High-temperature-resistant laccase as well as preparation and application thereof

Abstract

The invention belongs to the technical field of gene engineering of enzymes, and particularly relates to high-temperature-resistant laccase as well as preparation and application thereof. According to the present invention, the laccase gene derived from Priesta aryabhattai is subjected to molecular modification through the rational design so as to obtain the S281E mutant and the S281E / N387D mutant; the enzyme activities of the mutants S281E and S281E / N387D are respectively improved by 72.7% and 40.4% compared with the enzyme activities of the wild type mutants S281E and S281E / N387D; under the conditions that the temperature is 70 DEG C and the temperature is kept 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%.

Description

Technical Field

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

[0002] Agricultural 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 slow decomposition. Therefore, it is particularly important to convert lignocellulose into commercial products that can be effectively utilized. As a renewable matrix, lignocellulose is widely used in the production of reducing sugars and biofuels. Its main components include hemicellulose, cellulose and lignin.

[0003] In order to effectively release cellulose and hemicellulose, lignin removal pretreatment is necessary. At present, the commonly used industrial lignin removal processes include strong alkali high temperature pulping technology and strong acid high temperature xylose (or furfural) technology. However, these processes face challenges of restrictions and prohibitions on development in terms of environmental pollution. Therefore, bioenzymatic method, as a lignin degradation method that replaces traditional chemical processes, can effectively reduce pollution and improve economic benefits.

[0004] In lignin degradation, laccase (EC1.10.3.2) has attracted much attention due to its unique catalytic properties. Laccase was first discovered in the sap of Rhus verniciflua and is a metal-containing oxidase that can catalyze the oxidation of more than 200 substrates. Laccase oxidizes phenolic compounds to open the phenolic ring in the lignin molecule, resulting in the breaking of the lignin chain, thereby improving its degradability and availability.

[0005] Laccases come from a wide range of sources, including plants, insects, fungi, and bacteria. Among them, 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, optimal pH 3.0-6.0), their application in high temperature and extreme pH environments is limited. In contrast, bacterial laccases, especially laccases from Bacillus, have better thermal stability and pH stability, and are suitable for use in the fields of lignocellulose degradation, pulp bleaching, and wastewater treatment.

[0006] Bacillus expression systems have many advantages in industrial applications, including efficient protein secretion, non-pathogenicity, rapid growth, and low requirements for nutrients. These characteristics make Bacillus an ideal choice for the production of enzyme preparations, further promoting its potential in lignocellulose degradation and other industrial applications. Pichia pastoris also has many advantages for protein production, including a strong and strictly regulated alcohol oxidase (AOX1) promoter that can be induced for expression; a secretion pathway similar to that of higher eukaryotes, which can fold and post-translationally modify proteins entering the secretory channel.

[0007] In terms of enzyme modification, protein irrational molecular design and rational molecular design are two main methods. In the absence of information on the three-dimensional structure of the protein, irrational design obtains mutants with desired characteristics through gene mutation and high-throughput screening; while rational design is based on the known protein structure and conducts directed mutations on specific amino acid residues to construct new protein molecules. Compared with irrational design, rational design requires less work and is easier to obtain effective mutants.

[0008] In summary, laccase can degrade lignin in agricultural and industrial waste into lignocellulose, and can improve the conversion efficiency of reducing sugars when used in conjunction with cellulases and hemicellulases. Through bioenzymatic methods, especially the degradation technology using laccase, these wastes can be effectively converted, reducing environmental pollution and achieving sustainable utilization of resources. Future research should focus on improving the stability and catalytic efficiency of enzymes to promote their widespread application in industry.

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

[0010] The purpose of the present invention is to perform mutation modification on the existing laccase based on the problem of high temperature application scenarios, so as 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, and the mutants were screened using ABTS to screen out 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: right Priestia aryabhattaiThe laccase gene of the source was rationally mutated, and two mutants S281E and S281E / N387D with good thermal stability were screened using the Escherichia coli BL21 expression system. After incubation at 70°C for 60 minutes, the residual enzyme activity of WT was 25.2%, the residual enzyme activity of S281E was 45.1%, and the residual enzyme activity of S281E / N387D was 80.2%. The mutant laccase was then efficiently prepared using Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Pichia pastoris expression systems.

[0012] One of the technical solutions provided by the present invention is a laccase mutant, wherein the mutant is obtained by causing at least one of S281E and N387D mutations on the basis of the wild-type laccase shown in SEQ ID NO.1; Further, the laccase mutant is an S281E mutant, and the amino acid sequence is shown in SEQ ID NO.3; Furthermore, the coding gene of the S281E mutant lacm1 , the nucleotide sequence is shown in SEQ ID NO.4; Further, the laccase mutant is a S281E / N387D mutant, and the amino acid sequence is shown in SEQ ID NO.5; Furthermore, the coding gene of the S281E / N387D mutant lacm2 , the nucleotide sequence is shown in SEQ ID NO.6.

[0013] The second technical solution provided by the present invention is a recombinant plasmid or recombinant strain containing the above mutant encoding gene; Furthermore, the host cell used by the recombinant strain is Escherichia coli, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis or Pichia pastoris; 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; Furthermore, the expression vector used in the recombinant plasmid is: pET-28a (+), pBSA43 or pPIC9K; 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.

[0014] 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.

[0015] 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.

[0016] Beneficial effects: 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 condition 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

[0017] Figure 1 The electrophoresis diagram of the PCR amplification of the wild-type laccase gene of the present invention is Among them: M is DNA Marker, 1 is laccase gene.

[0018] Figure 2 The recombinant plasmid pET- lac Enzyme cutting verification diagram Where: M is DNA Marker, 1 is the recombinant plasmid pET- lac through Nco I and XOt I Double enzyme digestion electrophoresis; Figure 3 The SDS-PAGE image of the mutants S281E and S281E / N387D after purification of the present invention is Wherein: M is protein marker, 1 is S281E purified sample, and 2 is S281E / N387D purified sample. DETAILED DESCRIPTION

[0019] The technical content of the present invention is 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.

[0020] 1. Some of the culture media and solutions used in the embodiments of the present invention are as follows: LB medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, and the rest is water.

[0021] LBS medium: 91.1 g sorbitol, 10 g NaCl, 5 g yeast extract, 10 g tryptone, dissolved in 1 L water.

[0022] The solid medium of the above medium was supplemented with 2% agar.

[0023] 20 mM Phosphate Buffer: 38 mL 0.2 M NaH2PO4, 62 mL 0.2M Na2HPO4, pH 7.0.

[0024] 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 continue to add water to make up to 1 L after complete dissolution.

[0025] SP I medium (200 mL): 195.2 mL of SP salt solution, 0.8 mL of 5% casein hydrolyzate, 2 mL of 10% yeast juice, and 2 mL of 5% glucose solution. Mix well and take 5 mL of each aliquot into sterilized empty test tubes and store at 4 ℃.

[0026] SP II medium: 292.8 mL of SP salt solution, 1.2 mL of 5% casein hydrolysate, 3 mL of 10% yeast juice, 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 and store at 4 ℃.

[0027] Wash Buffer (mM): Weigh 29.25 g NaCl, 2.42 g Tris, and 3.5 g imidazole and dissolve them in ultrapure water to make up to 1 L. Filter through a 0.22 µm microporous membrane to remove impurities and store in a refrigerator at 4 ℃ away from light.

[0028] Elution Buffer (mM): Weigh 29.25 g NaCl, 2.42 g Tris, and 13.6 g imidazole and dissolve them in ultrapure water to make up to 1 L. Filter through a 0.22 µm microporous membrane to remove impurities and store in a refrigerator at 4 ℃ away from light.

[0029] Lysis Buffer (mM): Weigh 29.25 g NaCl, 2.42 g Tris, and 1.4 g imidazole and fully dissolve them in ultrapure water. Make up to 1 L. Filter with a 0.22 µm microporous membrane to remove impurities and store in a refrigerator at 4 ℃ away from light.

[0030] 2. The following definitions are adopted in the present invention: (1) Nomenclature of amino acid and DNA sequences 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.

[0031] (2) Identification of laccase mutants The mutated amino acid in the mutant is represented by "original amino acid + position + replaced amino acid". For example, S281E means that the amino acid at position 281 is replaced by Glu from the wild-type Ser; N387D means that the amino acid at position 387 is replaced by Asp from the wild-type Asn. The position number corresponds to the amino acid sequence number of the wild-type laccase in SEQ ID NO.1.

[0032] In the present invention, lowercase italics lac Indicates the gene encoding wild-type laccase, in lowercase italics lacm1 Indicates the gene encoding the mutant S281E, in lowercase italics lacm2 The encoding gene of the mutant S281E / N387D is shown in the following table.

[0033]

[0034] In the present invention, the wild-type laccase amino acid sequence is shown in SEQ ID NO.1: MELKKFVDILPIPSIIRPKGMYKGRPFYDVCMIETLHKFHRDLPKTKVWGYNGLVPGPTFNVEKNQNQVRWANNLPEKHFLPVDKTIHGASHNPEVRTVVHLHGSPSEPASDGHPEAWFTRGF RQTGPHFMKEIYHYTNGERATALWYHDHALGVTRLNVYAGLAGLYFIRDEQERSLSLPKGKYEVPLIIQDKTFNPDGSLFYPAQPDNPSPNLPYPSIVPSFLGDTITVNGKVWPFLKVEPRKYRF RLLNASNTRTYQFQLSNLRPFALIGTDGGLLSRPIKVKSLDVSPAERIDIVIDFSGLEGKNVILQDGFESENPTGEIMEFQVTKFLSCPDQSRLPPLLSHIDRIPLNKVKKIRRLTLNDSQDEF GRLMLLFDDKEWMNPATETPLLNSVEIWELVNLTPGIHPIHVHLVNFRVLDRYDQNGNLVAPLPADFGLKDTVLVGPGETVRIIMKFQPFSGDYVWHCHRLEHEDHDMMRPLKIIPSNRNKYKSN In the present invention, the amino acid sequence of the laccase mutant S281E is shown in SEQ ID NO.3: MELKKFVDILPIPSIIRPKGMYKGRPFYDVCMIETLHKFHRDLPKTKVWGYNGLVPGPTFNVEKNQPIYVRWANNLPEKHFLPVDKTIHGASHNPEVRTVVHLHGSPSEPASDGHPEAWFTRGFRQTGPHFMKEIYHYTNGERATALWYHDHALGVTRLNVYAGLAGLYFIRDEQERSLSLPKGKYEVPLIIQDKTFNPDGSLFYPAQPDNPSPNLPYPSIVPSFLGDTITVNGKVWPFLKVEPRKYRFRLLNASNTRTYQFQLSNLRPFALIGTDGGLLERPIKVKSLDVSPAERIDIVIDFSGLEGKNVILQDGFESENPTGEIMEFQVTKFLSCPDQSRLPPLLSHIDRIPLNKVKKIRRLTLNDSQDEFGRLMLLFDDKEWMNPATETPLLNSVEIWELVNLTPGIHPIHVHLVNFRVLDRYDQNGNLVAPLPADFGLKDTVLVGPGETVRIIMKFQPFSGDYVWHCHRLEHEDHDMMRPLKIIPSNRNKYKSN In the present invention, the amino acid sequence of the laccase mutant S281E / N387D is as shown in SEQ ID NO.5: MELKKFVDILPIPSIIRPKGMYKGRPFYDVCMIETLHKFHRDLPKTKVWGYNGLVPGPTFNVEKNQNQVRWANNLPEKHFLPVDKTIHGASHNPEVRTVVHLHGSPSEPASDGHPEAWFTRGF RQTGPHFMKEIYHYTNGERATALWYHDHALGVTRLNVYAGLAGLYFIRDEQERSLSLPKGKYEVPLIIQDKTFNPDGSLFYPAQPDNPSPNLPYPSIVPSFLGDTITVNGKVWPFLKVEPRKYRF RLLNASNTRTYQFQLSNLRPFALIGTDGGLLERPIKVKSLDVSPAERIDIVIDFSGLEGKNVILQDGFESENPTGEIMEFQVTKFLSCPDQSRLPPLLSHIDRIPLNKVKKIRRLTLNDSQDEF GRLMLLFDDKEWMDPATETPLLNSVEIWELVNLTPGIHPIHVHLVNFRVLDRYDQNGNLVAPLPADFGLKDTVLVGPGETVRIIMKFQPFSGDYVWHCHRLEHEDHDMMRPLKIIPSNRNKYKSN The present invention will be further explained below through specific examples.

[0035] Example 1: Obtaining wild-type laccase gene 1. The wild-type laccase gene comes from the laboratory preserved Priestia aryabhattai The genome of TCCC 11368 strain was extracted using Bacterial DNA Kit D3350-02 from OMEGA, USA, according to the instructions.

[0036] (1) Strain activation: Use an inoculation loop to take bacterial liquid from the glycerol tube, inoculate it on a solid culture medium plate, draw three zones, and culture at 37°C for 12 h. (2) Transfer: Pick a single colony from the culture plate and inoculate it into 5 mL of liquid LB medium. Cultivate at 220 rpm and 37 °C for 12 h. (3) Collecting bacteria: Take an appropriate amount of culture medium and dispense it into a 1.5 mL EP tube. Centrifuge at 12,000 rpm for 2 min and discard the supernatant. (4) Add 250 µL ddH2O to resuspend the cells, and add 50 µL 50 mg / mL lysozyme, and incubate at 37 °C for 20 min. (5) Add 100 µL BTL Buffer and 20 µL Proteinase K and vortex. (6) Incubate at 55°C in a water bath for 40-50 min, oscillating and mixing every 20-30 min. (7) Add 5 µL of RNase, invert several times to mix, and leave at room temperature for 5 min; (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; (9) Add 220 µL BDL Buffer, vortex to mix, and incubate in a 65 °C water bath for 10 min. (10) Add 220 µL of anhydrous ethanol and mix by pipetting; (11) Transfer the liquid in the EP tube to the adsorption column, let it stand for 1 min, centrifuge at 12,000 rpm for 1 min, pour the filtrate back into the recovery column, repeat twice, and discard the waste liquid; (12) Add 500 µL HBC Buffer, centrifuge at 12,000 rpm for 1 min, and discard the filtrate; (13) Add 700 µL DNA Wash Buffer, centrifuge at 12,000 rpm for 1 min, and discard the filtrate; (14) Add 500 µL DNA Wash Buffer, centrifuge at 12,000 rpm for 1 min, and discard the filtrate; (15) Centrifuge at 12,000 rpm for 2 min, discard the waste tube, place the recovery column in a new EP tube, place in a 55 °C metal bath for 10 min, and air dry; (16) Add 40 µL of 55 °C ddH2O for elution, discard the recovery column, and store the genome at -20 °C.

[0037] 2. Amplification of the wild-type laccase gene Using the genome obtained in step 1 as a template, amplify the coding gene of wild-type laccase lac.

[0038] A pair of primers were designed upstream and downstream of the ORF frame to introduce restriction enzyme sites respectively. Nco I. XOt I. Wild-type laccase encoding gene lac The primer sequences (upstream primer LAC-F, downstream primer LAC-R) are as follows: LAC-F: ATA CCATGG GCGAGTTAAAAAAATTTGTTGATATC (the underlined part is Nco I restriction site) LAC-R: GTG CTCGAG GTTTGATTTGTATTTATTACGATTGGA (the underlined part is XOt I restriction site); 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:

[0039] 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; extension: 72 °C for 10 min.

[0040] The PCR amplification product was subjected to 0.8% agarose gel electrophoresis, and a 1494 bp band was obtained ( Figure 1 ), using a small amount of DNA recovery kit to recover the PCR product, and obtain the wild-type laccase gene of the present invention. lac( SEQ ID NO.2). lac pET-28a(+) plasmid were cleaved with restriction endonucleases Nco I and XOt I was double-digested, and the gel-recovered lac Connect 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 .

[0041] Example 2: Construction of laccase mutants According to the results of protein molecular dynamics prediction, sites with greater impact on protein thermal stability were selected for mutation, including S281E and S281E / N387D. The mutation primers were designed as follows: f-S281E:TGGCCTTCTTGAACGTCC r-S281E: GGACGTTCAA GAAGGCCA f-N387D:GGAGTGGATGGATCCAGC r-N387D:GCTGGATCCA TCCACTCC The mutant S281E / N387D can be obtained by performing N387D mutation on the mutant S281E.

[0042] 1. In the first step of overlapping PCR, the upper and lower arms are carried out separately. Taking S281E as an example, the upstream reaction system is:

[0043] The downstream reaction system is:

[0044] 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, extension at 72°C for 5 s, 30 cycles; and extension at 72°C for 10 min.

[0045] 2. After gel excision and recovery of upstream and downstream fragments, PCR was performed. The reaction system was:

[0046] 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, extension at 72°C for 8 s, 5 cycles; and extension at 72°C for 10 min.

[0047] 3. After the PCR, 2 μL of primers LAC-F and LAC-R were added to the system, and the PCR 3 amplification program was 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 of reaction; 72°C extension for 10 minutes. The PCR amplification product was subjected to 0.8% agarose gel electrophoresis, and the PCR product was recovered using a small amount of DNA recovery kit to obtain the coding gene of the laccase site-directed mutant S281E. lacm1 .

[0048] 4. Site-directed mutant gene of laccase lacm1 After being connected 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 Transformed into E. coli BL21. Activated the BL21 transformed transformant onto a new zoned Kan plate, inverted and cultured at 37°C for 12 hours, then picked a single mutant colony and inoculated it into liquid LB medium containing Kan resistance under sterile conditions, and the plasmid was extracted and sent for sequencing to confirm the S281E mutant gene. lacm1 The positive transformant obtained was named BL21 / pET- lacm1 .

[0049] 5. Recombinant plasmid pET- lacm1 As a template, LAC-F, r-N387D, LAC-R, f-N387D were used as upstream and downstream primers to repeat the above overlapping PCR and transfection verification steps to obtain the coding gene of the mutant S281E / N387D. lacm2 , and the recombinant plasmid pET- lacm2 , and recombinant bacteria BL21 / pET- lacm2 .

[0050] 6. The wild-type laccase recombinant bacteria BL21 / pET- lac .

[0051] Example 3 Expression and purification of laccase 1. Inducible expression of recombinant strains (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 placed in a shaker at 37 °C for 12 h. (2) Transfer the recombinant bacterial suspension into 250 mL LB medium (final concentration of 50 μg / mL Kan) and culture in a shaker at 37°C until the OD600 is about 0.6; (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; (4) Purify the fermentation broth to prepare wild-type laccase and various laccase mutants.

[0052] 2. Ni column purification of recombinant protein (1) Disruption of bacterial cells 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 blow the bacteria away. Ultrasonic waves were used to break the bacteria, destroy the cell wall and release the protein inside the bacteria.

[0053] 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.

[0054] (2) Binding of WT, S281E and S281E / N387D to nickel column a. Before the nickel column purification process, add an appropriate amount of ddH2O to the purification column and add two times the column volume of Lysis buffer to balance the resin; b. Mix the equilibrated resin and bacterial supernatant, place in a magnetic stirrer, and stir at 80-100 r / min for 1 h, while keeping the temperature low (4 °C) throughout the process.

[0055] (3) Protein purification a. Add the binding solution to the purification column in 2-3 times in the chromatography cabinet; b. After the binding solution is completely filtered out, add 10 mL of Wash buffer to elute the impurities bound to the resin; c. Finally, add 10 mL of pre-cooled Elution Buffer to the purification column to elute all the target protein bound to the resin and collect the filtrate; d. Transfer all the eluate to an ultrafiltration centrifuge tube, centrifuge and ultrafilter until 1 mL of the solution remains, add pre-cooled 50 mM, pH 7.0 Tris-HCl buffer, and repeat the replacement twice. The purified WT, S281E and S281E / N387D proteins were obtained. The mutants S281E and S281E / N387D were purified by SDS-PAGE. Figure 3 shown.

[0056] Example 4 Determination of laccase activity and temperature stability 1. ABTS determination of laccase activity: (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; (2) Add 10 μL of purified laccase protein diluted to an appropriate concentration and mix well, then place in an 80 °C water bath for 1 min; (3) Add 30 µL of ABTS (50 mM) and mix by pipetting. Incubate the mixture in a constant temperature water bath at 80 °C for 10 min. Record the initial and final OD values ​​at 420 nm.

[0057]

[0058] Where: △OD = OD end - OD start; V1 represents the total volume of the reaction system (µL); △t represents the reaction time (min); V2: volume of enzyme solution added (µL); ε represents the molar absorption coefficient of the product at 420 nm, 36 mM -1 cm -1 ; d represents the inner diameter / optical path thickness of the 96-well ELISA plate (cm).

[0059] Enzyme specific activity (U / mg) = enzyme activity / protein concentration.

[0060] By using the above enzyme activity measurement method, the specific activities of the three purified laccases, WT, S281E and S281E / N387D in Example 2, were measured, and the values ​​are shown in the following table:

[0061] 2. Determination of thermal stability of laccase The purified laccase protein in Example 2 was appropriately diluted with a pH 7 phosphate buffer and incubated at 70° C. for 60 min. 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:

[0062] Example 5 Expression and preparation of laccase mutants in Bacillus subtilis 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 ; The recombinant plasmids were transferred into Bacillus subtilis WB600, and the mutant recombinant bacteria WB600 / pBSA43- lacm1 、WB600 / pBSA43- lacm2 and wild-type laccase recombinant strain WB600 / pBSA43- lac .

[0063] The recombinant strain WB600 / pBSA43- lacm1 、WB600 / pBSA43- lacm2 and WB600 / pBSA43- lac The samples were inoculated into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL) respectively, cultured at 37°C, 220 r / min overnight, transferred to 50 mL of fresh fermentation medium (containing kanamycin, 50 µg / mL) at a 2% inoculum amount, and cultured at 37°C, 220 r / min for 48 h (fermentation medium (g / L): corn flour 64, soybean cake powder 40, 2.7 amylase, Na2HPO44, KH2PO40.3, and the rest was water; kept at 90°C for 30 min and sterilized at 121°C for 20 min).

[0064] The ATBS method in Example 4 was used to determine the laccase activity in the fermentation of Bacillus subtilis (the fermentation broth was centrifuged and the supernatant was taken as the crude enzyme solution for enzyme activity determination). In Bacillus subtilis, the wild-type enzyme activity was 943.25 U / mL, the fermentation broth enzyme activity of S281E was 1620.2 U / mL, and the fermentation broth enzyme activity of S281E / N387D was 1325.5 U / mL.

[0065] Example 6 Expression and preparation of laccase mutants in recombinant strains of Bacillus amyloliquefaciens 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 ; The recombinant plasmids were respectively transferred into Bacillus amyloliquefaciens CGMCC No.11218, and the mutant recombinant bacteria CGMCC No.11218 / pBSA43- lacm1 、CGMCC No.11218 / pBSA43- lacm2 and wild-type laccase recombinant strain CGMCC No.11218 / pBSA43- lac .

[0066] The recombinant strain CGMCC No.11218 / pBSA43- lacm1 、CGMCC No.11218 / pBSA43- lacm2 and CGMCC No.11218 / pBSA43- lac The samples were inoculated into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL) respectively, cultured at 37°C, 220 r / min overnight, transferred to 50 mL of fresh fermentation medium (containing kanamycin, 50 µg / mL) at a 2% inoculum amount, and cultured at 37°C, 220 r / min for 48 h (fermentation medium (g / L): corn flour 64, soybean cake powder 40, 2.7 amylase, Na2HPO44, KH2PO40.3, and the rest was water; kept at 90°C for 30 min and sterilized at 121°C for 20 min).

[0067] The ATBS method in Example 4 was used to determine the activity of the laccase obtained by fermentation of Bacillus amyloliquefaciens (the fermentation broth was centrifuged and the supernatant was taken as the crude enzyme solution for enzyme activity determination). In Bacillus amyloliquefaciens, the wild-type enzyme activity was 1559.25 U / mL, the fermentation broth enzyme activity of S281E was 2638.6 U / mL, and the fermentation broth enzyme activity of S281E / N387D was 2114.8 U / mL.

[0068] Example 7 Expression and preparation of laccase mutants in recombinant strains of Bacillus licheniformis 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 ; The recombinant plasmids were transferred into Bacillus licheniformis 2709, and the mutant recombinant bacteria 2709 / pBSA43- lacm1 、2709 / pBSA43- lacm2 and wild-type laccase recombinant strain 2709 / pBSA43- lac .

[0069] The recombinant strain 2709 / pBSA43- lacm1 、2709 / pBSA43- lacm2 and 2709 / pBSA43- lac The samples were inoculated into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL) respectively, cultured at 37°C, 220 r / min overnight, transferred to 50 mL of fresh fermentation medium (containing kanamycin, 50 µg / mL) at a 2% inoculum amount, and cultured at 37°C, 220 r / min for 48 h (fermentation medium (g / L): corn flour 64, soybean cake powder 40, 2.7 amylase, Na2HPO44, KH2PO40.3, and the rest was water; kept at 90°C for 30 min and sterilized at 121°C for 20 min).

[0070] The ATBS method in Example 4 was used to determine the activity of the laccase obtained by fermentation of Bacillus licheniformis (the fermentation broth was centrifuged and the supernatant was taken as the crude enzyme solution for enzyme activity determination). In Bacillus licheniformis, the wild-type enzyme activity was 2271.5 U / mL, the fermentation broth enzyme activity of S281E was 4050.0 U / mL, and the fermentation broth enzyme activity of S281E / N387D was 3253.5 U / mL.

[0071] Example 8 Expression and preparation of laccase mutants in Pichia pastoris GS115 recombinant strain 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 ; Use restriction enzyme Sal I pairs 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 bacteria GS115 / pPIC9K- lac .

[0072] (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; (2) Pipette 1 mL of bacterial solution and inoculate it into BMGY enrichment medium. Cultivate at 30 °C and 220 rpm for 16-19 h. (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 and collect the bacteria by centrifugation. Repeat once; add 10 mL of BMMY medium to resuspend the bacteria and transfer 1 mL of the bacterial liquid to 50 mL of BMMY medium using a pipette. Add 0.25 mL of methanol every 12 h and ferment for 6 days.

[0073] The activity of the laccase obtained by fermentation of Pichia pastoris was determined using the ATBS method in Example 4 (the fermentation broth was centrifuged and the supernatant was taken as the crude enzyme solution for enzyme activity determination). In Pichia pastoris GS115, the enzyme activity of the wild-type laccase fermentation broth was 570.5 U / mL, the enzyme activity of the fermentation broth of S281E was 1010.6 U / mL, and the enzyme activity of the fermentation broth of S281E / N387D was 813.8 U / mL.

[0074] 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 mutant is obtained by causing at least one of S281E and N387D mutations to occur on the wild-type laccase shown in SEQ ID NO.

1.

2. The laccase mutant according to claim 1, characterized in that The laccase mutant is an S281E mutant, and the amino acid sequence is shown in SEQ ID NO.

3.

3. The laccase mutant according to claim 1, characterized in that The laccase mutant is a S281E / N387D mutant, and the amino acid sequence is shown in SEQ ID NO.

5. The gene encoding the laccase mutant according to claim 1 .

5. A recombinant plasmid or recombinant strain comprising the mutant encoding gene according to claim 4.

6. The recombinant plasmid according to claim 5, characterized in that The expression vector used by the recombinant plasmid is: pET-28a(+), pBSA43 or pPIC9K.

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

8. Use of the recombinant plasmid or recombinant strain according to claim 5 in producing the laccase mutant according to claim 1.

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

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