation medium (containing kanamycin, 50 μg / mL) at a 2% inoculum volume. The culture was continued at 37°C, 220 rpm for 48 h (fermentation medium (g / L): corn flour 64, soybean meal 40, 2.7 amylase, Na2HPO44, KH2PO40.3, and the rest was water; kept at 90°C for 30 min and then sterilized at 121°C for 20 min).

[0155] The ATBS method described in Example 4 was used to determine the activity of laccases obtained from Bacillus licheniformis fermentation (the fermentation broth was centrifuged and the supernatant was used as the crude enzyme solution for enzyme activity determination). In Bacillus licheniformis, the wild-type enzyme had an activity of 2271.5 U / mL, the fermentation broth of S281E had an activity of 4050.0 U / mL, and the fermentation broth of S281E / N387D had an activity of 3253.5 U / mL.

[0156] Example 8 Expression and Preparation of Laccase Mutants in Pichia pastoris GS115 Recombinant Strain

[0157] The genes encoding the laccase mutants S281E and S281E / N387D lacm1 and lacm2 and wild-type laccase encoding gene lac The new recombinant plasmid pPIC9K- lacm1 、pPIC9K- lacm2 and pPIC9K- lac ;

[0158] Use restriction enzymes Sal I pair of recombinant plasmid pPIC9K- lacm1 、pPIC9K- lacm2 and pPIC9K- lac Linearization, the linearized recombinant plasmids were electroporated into Pichia pastoris GS115, and the mutant recombinant bacteria GS115 / pPIC9K- lacm1 、GS115 / pPIC9K- lacm2 and wild-type laccase recombinant strain GS115 / pPIC9K- lac .

[0159] (1) Select the recombinant expression strain GS115 / pPIC9K- lacm1 、GS115 / pPIC9K- lacm2 and GS115 / pPIC9K- lac A single colony was inoculated into a 5 mL YPD tube containing 50 μg / mL Kan and cultured at 30 °C, 200 rpm for 24 h;

[0160] (2) Pipette 1 mL of bacterial solution and inoculate it into BMGY enrichment medium. Incubate at 30 °C and 220 rpm for 16-19 h.

[0161] (3) Collect the bacterial liquid in the BMGY medium in a 50 mL centrifuge tube and collect the bacteria by centrifugation; add 20 mL of BMMY medium to resuspend the bacteria, centrifuge and collect the bacteria, and repeat once; add 10 mL of BMMY medium to resuspend the bacteria, transfer 1 mL of the bacterial liquid to 50 mL of BMMY medium with a pipette, add 0.25 mL of methanol every 12 hours, and ferment for 6 days.

[0162] The ATBS method described in Example 4 was used to determine the activity of laccases obtained from Pichia pastoris fermentation (the fermentation broth was centrifuged and the supernatant was used as the crude enzyme solution for enzyme activity determination). The activity of the wild-type laccase in the fermentation broth of Pichia pastoris GS115 was 570.5 U / mL, the activity of the S281E laccase in the fermentation broth was 1010.6 U / mL, and the activity of the S281E / N387D laccase in the fermentation broth was 813.8 U / mL.

[0163] The above-described embodiments merely represent several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that, without departing from the concept of this patent, a person skilled in the art would be able to make various variations, combinations, and improvements to the above-described embodiments, all of which fall within the scope of protection of this patent. Therefore, the scope of protection of this patent shall be determined by the claims.

Claims

1. A laccase mutant, characterized in that: The mutant is an S281E mutant obtained by causing S281E mutation on the basis of the wild-type laccase shown in SEQ ID NO.1, and the amino acid sequence of the S281E mutant is shown in SEQ ID NO.3; or, The mutant is an S281E / N387D mutant obtained by causing S281E and N387D mutations on the basis of the wild-type laccase shown in SEQ ID NO.

1. The amino acid sequence of the S281E / N387D mutant is shown in SEQ ID NO.

5.

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

3. A recombinant plasmid or recombinant strain comprising the gene encoding the mutant according to claim 2.

4. The recombinant plasmid according to claim 3, wherein The expression vector used by the recombinant plasmid is: pET-28a(+), pBSA43 or pPIC9K.

5. The recombinant strain according to claim 3, characterized in that The host cell used by the recombinant strain is Escherichia coli, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis or Pichia pastoris.

6. Use of the recombinant plasmid or recombinant strain according to claim 3 in producing the laccase mutant according to claim 1.

7. Use of the laccase mutant according to claim 1 in lignin degradation.

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