A laccase mutant and use thereof

Abstract

The application provides a laccase mutant and application thereof, relates to the technical field of enzyme engineering, and the laccase mutant is obtained by at least one of Q428K, F334K and N387K mutations on the basis of wild-type laccase. Rational mutation is carried out on the wild-type laccase by rational molecular design, and the mutant Q428K, Q428K / F334K and Q428K / F334K / N387K with improved enzyme activity and thermal stability at 80 DEG C are obtained. The enzyme activity of the mutant Q428K, Q428K / F334K and Q428K / F334K / N387K is 1.7 times, 2.9 times and 3.6 times that of the wild-type laccase respectively.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically to a laccase mutant and its application. Background Technology

[0002] Lacase (EC1.10.3.2), also known as polyphenol oxidase, is a copper-containing polyphenol oxidase widely distributed in fungi, plants, insects, and bacteria. Its active site contains four copper ions, catalyzing the oxidation of various phenolic and non-phenolic compounds through a single-electron transfer mechanism, while simultaneously reducing oxygen to water. The reaction process does not require the participation of hydrogen peroxide. This enzyme drives key biological processes in nature, such as lignin degradation, plant disease resistance, and insect epidermal hardening. Due to its broad substrate spectrum, environmental friendliness, and mild reaction conditions, it is widely used in industrial and environmental fields such as paper bleaching, textile dyeing wastewater decolorization, pollutant biodegradation, and biosensor development. Lacases from different sources exhibit significant differences in pH adaptability, thermal stability, and catalytic efficiency.

[0003] Lignin is the second most abundant natural aromatic polymer in plant cell walls after cellulose. It is a complex three-dimensional network structure formed by three phenylpropane monomers (guaiacol G, syringyl S, and p-hydroxyphenyl H) cross-linked through ether and carbon-carbon bonds, accounting for 15-30% of the dry weight of lignocellulose biomass. Its hydrophobicity, chemical inertness, and persistent cross-linking properties (containing over 3000 bond types) constitute a major barrier against biodegradation in plants (i.e., "resistance"), limiting not only the efficiency of cellulose enzymatic hydrolysis but also being a core bottleneck in the utilization of biomass resources. However, lignin's complex structure and high stability make it difficult to effectively convert and utilize. Traditional methods of lignin degradation require high temperature, high pressure, strong acid, or strong alkali conditions, which are not only energy-intensive and environmentally unfriendly but also cause non-selective degradation of lignin, increasing costs.

[0004] Therefore, there is an urgent need for a highly active and thermally stable laccase to achieve highly selective and stable degradation of lignin.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a laccase mutant with high activity and high thermal stability and its application. By using rational molecular design to rationally mutate the amino acid sequence of wild-type laccase, a laccase mutant with higher activity and high temperature resistance can be prepared.

[0007] To achieve the above-mentioned objectives of this invention, the following technical solution is adopted:

[0008] A laccase mutant is obtained by the following mutation on the basis of wild-type laccase: the mutation is Q428K or Q428K / F334K or Q428K / F334K / N387K; wherein the amino acid sequence of the wild-type laccase is shown in SEQ ID NO.1.

[0009] The amino acid sequence of wild-type laccase, SEQ ID NO.1:

[0010] MELKKFVDILPIPSIIRPKGMYKGRPFYDVCMIETLHKFHRDLPKTKVWGYNGLVPGPTFNVEKNQNQVRWANNLPEKHFLPVDKTIHGASHNPEVRTVVHLHGSPSEPASDGHPEAWFTRGF RQTGPHFMKEIYHYTNGERATALWYHDHALGVTRLNVYAGLAGLYFIRDEQERSLSLPKGKYEVPLIIQDKTFNPDGSLFYPAQPDNPSPNLPYPSIVPSFLGDTITVNGKVWPFLKVEPRKYRF RLLNASNTRTYQFQLSNLRPFALIGTDGGLLSRPIKVKSLDVSPAERIDIVIDFSGLEGKNVILQDGFESENPTGEIMEFQVTKFLSCPDQSRLPPLLSHIDRIPLNKVKKIRRLTLNDSQDEFG RLMLLFDDKEWMNPATETPLLNSVEIWELVNLTPGIHPIHVHLVNFRVLDRYDQNGNLVAPLPADFGLKDTVLVGPGETVRIIMKFQPFSGDYVWHCHRLEHEDHDMMRPLKIIPSNRNKYKSN.

[0011] Preferably, the amino acid sequence of the Q428K mutant is shown in SEQ ID NO.3; the amino acid sequence of the Q428K / F334K mutant is shown in SEQ ID NO.5; and the amino acid sequence of the Q428K / F334K / N387K mutant is shown in SEQ ID NO.7.

[0012] SEQ ID NO.2 is the nucleotide sequence of wild-type laccase; SEQ ID NO.4 is the nucleotide sequence of the Q428K mutant; SEQ ID NO.6 is the nucleotide sequence of the Q428K / F334K mutant; SEQ ID NO.8 is the nucleotide sequence of the Q428K / F334K / N387K mutant.

[0013] A polynucleotide encoding the above-mentioned laccase mutant.

[0014] A recombinant plasmid containing the aforementioned polynucleotides.

[0015] Preferably, the expression vector of the recombinant plasmid is any one of pET-28a(+), pBSA43, or pPIC9K.

[0016] A host cell containing the aforementioned polynucleotides or recombinant plasmids.

[0017] Preferably, the host cell is a prokaryotic cell or a eukaryotic cell.

[0018] Preferably, the host cell is any one or more of Escherichia coli, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, or Pichia pastoris;

[0019] Preferably, the host cells are Bacillus subtilis WB600 (purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd., catalog number SICC354844), Bacillus amyloliquefaciens CGMCC No.11218 (disclosed in Chinese patent CN 105087448B), Bacillus licheniformis 2709 (disclosed in Chinese patent CN 109022476 B), and Pichia pastoris GS115 (purchased from...). north Jingbai Aolaibo Technology Co., Ltd. Product code BTN12-154y) or E. coli BL2 (purchased from Beijing Bio-Lab Technology Co., Ltd. Limited Company Any one of the following (item number BTN12-174y);

[0020] Preferably, the host cell is Escherichia coli BL2.

[0021] A genetically engineered bacterium, comprising the above-mentioned polynucleotides or the above-mentioned recombinant plasmids.

[0022] Application of a laccase mutant in any one of papermaking bleaching, textile dyeing wastewater decolorization, or biodegradation of pollutants.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] This invention utilizes rational molecular design to rationally mutate wild-type laccase, obtaining mutants Q428K, Q428K / F334K, and Q428K / F334K / N387K with improved enzyme activity and thermostability at 80℃ compared to the wild type. The enzyme activities of mutants Q428K, Q428K / F334K, and Q428K / F334K / N387K are increased to 1.7 times, 2.9 times, and 3.6 times that of wild-type laccase, respectively. Attached Figure Description

[0025] Figure 1 : This is an electrophoresis diagram of PCR amplification of wild-type laccase; where M is the DNA Marker and lane 1 is the laccase gene lac;

[0026] Figure 2 : This is a verification image of pET-lac plasmid digestion, where M is the DNA Marker and 1 is a double digestion image of pET-lac with NcoI and XhoI.

[0027] Figure 3 : Bar chart showing residual enzyme activity of wild-type and mutant Q428K / F334K / N387K laccase. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0029] Example 1

[0030] The solutions and culture media used in the embodiments of this invention are as follows:

[0031] Lysis buffer (mM): Tris 20, NaCl 500, dithiothreitol 1, imidazole 20.

[0032] Wash buffer (mM): Tris 20, NaCl 500, dithiothreitol 1, imidazole 100.

[0033] Elution Buffer (mM): Tris 20, NaCl 500, dithiothreitol 1, imidazole 500.

[0034] LB medium (g / L): yeast extract 5.0, tryptone 10.0, NaCl 10.0, the remainder being water. Solid medium supplemented with 2% agar.

[0035] BMGY medium (g / L): peptone 20.0, yeast extract 10.0, YNB 13.4, 4×10 -5% Biotin, 10% 1 mmol / L potassium phosphate buffer (pH 6.0), 0.5% glycerol.

[0036] BMMY medium (g / L): peptone 20.0, yeast extract 10.0, YNB 13.4, 4×10 -5 % Biotin, 10% 1 mmol / L potassium phosphate buffer (pH 6.0), 0.5% methanol.

[0037] Fermentation medium (g / L): corn flour 64, soybean meal 40, with 2.7 amylase, 4 Na2HPO4, 0.3 KH2PO4 added, and the remainder being water; incubate at 90℃ for 30 min and then sterilize at 121℃ for 20 min.

[0038] Obtaining wild-type laccase genes

[0039] 1. Genomic DNA was extracted from Bacillus megaterium TCCC 111368 using the OMEGA Bacterial DNA Kit. The extraction steps are as follows:

[0040] (1) Inoculate the strain onto LB solid plates with an inoculation loop and incubate overnight at 37 °C.

[0041] (2) Pick a single colony from the culture plate and inoculate it into a liquid test tube culture medium. Incubate overnight at 37°C with shaking at 220 r / min.

[0042] (3) Take 3 mL-5 mL of bacterial solution and place it in a sterilized EP tube. Centrifuge at 12000 r / min for 2 min and discard the supernatant.

[0043] (4) Add 200 μL of sterile water to the EP tube to resuspend the bacterial cells, then add 50 μL of lysozyme, mix by blowing and aspiration, and keep warm at 37°C for 20 min.

[0044] (5) Add 100 μL of BTL buffer and 20 μL of proteinase K to the EP tube, vortex to mix, incubate at 55 °C for 40 min, and vortex to mix every 20 min.

[0045] (6) Add 5 μL of RNase, invert and mix several times, and let stand at room temperature for 10 min.

[0046] (7) Centrifuge at 12000 r / min for 2 min, remove the undigested part, transfer the supernatant to a new EP tube, add 220 μL BDL buffer, and incubate in a 65℃ water bath for 15 min.

[0047] (8) Add 220 μL of anhydrous ethanol and mix by blowing and sucking.

[0048] (9) Transfer the liquid in the EP tube to the recovery column and let it stand for 1 min. Centrifuge at 12000 r / min for 1 min. Pour the filtrate back into the recovery column and repeat twice. Discard the waste liquid.

[0049] (10) Add 500 μL HBC buffer, centrifuge at 12000 r / min for 1 min, and discard the filtrate.

[0050] (11) Add 700 μL DNA wash buffer, let stand for 1 min, centrifuge at 12000 r / min for 1 min, and discard the filtrate.

[0051] (12) Add 500 μL DNA wash buffer, let stand for 1 min, centrifuge at 12000 r / min for 1 min, and discard the filtrate.

[0052] (13) 12000 r / min for 2 min, discard the waste liquid tube, and put the recovery column into a new EP tube.

[0053] (14) Dry in a 55 ℃ metal bath for 10 min.

[0054] (15) Add 50 μL of sterile water at 55℃, let stand at room temperature for 5 min, centrifuge at 12000 r / min for 2 min, discard the recovery column, and the liquid in the EP tube is the genome.

[0055] 2. Using the extracted Bacillus megaterium genome as a template, a pair of primers were designed upstream and downstream of the ORF frame to introduce restriction enzyme sites NcoI and XhoI, respectively. The amplification primers for the laccase gene lac in this invention are as follows:

[0056] Upstream primer P1:

[0057] 5'-AGATATA CCATGG GCGAGTTAA-3' (underlined part is the NcoI restriction site)

[0058] Downstream primer P2:

[0059] 5'-GTGGTG CTCGAG GTTTGA-3' (underlined part is the XhoI restriction site);

[0060] Using P1 and P2 as upstream and downstream primers, amplification was performed using the Bacillus megaterium laccase genome as a template.

[0061] The amplification reaction system is shown in Table 1:

[0062] Table 1: Amplification reaction system

[0063] upstream primer P1 2.0 μL Downstream primer P2 2.0 μL Genomic DNA as template 2.0 μL Primer Star Max Enzyme 25 μL <![CDATA[ddH2O]]> 19 μL

[0064] The amplification program was as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 54℃ annealing for 20 s, 72℃ extension for 8 s, for 30 cycles; 72℃ extension for 10 min. The PCR amplification products were subjected to 0.8% agarose gel electrophoresis, yielding a 1530 bp band. Figure 1 The PCR product was recovered using a small-volume DNA recovery kit to obtain the wild-type laccase gene lac (SEQ ID NO.2) of this invention. The pET-28a(+) plasmid was double-digested with restriction endonucleases NcoI and XhoI. The recovered lac was homologously ligated with the pET vector to obtain the recombinant plasmid pET-lac. Enzyme digestion verification was performed as follows. Figure 2 As shown, it was transformed into Escherichia coli BL21 and named the recombinant strain BL21 / pET-lac.

[0065] Example 2: Construction of laccase mutants

[0066] Based on the protein molecular dynamics predictions, sites that significantly affect protein thermal stability were selected for mutation, including Q428K, Q428K / F334K, and Q428K / F334K / N387K. The mutation primers were designed as follows:

[0067] f-Q428K:GATCGTTATGATAAAAATGGAAACTT

[0068] r-Q428K: AAGTTTCCAT TTTTATCATA ACGATC

[0069] f-F334K: AAGTAACTAAAAAAATTATCTTGTCCTGATC

[0070] r-F334K: GATCAGGACA AGATAATTTT TTAGTTACTT

[0071] f-N387K:GAGTGGATGAAACCAGCAAC

[0072] r-N387K:GTTGCTGGTT TCATCCACTC

[0073] The mutant Q428K / F334K can be obtained by mutating F334K into the mutant Q428K, and the mutant Q428K / F334K / N387K can be obtained by mutating N387K into the mutant Q428K / F334K.

[0074] 1. In the first step of overlapping PCR, the upper and lower arms are performed separately. Taking Q428K as an example, the upstream reaction system is shown in Table 2, and the downstream reaction system is shown in Table 3:

[0075] Table 2: Reaction system of upstream reactants

[0076] Upstream primer LAC-F 2.0μL Downstream primer r-Q428K 2.0μL Wild-type laccase gene (pET-lac) 2.0μL Primer Star Max Enzyme 25μL <![CDATA[ddH2O]]> 19μL

[0077] Table 3: Reaction system of downstream reactants

[0078] Upstream primer f-Q428K 2.0μL Downstream primer LAC-R 2.0μL Wild-type laccase gene (pET-lac) 2.0μL Primer Star Max Enzyme 25μL <![CDATA[ddH2O]]> 19μL

[0079] The sequence of the upstream primer LAC-F is: AGATATA CCATGG GCGAGTTAA

[0080] The sequence of the downstream primer LAC-R is: GTGGTG CTCGAG GTTTGA

[0081] The amplification program was as follows: 98℃ pre-denaturation for 30 min; 98℃ denaturation for 10 s, 54℃ annealing for 20 s, 72℃ extension for 5 s, for 30 cycles; 72℃ extension for 10 min.

[0082] 2. After gel extraction and recovery of upstream and downstream fragments, PCR was performed. The reaction system is shown in Table 4.

[0083] Table 4: Reaction system for PCR after gel extraction and recovery of upstream and downstream fragments

[0084] Upstream segment 2.0μL Downstream segments 2.0μL Primer Star Max Enzyme 25μL <![CDATA[ddH2O]]> 21μL

[0085] The amplification program was as follows: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 54℃ annealing for 20s, 72℃ extension for 8s, for 5 cycles; 72℃ extension for 10min.

[0086] 3. After PCR, add 2 μL each of primers LAC-F and LAC-R to the system. The PCR amplification program is as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 54℃ annealing for 20 s, 72℃ extension for 10 s, for 30 cycles; 72℃ extension for 10 min. The PCR amplification products are subjected to 0.8% agarose gel electrophoresis and recovered using a small-volume DNA recovery kit to obtain the gene encoding lacm1, which is the site-directed mutant of laccase Q428K.

[0087] 4. The laccase site-directed mutant gene *lacm1* was ligated into the expression vector *pET* and transformed into JM109. The plasmid was then extracted to obtain the recombinant plasmid *pET-lacm1*, which was subsequently transformed into *E. coli* BL21. The transformed strains from BL21 were activated onto a newly streaked Kan plate and incubated upside down at 37°C for 12 hours. Under aseptic conditions, single colonies of the mutant were picked and inoculated into liquid LB medium containing Kan resistance. The plasmid was extracted and sequenced to confirm the sequence of the Q428K mutant gene *lacm1*. The obtained positive transformant was named BL21 / pET-lacm1.

[0088] 5. Using recombinant plasmid pET-lacm1 as a template, repeat the above overlapping PCR and chemical transformation verification steps with LAC-F, r-F334K, LAC-R, and f-F334K as upstream and downstream primers to obtain the gene encoding lacm2 of mutant Q428K / F334K, as well as recombinant plasmid pET-lacm2 and recombinant bacteria BL21 / pET-lacm2.

[0089] 6. Using recombinant plasmid pET-lacm2 as a template, repeat the above overlapping PCR and chemical transformation verification steps with LAC-F, r-N387D, LAC-R, and f-N387D as upstream and downstream primers to obtain the encoding gene lacm3 of the mutant Q428K / F334K / N387K, as well as the recombinant plasmid pET-lacm3 and the recombinant bacterium BL21 / pET-lacm3.

[0090] 7. And obtain wild-type recombinant laccase BL21 / pET-lac using the same method as above.

[0091] Example 3: Determination of wild-type laccase activity and temperature stability

[0092] ABTS assay for wild-type laccase activity

[0093] Measurement method:

[0094] (1) Take 200 µL of pH 5.5 citrate-disodium hydrogen phosphate buffer (50 mM, containing 5 mM Cu). 2+ In a 96-well microplate, incubate at 80°C for 1 min.

[0095] (2) Add 10 µL of LAC / LACM diluted to an appropriate concentration and mix well. Then place it in an 80℃ water bath for 1 min.

[0096] (3) Add 30 µL of ABTS (50 mM) and mix by blowing and suction. React in a constant temperature water bath at 80℃ for 10 min. Record the OD values ​​at the beginning and end of the reaction at 420 nm.

[0097] Calculate enzyme activity:

[0098]

[0099] In the formula: △OD = OD_end - OD_initial;

[0100] V1 represents the total volume of the reaction system (µL);

[0101] △t represents the reaction time (min);

[0102] V2. Add enzyme solution volume (µL);

[0103] ε represents the molar absorptivity of the product at 420 nm, which is 36 mM. -1 cm -1 ;

[0104] d represents the inner diameter / optical path thickness (cm) of the 96-well microplate.

[0105] Enzyme specific activity (U / mg) = enzyme activity / protein concentration.

[0106] Temperature stability determination of wild-type laccase

[0107] Wild-type laccase was placed in a pH 7.0 buffer solution and incubated at 80°C for different times. Samples were taken at regular intervals. After incubation, the residual enzyme activity was measured at pH 5.0 and 80°C. The enzyme activity without incubation was taken as 100%. The residual enzyme activity was calculated, and the enzyme activity change curve was plotted. The half-life was then calculated.

[0108] Example 4: Expression, purification, and enzyme activity assay of WT, Q428K, Q428K / F334K, and Q428K / F334K / N387K.

[0109] 1. Induced expression of recombinant strains

[0110] (1) On LB plates, pick one single colony of BL21 / pET-lacm3, BL21 / pET-lacm2, BL21 / pET-lacm1 and BL21 / pET-lac respectively, inoculate them into 5 mL LB test tubes (containing 50 µg / mL Kan), and place them in a shaker at 37℃ for 10 h;

[0111] (2) Transfer the recombinant bacterial culture to 250 mL LB medium (final concentration of 50 µg / mL Kan) and place it in a shaker at 37 ℃ for 2-2.5 h;

[0112] (3) Add 125 µL of IPTG (final concentration 0.5 mmol / L) and incubate in a shaker at 16 ℃ for 16-20 h;

[0113] (4) The fermentation broth was purified to prepare wild-type laccase and laccase mutant.

[0114] 2. Ni column purification of recombinant proteins

[0115] (1) Broken bacterial cells

[0116] Collect the fermentation broth using a centrifuge cup, centrifuge at 8000 rpm for 10 min, discard the supernatant, add 20 mL of Lysisbuffer to aspirate the bacterial cells, and use ultrasound to break down the bacterial cells, destroy the cell wall and release the intracellular proteins.

[0117] After disruption, the bacterial culture was poured into a 50 mL centrifuge tube and centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was then collected.

[0118] WT, Q428K, Q428K / F334K and Q428K / F334K / N387K are bonded with nickel pillars.

[0119] a. Before performing the nickel column purification process, add an appropriate amount of ddH2O to the purification column and add twice the column volume of Lysis buffer to balance the resin.

[0120] b. Mix the supernatant of the equilibrated resin and bacterial cells, place them in a magnetic stirrer, and combine at a speed of 80-100 r / min for 1 h, while maintaining a low temperature (4℃) throughout the process.

[0121] (3) Protein purification

[0122] a. Add the binding buffer to the purification column in 2-3 fractions in the chromatography cabinet;

[0123] b. After the binding buffer has been completely filtered out, add 10 mL of wash buffer to elute any extraneous proteins bound to the resin;

[0124] 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;

[0125] d. Transfer all eluent to an ultrafiltration centrifuge tube and centrifuge until 1 mL of solution remains. Add pre-chilled 50 mM, pH 7.0 Tris-HCl buffer and repeat the replacement twice. The purified WT, Q428K, Q428K / F334K, and Q428K / F334K / N387K proteins are then obtained.

[0126] The specific activity was determined according to Example 3, and the final calculated specific activities of wild-type laccase and mutant LACM3 are shown in Table 5.

[0127] Table 5: Specific activities of wild-type laccase and mutant LACM3

[0128] Laccase Enzyme activity (U / mg) WT 197.3 Q428K 333.6 Q428K / F334K 587.1 Q428K / F334K / N387K 713.7

[0129] Temperature stability was determined according to step (2) of Example 3, and the thermal stability at 80°C was obtained (i.e., the enzyme activity of WT and Q428K, Q428K / F334K and Q428K / F334K / N387K mutants was determined after incubation at 80°C for 30 min according to step (2) of Example 3). Enzyme activity change curves were plotted, and the residual enzyme activities of wild-type laccase and each mutant were finally calculated as shown in Table 6. Figure 3 As shown.

[0130] Table 6: Residual enzyme activity of wild-type laccase and each mutant

[0131] Laccase Residual enzyme activity (%) WT 0 Q428K 33 Q428K / F334K 31 Q428K / F334K / N387K 53

[0132] Example 5 Expression and preparation of laccase mutant in Bacillus subtilis

[0133] The genes encoding lacm1, lacm2, and lacm3 of the laccase mutants Q428K, Q428K / F334K, and Q428K / F334K / N387K, as well as the wild-type laccase gene lac, were ligated with the expression plasmid pBSA43 to obtain new recombinant plasmids PBSA43-lacm1, PBSA43-lacm2, PBSA43-lacm3, and PBSA43-lac;

[0134] The recombinant plasmids were transformed into Bacillus subtilis WB600, and after screening for kanamycin (Kan) resistance and enzyme digestion verification, mutant recombinant bacteria WB600 / PBSA43-lacm1, WB600 / PBSA43-lacm2, WB600 / PBSA43-lacm3 and wild-type laccase recombinant bacteria WB600 / PBSA43-lac were obtained.

[0135] Recombinant strains WB600 / PBSA43-lacm1, WB600 / PBSA43-lacm2, WB600 / PBSA43-lacm3, and WB600 / PBSA43-lac were inoculated into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL) and cultured overnight at 37°C and 220 rpm. Then, at a 2% inoculation rate, they were transferred to 50 mL of fresh fermentation medium (containing kanamycin, 50 µg / mL) and cultured for another 48 h at 37°C and 220 rpm. The fermentation medium (g / L) consisted of 64 g corn flour, 40 g soybean meal, 2.7 g amylase, 4 g Na₂HPO₄, 0.3 g KH₂PO₄, and the remainder was water. The culture was incubated at 90°C for 30 min and then sterilized at 121°C for 20 min.

[0136] The ATBS method described in Example 3 was used to determine the laccase activity obtained from Bacillus subtilis fermentation (the supernatant was collected after centrifugation of the fermentation broth for enzyme activity determination). The wild-type enzyme activity in Bacillus subtilis was 938.77 U / mL, while the fermentation enzyme activities of Q428K, Q428K / F334K, and Q428K / F334K / N387K were 1588.6 U / mL, 2791.8 U / mL, and 4702.55 U / mL, respectively.

[0137] Example 6 Expression and preparation of laccase mutant in recombinant Bacillus amyloliquefaciens strain

[0138] The genes encoding lacm1, lacm2, and lacm3 of the laccase mutants Q428K, Q428K / F334K, and Q428K / F334K / N387K, as well as the wild-type laccase gene lac, were ligated with the expression plasmid PBSA43 to obtain new recombinant plasmids PBSA43-lacm1, PBSA43-lacm2, PBSA43-lacm3, and PBSA43-lac.

[0139] The recombinant plasmids were transformed into Bacillus amyloliquefaciens CGMCCNo.11218, and after screening for kanamycin (Kan) resistance and enzyme digestion verification, mutant recombinant bacteria CGMCCNo.11218 / PBSA43-lacm1, CGMCCNo.11218 / PBSA43-lacm2 and CGMCCNo.11218 / PBSA43-lacm3 and wild-type laccase recombinant bacteria CGMCCNo.11218 / PBSA43-lac were obtained.

[0140] Recombinant strains CGMCCNo.11218 / PBSA43-lacm1, CGMCCNo.11218 / PBSA43-lacm2, CGMCCNo.11218 / PBSA43-lacm3, and CGMCCNo.11218 / PBSA43-lac were inoculated into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL) and cultured overnight at 37°C and 220 rpm. Then, at a 2% inoculation rate, the culture was transferred to 50 mL of fresh fermentation medium (containing kanamycin, 50 µg / mL) and cultured for another 48 h at 37°C and 220 rpm. (Fermentation medium (g / L): corn flour 64, soybean meal 40, with 2.7 g amylase, Na2HPO4 4, KH2PO4 0.3, and the remainder water; incubated at 90°C for 30 min and then sterilized at 121°C for 20 min).

[0141] The ATBS method described in Example 3 was used to determine the laccase activity obtained from Bacillus amyloliquefaciens fermentation (fermentation broth was centrifuged, and the supernatant was collected for enzyme activity determination). The wild-type Bacillus amyloliquefaciens enzyme activity was 1558.78 U / mL, while the fermentation enzyme activities of Q428K, Q428K / F334K, and Q428K / F334K / N387K were 2641.08 U / mL, 4637.88 U / mL, and 5661.24 U / mL, respectively.

[0142] Example 7: Expression and preparation of laccase mutants in recombinant Bacillus licheniformis strains

[0143] The genes encoding lacm1, lacm2, and lacm3 of the laccase mutants Q428K, Q428K / F334K, and Q428K / F334K / N387K, as well as the wild-type laccase gene lac, were ligated with the expression plasmid pBSA43 to obtain new recombinant plasmids pBSA43-lacm1, pBSA43-lacm2, pBSA43-lacm3, and pBSA43-lac;

[0144] The recombinant plasmids were transformed into Bacillus licheniformis 2709, and after screening for kanamycin (Kan) resistance and enzyme digestion verification, mutant recombinant bacteria 2709 / pBSA43-lacm1, 2709 / pBSA43-lacm2, 2709 / pBSA43-lacm3 and wild-type laccase recombinant bacteria 2709 / pBSA43-lac were obtained.

[0145] Recombinant strains 2709 / pBSA43-lacm1, 2709 / pBSA43-lacm2, 2709 / pBSA43-lacm3, and 2709 / pBSA43-lac were inoculated into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL) and cultured overnight at 37°C and 220 rpm. The inoculum was then transferred at a 2% inoculation rate to 50 mL of fresh fermentation medium (containing kanamycin, 50 µg / mL) and cultured for another 48 h at 37°C and 220 rpm. (Fermentation medium (g / L): corn flour 64 g, soybean meal 40 g, with 2.7 g amylase, Na2HPO4 4 g, KH2PO4 0.3 g, and the remainder water; incubated at 90°C for 30 min and then sterilized at 121°C for 20 min).

[0146] The ATBS method described in Example 3 was used to determine the laccase activity obtained from Bacillus licheniformis fermentation (the supernatant was collected after centrifugation of the fermentation broth as crude enzyme solution for enzyme activity determination). The wild-type enzyme activity in Bacillus licheniformis was 2269.8 U / mL, while the fermentation enzyme activities of Q428K, Q428K / F334K, and Q428K / F334K / N387K were 3847.31 U / mL, 6802.1 U / mL, and 8216.7 U / mL, respectively.

[0147] Example 8: Expression and preparation of laccase mutant in Pichia pastoris GS115 recombinant strain

[0148] The genes encoding lacm1, lacm2, and lacm3 of the laccase mutants Q428K, Q428K / F334K, and Q428K / F334K / N387K, as well as the wild-type laccase gene lac, were ligated with the expression plasmid pPIC9K to obtain new recombinant plasmids pPIC9K-lacm1, pPIC9K-lacm2, pPIC9K-lacm3, and pPIC9K-lac.

[0149] The recombinant plasmids pPIC9K-lacm1, pPIC9K-lacm2, pPIC9K-lacm3, and pPIC9K-lac were linearized using the restriction endonuclease SalI. The linearized recombinant plasmids were then electroporated into Pichia pastoris GS115. After screening for kanamycin (Kan) resistance, the mutant recombinant strains GS115 / pPIC9K-lacm1, GS115 / pPIC9K-lacm2, GS115 / pPIC9K-lacm3 and the wild-type laccase recombinant strain GS115 / pPIC9K-lac were obtained by restriction enzyme digestion verification.

[0150] (1) Select single colonies of recombinant expression strains GS115 / pPIC9K-lacm1, GS115 / pPIC9K-lacm2, GS115 / pPIC9K-lacm3 and GS115 / pPIC9K-lac, and inoculate them into 5 mL YPD tubes containing 50 µg / mL Kan. Incubate at 30 °C and 200 r / min for 24 h.

[0151] (2) Take 1 mL of bacterial culture and inoculate it into BMGY enrichment medium, and incubate at 30℃ and 220 r / min for 16-19 h;

[0152] (3) Collect the bacterial culture in BMGY medium with a 50 mL centrifuge tube, and collect the bacterial cells by centrifugation; add 20 mL of BMMY medium to resuspend the bacterial cells, centrifuge to collect the bacteria, and repeat once; add 10 mL of BMMY medium to resuspend the bacterial cells, transfer the bacterial culture to BMMY medium with a pipette, add methanol to a final concentration of 0.5% every 12 h, and ferment for 6 days.

[0153] The ATBS method described in Example 3 was used to determine the laccase activity obtained from Pichia pastoris fermentation (the supernatant was collected after centrifugation of the fermentation broth for enzyme activity determination). In Pichia pastoris GS115, the wild-type laccase activity was 572.2 U / mL, while the fermentation activities of Q428K, Q428K / F334K, and Q428K / F334K / N387K were 967.8 U / mL, 1702.8 U / mL, and 2075.9 U / mL, respectively.

[0154] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A laccase mutant, characterized in that, The wild-type laccase was obtained by undergoing the following mutations: Q428K, Q428K / F334K, or Q428K / F334K / N387K; wherein the amino acid sequence of the wild-type laccase is shown in SEQ ID NO.

1. The amino acid sequence of the Q428K mutant is shown in SEQ ID NO.3; the amino acid sequence of the Q428K / F334K mutant is shown in SEQ ID NO.5; and the amino acid sequence of the Q428K / F334K / N387K mutant is shown in SEQ ID NO.

7.

2. A polynucleotide, characterized in that, Encodes the laccase mutant according to claim 1.

3. A recombinant plasmid, characterized in that, It comprises the polynucleotide as described in claim 2.

4. The recombinant plasmid according to claim 3, characterized in that, The expression vector for the recombinant plasmid is any one of pET28a(+), pBSA43, or pPIC9K.

5. A host cell, characterized in that, The host cell comprises the polynucleotide of claim 2 and is not an animal or plant species.

6. A host cell, characterized in that, The recombinant plasmid comprising any one of claims 3-4, wherein the host cell is not an animal or plant species.

7. The host cell according to claim 5 or 6, characterized in that, The host cell is a prokaryotic cell or a eukaryotic cell.

8. The host cell according to claim 7, characterized in that, The host cell is any one or more of Escherichia coli, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, or Pichia pastoris.

9. A genetically engineered bacterium, characterized in that, It comprises the polynucleotide as described in claim 2.

10. A genetically engineered bacterium, characterized in that, It includes the recombinant plasmid according to any one of claims 3-4.

11. The application of the laccase mutant as described in claim 1 in any one of papermaking bleaching, textile dyeing wastewater decolorization, or biodegradation of pollutants.

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