High-temperature-resistant laccase mutant, gene, engineering bacteria, preparation method and application thereof

Abstract

The application belongs to the technical field of bioengineering, and particularly relates to a high-temperature-resistant laccase mutant obtained by site-directed mutation through an overlapping PCR technique, and preparation and application thereof. The laccase mutant is obtained on the basis of a wild-type laccase shown in SEQ ID NO. 1, and at least one of D221Y, E231D, Y441H or Y441F mutations occurs, and the mutant enzyme activity and the thermal stability at 80 DEG C are both improved to different degrees relative to the wild type, which is beneficial to expand the application in the industrial fields of degrading lignocellulose, paper pulp bleaching and the like.

Description

Technical fields:

[0001] This invention belongs to the field of bioengineering technology, specifically relating to the preparation and application of heat-resistant laccase mutants obtained by site-directed mutagenesis using overlapping PCR technology. Background technology:

[0002] Laccase is a polyphenol oxidase that catalyzes the degradation of phenols, amines, and aromatic compounds. Its structure and function are similar to ascorbic acid oxidase and ceruloplasmin, and it belongs to the multi-copper oxidase (MCOs) family. In the late 19th century, Benfield first isolated and named it laccase from the solidified form of raw lacquer. Laccase contains four copper atoms, enabling it to undergo electron transfer reactions without generating other byproducts. Therefore, laccase is widely used in industries such as the degradation of lignocellulose, pulp bleaching, and the food industry.

[0003] Laccase has a low redox potential, enabling it to directly oxidize some phenolic substrates, but it cannot directly oxidize non-phenolic substrates with higher redox potentials. Therefore, to broaden the range of laccase oxidation, certain low-molecular-weight compounds (mediators) need to be added as aids. The mediators are oxidized by laccase into free radicals during the cycle. These free radicals act as electron carriers, reacting with phenols and other compounds to oxidize them. This reaction system is called the laccase-mediator system (LMS).

[0004] Laccases have a wide range of sources, primarily originating from plants, insects, and microorganisms in nature. Plant laccases are mainly isolated from lacquer trees, tea trees, cotton, rice, and pears, playing important roles in resisting various adverse environments, increasing crop yields, and in plant lignin synthesis. Insect laccases are the main research focus for animal laccases; laccase activity has been found in insects such as the tobacco hawk moth, mosquitoes, cotton bollworms, and locusts. Fungal laccases are widely distributed in species such as *Basidiomyeetes*, *Asomyeetes*, *Neurospora*, *Aspergillus*, and *Phanerochaetc. chrysosporium*, with white-rot fungi being the most studied. Bacterial laccases have been successively discovered in genera such as *Bacillus*, *Strepotomyces*, *Marinomonas*, *Enterobacter*, *Proteobacterium*, and *Alteromonas*.

[0005] Currently, most commercially available laccases are fungal in origin. Most fungal laccases function better under mild conditions, with an optimal temperature between 30-60℃ and an optimal pH between 3.0-6.0. The application of fungal laccases in high-temperature and low-pH environments is limited. Bacterial laccases, on the other hand, exhibit better thermal and pH stability. Among bacterial laccases, Bacillus-derived laccases possess even better thermal and pH stability, thus enabling their application in important fields such as lignocellulose degradation, pulp bleaching, and wastewater treatment. Therefore, further improving the stability of bacterial laccases would be beneficial for their industrial applications.

[0006] Irrational protein molecular design involves inducing numerous mutations in a gene under certain conditions without knowing the protein's three-dimensional structure and mechanism of action, followed by targeted selection of mutants with desired characteristics through multiple rounds of high-throughput screening. Rational molecular design, on the other hand, involves targeted mutations of a gene sequence most likely to affect a protein's function and properties, based on known three-dimensional structure and function. This aims to intentionally alter one or two amino acid residues or modules of the protein, thereby constructing new protein molecules. Compared to irrational design, rational design has the advantages of requiring less work and more readily obtaining effective mutants.

[0007] Bacillus expression systems have the following advantages: 1. They can efficiently secrete various proteins; 2. Many Bacillus species have been used in the fermentation industry for a considerable period of time, are non-pathogenic, and do not produce any endotoxins; 3. The genetic background of Bacillus microorganisms is well understood, and they grow rapidly with no special requirements for nutrients; 4. They do not exhibit significant codon bias; 5. The fermentation process is simple. Bacillus is an aerobic bacterium, so anaerobic fermentation equipment is not required. After fermentation, the fermentation broth and bacterial cells can be easily separated to proceed to the separation, purification, and recovery stage of the target protein; 6. They are resistant to stress and can produce a variety of heat-resistant enzyme preparations.

[0008] Therefore, in this invention, a thermostable laccase mutant gene was obtained by rationally designing and screening mutations in a laccase gene derived from Bacillus Safensis. Subsequently, a heat-resistant mutant laccase was prepared by expressing the gene using a Bacillus expression system. Summary of the Invention:

[0009] To address the challenges of high-temperature applications of laccase, and to obtain laccase with improved thermostability, further modifications to its existing properties are necessary. This invention aims to provide a thermostable laccase mutant. The laccase gene (lac) from *Bacillus safensis* was expressed in *Escherichia coli* BL21. Overlap PCR was used to mutate the *lac* gene, and the mutants were screened using ABTS to select those with improved thermostability. This resulted in the expression and preparation of laccase in *Bacillus subtilis* WB600 and *Bacillus amyloliquefaciens* CGMCC No. 11218.

[0010] The technical approach to achieving the objective of this invention is summarized as follows:

[0011] The laccase gene *lac* from *Bacillus safensis* was mutated, and mutants D221Y, E231D, Y441H, Y441F, E231D / Y441H, D221Y / Y441H, D221Y / Y441F, E231D / Y441F, and D221Y / E231D were obtained using an *E. coli* expression system. These mutants showed increased half-lives at 80°C to 192%, 143%, 168%, 140%, 237%, 158%, 166%, 167%, and 168% of the wild-type laccase (LAC), respectively. The encoding genes *lacm1*, *lacm2*, *lacm3*, *lacm4*, *lacm5*, *lacm6*, *lacm7*, *lacm8*, and *lacm9* were also obtained. The optimal mutant laccase E231D / Y441H was efficiently prepared using Bacillus subtilis WB600 and Bacillus amyloliquefaciens CGMCC No.11218.

[0012] One of the technical solutions provided by the present invention is a laccase mutant, which is obtained by undergoing at least one of the following mutations, namely D221Y, E231D, Y441H or Y441F, on the basis of the wild-type laccase shown in SEQ ID NO.1.

[0013] Furthermore, the laccase mutant is a D221Y mutant, and its amino acid sequence is shown in SEQ ID NO.3;

[0014] Furthermore, the nucleotide sequence of the gene lacm1 encoding the D221Y mutant is shown in SEQ ID NO.4;

[0015] Furthermore, the laccase mutant is the E231D mutant, with the amino acid sequence shown in SEQ ID NO.5;

[0016] Furthermore, the nucleotide sequence of the gene lacm2 encoding the E231D mutant is shown in SEQ ID NO.6;

[0017] Furthermore, the laccase mutant is the Y441H mutant, and its amino acid sequence is shown in SEQ ID NO.7;

[0018] Furthermore, the nucleotide sequence of the gene lacm3 encoding the Y441H mutant is shown in SEQ ID NO.8;

[0019] Furthermore, the laccase mutant is the Y441F mutant, and its amino acid sequence is shown in SEQ ID NO.9;

[0020] Furthermore, the nucleotide sequence of the gene lacm4 encoding the Y441F mutant is shown in SEQ ID NO.10;

[0021] Furthermore, the laccase mutant is an E231D / Y441H mutant, and its amino acid sequence is shown in SEQ ID NO.11;

[0022] Furthermore, the nucleotide sequence of the gene lacm5 encoding the E231D / Y441H mutant is shown in SEQ ID NO. 12;

[0023] Furthermore, the laccase mutant is a D221Y / Y441H mutant, and its amino acid sequence is shown in SEQ ID NO.13;

[0024] Furthermore, the nucleotide sequence of the gene lacm6 encoding the D221Y / Y441H mutant is shown in SEQ ID NO. 14;

[0025] Furthermore, the laccase mutant is a D221Y / Y441F mutant, with the amino acid sequence shown in SEQ ID NO. 15;

[0026] Furthermore, the nucleotide sequence of the coding gene lacm7 of the D221Y / Y441F mutant is shown in SEQ ID NO. 16;

[0027] Furthermore, the laccase mutant is an E231D / Y441F mutant, with the amino acid sequence shown in SEQ ID NO.17;

[0028] Furthermore, the coding gene lacm8 of the E231D / Y441F mutant has the nucleotide sequence shown in SEQ ID NO. 18;

[0029] Furthermore, the laccase mutant is a D221Y / E231D mutant, and its amino acid sequence is shown in SEQ ID NO.19;

[0030] Furthermore, the coding gene lacm9 of the D221Y / E231D mutant has the nucleotide sequence shown in SEQ ID NO. 20.

[0031] The second technical solution provided by the present invention is a recombinant plasmid or recombinant strain containing the above-mentioned mutant encoding gene;

[0032] Furthermore, the expression vector used was pET-28a(+), and the host was Escherichia coli;

[0033] Furthermore, the host cell is Escherichia coli BL21;

[0034] Furthermore, the host cell is Bacillus subtilis WB600, or the host cell is Bacillus amyloliquefaciens CGMCC No.11218;

[0035] Preferably, the recombinant strain is obtained by linking the mutant coding gene with the expression vector pET-28a(+) and then expressing it in host Escherichia coli.

[0036] The third technical solution provided by this invention is the application of the above-mentioned recombinant plasmid or recombinant strain in the laccase mutant described in the first technical solution.

[0037] The fourth technical solution provided by this invention is the application of the laccase mutant described in technical solution one, particularly in the industrial fields of degrading lignocellulose and pulp bleaching; more specifically, it is the application of wild-type LAC or mutant laccase in synergistic degradation of corn stalks or bagasse using the mediator 1-hydroxybenzotriazole (HBT) with commercial cellulase.

[0038] In this invention, the following definitions are used:

[0039] 1. Nomenclature of amino acids and DNA nucleic acid sequences

[0040] The IUPAC nomenclature, a recognized system for naming amino acid residues, is used, employing single-letter or three-letter codes. DNA nucleic acid sequences are named using the IUPAC nomenclature.

[0041] 2. Identification of laccase mutants

[0042] The term "amino acid replaced at the original amino acid position" is used to represent the mutated amino acid in the LAC mutant. For example, E231D indicates that the amino acid at position 231 is replaced by Asp from Glu in wild-type LAC, and the position number corresponds to the amino acid sequence number of the mature peptide of wild-type LAC in SEQ ID NO. 1.

[0043] In this invention, LAC represents wild-type laccase, LACM represents laccase mutant, lowercase italic lac represents the coding gene of wild-type laccase LAC, lowercase italic lacm1 represents the coding gene of mutant D221Y, and lowercase italic lacm2, lacm3...lacm9 represent the coding genes of mutants E231D, Y441H...D221Y / E231D, respectively. Specific information is shown in the table below.

[0044]

[0045] Beneficial effects:

[0046] This invention utilizes overlap PCR technology to mutate wild-type LAC, obtaining mutants D221Y, E231D, Y441H, Y441F, E231D / Y441H, D221Y / Y441H, D221Y / Y441F, E231D / Y441F, and D221Y / E231D with improved thermostability at 80℃ compared to the wild-type laccase (LAC). The half-life at 80℃ is increased to 192%, 143%, 168%, 140%, 237%, 158%, 166%, 167%, and 168% of that of the wild-type laccase (LAC), respectively. Attached image description:

[0047] Figure 1 The image shows the PCR amplification electrophoresis diagram of wild-type laccase, where M is the DNA Marker, lane 1 is the pET-28a(+) PCR product, and lane 2 is the laccase gene lac.

[0048] Figure 2 Image for pET-lac plasmid digestion verification

[0049] Where: M is DNA Marker, and 1 is pET-lac digestion map after double digestion with NcoI and XhoI. Detailed implementation method:

[0050] The technical content of the present invention will be further described below with reference to the embodiments. However, the present invention is not limited to these embodiments, and the scope of protection of the present invention cannot be limited by the following embodiments.

[0051] The solutions and culture media used in the embodiments of this invention are as follows:

[0052] Lysis buffer (mM): Tris 20, NaCl 500, dithiothreitol 1, imidazole 20.

[0053] Wash buffer (mM): Tris 20, NaCl 500, dithiothreitol 1, imidazole 100.

[0054] Elution Buffer (mM): Tris 20, NaCl 500, dithiothreitol 1, imidazole 500.

[0055] LB medium (g / L): yeast extract 5.0, tryptone 10.0, NaCl 10.0, the remainder being water. Solid medium supplemented with 2% agar.

[0056] Fermentation medium (g / L): corn flour 64, soybean meal 40, with the addition of 2.7 amylase, 4Na2HPO4, 0.3KH2PO4, and the remainder being water; incubate at 90℃ for 30 min and then sterilize at 121℃ for 20 min.

[0057] In this invention, the mature peptide sequence of wild-type laccase LAC is shown in SEQ ID NO.1: MNLEKFVDELPIPEVAEPVKKNPRQTYYEIAMEEVFLKVHRDLPPTKLWTYNGSLPGPTIKANRNEKVKVKWMNKLPLKHFLPVDHTIHSSHHDEPEVKTVVHLHGGVTPASSDGYPEAWFSRDFEATGPFFERETYVYPNHQQACTLWYHDHAMALTRLNVYAGLAGFYLISDAFEKSLELPKDDYDIPLMIMDRTFQEDGSLFYPSRPNDTPEDSDIPDPSIVPFFCGETILVNGKVWPYLEVEPRKYRF RILNASNTRTYELHLDNDATILQIGSDGGFLPRPVRHQSFSIAPAERFDVIIDFSAYENKTITLKNTAGCGQDVNPETDANIMQFKVTRPLKGRVPKTLRPIFKPLPPLRPSRADRERKLTLTGTQDKY GRPILLLDNHFWNDPVTENPRLGSLEVWSIVNPTRGTHPIHLHLVQFRVLDRRPFDTEVYQSTGEIVYTGPNEAPPLHEQGYKDTIQAHAGEVIRIVARFVPYSGRYVWHCHILEHEDYDMMRPMDIIQ.

[0058] In this invention, the mature peptide sequence of the E231D / Y441H mutant is shown in SEQ ID NO.11: MNLEKFVDELPIPEVAEPVKKNPRQTYYEIAMEEVFLKVHRDLPPTKLWTYNGSLPGPTIKANRNEKVKVKWMNKLPLKHFLPVDHTIHSSHHDEPEVKTVVHLHGGVTPASSDGYPEAWFSRDFEATGPFFERETYVYPNHQQACTLWYHDHAMALTRLNVYAGLAGFYLISDAFEKSLELPKDDYDIPLMIMDRTFQEDGSLFYPSRPNDTPEDSDIPDPSIVPFFCGDTILVNGKVWPYLEVEPRKYR FRILNASNTRTYELHLDNDATILQIGSDGGFLPRPVRHQSFSIAPAERFDVIIDFSAYENKTITLKNTAGCGQDVNPETDANIMQFKVTRPLKGRVPKTLRPIFKPLPPLRPSRADRERKLTLTGTQDKY GRPILLLDNHFWNDPVTENPRLGSLEVWSIVNPTRGTHPIHLHLVQFRVLDRRPFDTEVHQSTGEIVYTGPNEAPPLHEQGYKDTIQAHAGEVIRIVARFVPYSGRYVWHCHILEHEDYDMMRPMDIIQ.

[0059] The present invention will be further explained and illustrated below through specific embodiments.

[0060] Example 1: Obtaining the wild-type laccase gene

[0061] 1. Genomic DNA was extracted from Bacillus Safensis TCCC 111022 using the OMEGA Bacterial DNA Kit. The extraction steps are as follows:

[0062] (1) Inoculate the strain onto LB solid plates with an inoculation loop and incubate overnight at 37°C.

[0063] (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.

[0064] (3) Take 3-5 mL of bacterial solution and place it in a sterilized EP tube. Centrifuge at 12000 r / min for 2 min and discard the supernatant.

[0065] (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.

[0066] (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.

[0067] (6) Add 5 μL of RNase, invert and mix several times, and incubate at room temperature for 10 min.

[0068] (7) Centrifuge at 12000r / min for 2min to remove undigested portion, transfer the supernatant to a new EP tube, add 220μL BDL buffer, and incubate at 65℃ for 15min.

[0069] (8) Add 220 μL of anhydrous ethanol and mix by blowing and sucking.

[0070] (9) Transfer the liquid in the EP tube into 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.

[0071] (10) Add 500 μL HBC buffer, centrifuge at 12000 r / min for 1 min, and discard the filtrate.

[0072] (11) Add 700 μL DNA wash buffer, let stand for 1 min, centrifuge at 12000 r / min for 1 min, and discard the filtrate.

[0073] (12) Add 500 μL DNAwash buffer, let stand for 1 min, centrifuge at 12000 r / min for 1 min, and discard the filtrate.

[0074] (13) 12000r / min for 2min, discard the waste liquid tube, and put the recovery column into a new EP tube.

[0075] (14) Place in a 55℃ metal bath to dry for 10 minutes.

[0076] (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.

[0077] 2. Using the extracted genome of *Bacillus sabolicus* 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:

[0078] Upstream primer P1: 5'-CATGCCATGGATGAACCTAGAAAAATTTGTTGAC-3'

[0079] Downstream primer P2: 5'-CCGTCGAGCTGAATGATATCCATCGGTCT-3'

[0080] Using P1 and P2 as upstream and downstream primers, amplification was performed using the laccase genome of Bacillus salsa as a template.

[0081] The amplification reaction system is as follows:

[0082] upstream primer P1 2.0μL Downstream primer P2 2.0μL Genomic DNA as template 2.0μL Primer Star Max Enzyme 25μL <![CDATA[ddH2O]]> 19μL

[0083] 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. lac and the pET-28a(+) plasmid were double-digested with restriction endonucleases NcoI and XhoI, respectively. The lac recovered from the gel was ligated into 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 JM109 and Escherichia coli BL21.

[0084] Example 2: Construction of Laccase Mutants

[0085] Based on the protein kinetics simulation results, amino acid residues that have a significant impact on protein stability were selected for mutation, including D221Y, E231D, Y441H, and Y441F. Mutation primers were designed according to different mutation sites as shown in Table 1 below.

[0086] Table 1 lists some of the mutation primers:

[0087]

[0088] 1. Site-directed mutagenesis was performed using overlap PCR to construct the laccase mutant LACM. Taking the D221Y mutation as an example, the following mutation primers were designed:

[0089] Mutated upstream primer D221Y-F:

[0090] 5'-ACATACCACCGCCATCCAT-3'

[0091] Mutated downstream primer D221Y-R:

[0092] 5'-ATGGATGGCGGTGGTATGT-3'

[0093] In the first step of the overlap PCR reaction system, P1 and 221-R were used as upstream and downstream primers, respectively, and P2 and 221-F were used as upstream and downstream primers, respectively. Using plasmid pET-lac as a template, PCR1 reaction was performed to obtain the upstream and downstream fragments, respectively.

[0094] The reaction system for upstream fragment amplification is as follows:

[0095] P1 2μL 221-R 2μL Wild-type laccase gene 2μL Primer Star Max Enzyme 25μL <![CDATA[ddH2O]]> 19μL

[0096] The reaction system for downstream fragment amplification is as follows:

[0097] P2 2μL 221-F 2μL Wild-type laccase gene 2μL Primer Star Max Enzyme 25μL <![CDATA[ddH2O]]> 19μL

[0098] 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.

[0099] 2. After gel extraction and recovery of upstream and downstream fragments, PCR 2 was performed. The reaction system was as follows:

[0100] Upstream segment 2.0μL Downstream segments 2.0μL Primer Star Max Enzyme 25μL <![CDATA[ddH2O]]> 21μL

[0101] 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.

[0102] 3. After PCR 2, add 2 μL each of primers P1 and P2 to the system. The PCR 3 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 laccase site-directed mutant gene lacm1.

[0103] 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*. This recombinant plasmid was further transformed into *E. coli* BL21 to obtain the recombinant strain BL21 / pET-lacm1. The transformed strains from BL21 were activated onto a newly streaked Kan plate and incubated upside down at 37°C for 12 hours. Then, 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 D221Y mutant gene *lacm1*. The obtained positive transformant was named *E. coli* BL21 / pET-lacm1.

[0104] 5. The acquisition of the coding genes of the remaining mutants is the same as in steps 1-4, simply by replacing the corresponding primers according to Table 1. Thus, positive transformants E. coli BL21 / pET-lacm2 containing the E231D mutant gene lacm2; positive transformants E. coli BL21 / pET-lacm3 containing the Y441H mutant gene lacm3; and positive transformants E. coli BL21 / pET-lacm4 containing the Y441F mutant gene lacm4 were prepared.

[0105] Example 3: Expression, purification, and enzyme activity assay of LACM

[0106] 1. Induced expression of recombinant strains

[0107] (1) On an LB plate, pick one single colony of each E. coli BL21 / pET-lacm obtained in step 5 of Example 2, inoculate it into a 5 mL LB tube (containing 50 μg / mL Kan), and place it in a shaker at 37°C for 10 h.

[0108] (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.

[0109] (3) Add 125 μL of IPTG (final concentration 0.5 mmol / L) and incubate in a shaker at 16℃ for 16-20 h;

[0110] (4) At the same time, E. coli BL21 / pET-lac was cultured as a control. The fermentation broth was used to purify and prepare wild-type laccase and various laccase mutants.

[0111] 2. Ni column purification of recombinant proteins

[0112] (1) Broken bacterial cells

[0113] Collect the fermentation broth using a centrifuge cup, centrifuge at 10,000 rpm for 15 minutes, 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.

[0114] 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 collected, and samples were taken for SDS-PAGE analysis. The results were used to determine whether LAC / LACM was expressed.

[0115] (2) LAC / LACM bonded with nickel pillar

[0116] 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.

[0117] 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 hour, while maintaining a low temperature (4℃) throughout the process.

[0118] (3) Protein purification

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

[0120] b. After the binding solution has been 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-cooled Elution Buffer to the purification column to elute all the target protein bound to the resin and collect the filtrate;

[0122] d. Transfer all eluent to an ultrafiltration centrifuge tube and centrifuge until 1 mL of solution remains. Add pre-cooled 50 mM Tris-HCl buffer (pH 7.0) and repeat the replacement process twice. The purified LAC / LACM protein is then obtained.

[0123] 3. ABTS assay for LAC / LACM enzyme activity

[0124] Measurement method:

[0125] (1) Take 200 μL of citrate-disodium hydrogen phosphate buffer (50 mM, containing 5 mM Cu). 2+ In a 96-well microplate (pH 5.0), incubate at 80°C for 1 min.

[0126] (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.

[0127] (3) Add 30 μL of ABTS (50 mM) and mix by blowing and suction. React in a constant temperature water bath at 80 °C for 10 min. Record the OD values ​​at the beginning and end of the reaction at 420 nm.

[0128] Calculate enzyme activity:

[0129]

[0130] In the formula: △OD = OD_end - OD_initial;

[0131] V1 represents the total volume of the reaction system (μL);

[0132] △t represents the reaction time (min);

[0133] V2 is added to the enzyme solution in μL;

[0134] ε represents the molar absorptivity of the product at 420 nm, which is 36 mM. -1 cm -1 ;

[0135] d represents the inner diameter / optical path thickness (cm) of the 96-well microplate.

[0136] Enzyme specific activity (U / mg) = enzyme activity / protein concentration.

[0137] The final calculated specific activities of wild-type LAC and each single mutant are as follows:

[0138] Laccase Enzyme activity (U / mg) WT 6.6 D221Y 9.8 E231D 8.2 Y441H 12.4 Y441F 8.5

[0139] 4. Using ABTS as a substrate, the thermal stability of LACM was determined. The enzyme activity of LACM was measured after different time periods at 80℃. The activity of LACM without incubation was taken as 100%, and the corresponding residual enzyme activity was calculated.

[0140] Testing revealed that mutants D221Y, E231D, Y441H, and Y441F exhibited higher thermal stability at 80℃ than the wild type. The final calculated half-lives of the wild-type LAC and each mutant are as follows:

[0141] Laccase Half-life (min) WT 42.7 D221Y 82.2 E231D 61.1 Y441H 71.9 Y441F 59.8

[0142] Following the method in Example 2, combined mutations were performed on the aforementioned single mutants with improved thermal stability to obtain the coding genes for mutants E231D / Y441H, D221Y / Y441H, D221Y / Y441F, E231D / Y441F, and D221Y / E231D, as well as recombinant bacteria: a positive transformant *E. coli* BL21 / pET-lacm5 containing the *lacm5* gene of the E231D / Y441H mutant; a positive transformant *E. coli* BL21 / pET-lacm6 containing the *lacm6* gene of the D221Y / Y441H mutant; a positive transformant *E. coli* BL21 / pET-lacm7 containing the *lacm7* gene of the D221Y / Y441F mutant; and a positive transformant *E. coli* containing the *lacm8* gene of the E231D / Y441F mutant. BL21 / pET-lacm8; a positive transformant of E. coli BL21 / pET-lacm9 containing the D221Y / E231D mutant gene lacm9.

[0143] After expression and purification according to the method in Example 3, the specific enzyme activity of the combined mutant laccase was determined, and the specific enzyme activities of wild-type LAC and each combined mutant were calculated as follows:

[0144]

[0145]

[0146] 5. The above-mentioned combined mutants E231D / Y441H, D221Y / Y441H, D221Y / Y441F, E231D / Y441F, and D221Y / E231D were subjected to thermal stability testing according to Example 3. The final calculated half-life at 80°C for the wild type and each combined mutant is as follows:

[0147] Laccase Half-life (min) WT 42.7 E231D / Y441H 101.6 D221Y / Y441H 71.1 D221Y / Y441F 71.5 E231D / Y441F 72.0 D221Y / E231D 64.5

[0148] Example 4: Expression and preparation of laccase mutant in Bacillus subtilis

[0149] The laccase mutant E231D / Y441H encoding gene lacm5 and the wild-type laccase encoding gene lac were respectively ligated with expression plasmid pLY-3 to obtain new recombinant plasmids pLY-3-lacm5 and pLY-3-lac.

[0150] The recombinant plasmids were transformed into Bacillus subtilis WB600, and after screening for kanamycin (Kan) resistance and enzyme digestion verification, mutant recombinant strain WB600 / pLY-3-lacm5 and wild-type laccase recombinant strain WB600 / pLY-3-lac were obtained.

[0151] Recombinant strains WB600 / pLY-3-lacm5 and WB600 / pLY-3-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, soybean meal 40, with 2.7 g / L amylase, 4 g / L Na2HPO4, 0.3 g / L KH2PO4, and the remainder water; incubated at 90°C for 30 min and then sterilized at 121°C for 20 min).

[0152] The ATBS method described in Examples 3-3 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 34.3 U / mL, and the fermentation enzyme activity of E231D / Y441H was 79.5 U / mL.

[0153] Example 5: Expression and preparation of laccase mutants in recombinant Bacillus amyloliquefaciens strains

[0154] The laccase mutant E231D / Y441H encoding gene lacm5 and the wild-type laccase encoding gene lac were respectively ligated with expression plasmid pLY-3 to obtain new recombinant plasmids pLY-3-lacm5 and pLY-3-lac.

[0155] The recombinant plasmids were transformed into Bacillus amyloliquefaciens CGMCC No.11218, and after screening for kanamycin (Kan) resistance and enzyme digestion verification, the mutant recombinant strain CGMCC No.11218 / pLY-3-lacm5 and the wild-type laccase recombinant strain CGMCC No.11218 / pLY-3-lac were obtained.

[0156] Recombinant strains CGMCC No.11218 / pLY-3-lacm5 and CGMCC No.11218 / pLY-3-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, soybean meal 40, with 2.7% amylase, 4% Na2HPO4, 0.3% KH2PO4, and the remainder water; incubated at 90°C for 30 min and then sterilized at 121°C for 20 min).

[0157] The activity of alkaline protease obtained from Bacillus amyloliquefaciens fermentation was determined using the ATBS method described in Examples 3-3 (the supernatant was collected after centrifugation of the fermentation broth for enzyme activity determination). The enzyme activity of wild-type Bacillus amyloliquefaciens was 67.9 U / mL, and the enzyme activity of E231D / Y441H fermentation was 160.6 U / mL.

[0158] Example 6: Application of laccase mutant in the degradation of corn stalks and sugarcane bagasse

[0159] 1. Pre-treatment of corn stalks and sugarcane bagasse

[0160] The corn stalks and sugarcane bagasse are sourced from local companies in Jiangsu, China. First, they are washed with deionized water to remove surface dirt, then placed in a 60°C constant-temperature drying oven to dry them until the moisture content is below 2%. The dried raw materials are then pulverized using a grinder and filtered through a 40-mesh sieve to ensure they become uniform powder. Finally, they are left at room temperature for later use.

[0161] 2. Laccase-cellulase synergistic enzymatic saccharification

[0162] Treated corn stalks and sugarcane bagasse were used as samples to be degraded. Wild-type laccase and mutant laccase E231D / Y441H of this invention were added respectively. The saccharification efficiency of reducing sugars was measured with and without the mediator 1-hydroxybenzotriazole (HBT) and commercial cellulase (Cellic CTec2). Details are as follows:

[0163] The experiment was divided into three groups:

[0164] (1) Sample to be degraded + commercial cellulase;

[0165] (2) Sample to be degraded + WT / mutant laccase + commercial cellulase;

[0166] (3) Sample to be degraded + WT / mutant laccase + HBT + commercial cellulase;

[0167] The saccharification reaction was performed in a 20 mL system: Based on the experimental groups described above, each component was added / omitted according to the following dosages. 0.5 g of the sample to be degraded was placed in a 50 mL centrifuge tube, and 50 U of WT / mutant laccase, 5 mM mediator HBT, and 2.5 U of commercial cellulase were added. Then, a solution containing 5 mM Cu at pH 5.0 was added. 2+ The citrate-disodium hydrogen phosphate buffer solution was brought up to 20 mL, and the mixture was reacted in a water bath at 50 °C and 150 r / min for 6 h. After the reaction was completed, a sample was taken and boiled for 10 min to terminate the reaction. The supernatant was centrifuged and the reducing sugar concentration was determined using the dinitrosalicylic acid (DNS) method.

[0168] 1. Saccharification results in corn stalks

[0169] Compared with cellulase treatment alone, the addition of laccase and cellulase increased the reducing sugar content of mutant E231D / Y441H by 136% and WT by 24%. When HBT mediator was added, the reducing sugar content of mutant E231D / Y441H increased by 223% and WT by 158%.

[0170] Group Relative content of reducing sugars (%) Cellulase 100 Cellulase + WT 124 Cellulase + E231D / Y441H 236 Cellulase + WT + HBT 258 Cellulase + E231D / Y441H + HBT 323

[0171] 2. Saccharification results in sugarcane bagasse treatment

[0172] Compared with cellulase treatment alone, treatment with laccase and cellulase increased the reducing sugar content of mutant E231D / Y441H by 128% and WT by 20%. When HBT mediator was added, the reducing sugar content of mutant E231D / Y441H increased by 236% and WT by 132%.

[0173] Group Relative content of reducing sugars (%) Cellulase 100 Cellulase + WT 120 Cellulase + E231D / Y441H 228 Cellulase + WT + HBT 232 Cellulase + E231D / Y441H + HBT 336

[0174] The above results indicate that laccase, in conjunction with cellulase, can improve the enzymatic saccharification efficiency of straw and sugarcane bagasse. With or without the addition of a mediator, the saccharification efficiency of the mutant treated with cellulase on corn straw and sugarcane bagasse was higher than that of the WT.

[0175] The above-described embodiments are merely illustrative of several implementations of the present invention, and 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 those skilled in the art can make various modifications, combinations, and improvements to the above embodiments without departing from the concept of this patent, and these modifications and combinations all fall within the scope of protection of this patent. Therefore, the scope of protection of this patent should be determined by the claims.

Claims

1. A laccase mutant, characterized in that, The laccase mutant is obtained by one or two mutations of D221Y, E231D, Y441H or Y441F based on the wild-type laccase shown in SEQ ID NO.

1.

2. The laccase mutant of claim 1, wherein, the laccase mutant is a D221Y mutant, and the amino acid sequence is shown in SEQ ID NO. 3; the laccase mutant is an E231D mutant, and the amino acid sequence is shown in SEQ ID NO. 5; the laccase mutant is a Y441H mutant, and the amino acid sequence is shown in SEQ ID NO. 7; the laccase mutant is a Y441F mutant, and the amino acid sequence is shown in SEQ ID NO. 9; the laccase mutant is an E231D / Y441H mutant, and the amino acid sequence is shown in SEQ ID NO. 11; the laccase mutant is a D221Y / Y441H mutant, and the amino acid sequence is shown in SEQ ID NO. 13; the laccase mutant is a D221Y / Y441F mutant, and the amino acid sequence is shown in SEQ ID NO. 15; the laccase mutant is an E231D / Y441F mutant, and the amino acid sequence is shown in SEQ ID NO. 17; the laccase mutant is a D221Y / E231D mutant, and the amino acid sequence is shown in SEQ ID NO.

19.

3. A gene encoding the laccase mutant of claim 1 or 2.

4. A recombinant plasmid or a recombinant strain comprising the mutant gene of claim 3.

5. Use of the recombinant plasmid or the recombinant strain of claim 4 in the production of the laccase mutant of claim 1 or 2.

6. Use of the laccase mutant of claim 1 or 2 in the degradation of lignocellulose.

7. Use of the laccase mutant of claim 1 or 2 in the pulp bleaching industry.

8. Use according to claim 6 or 7, wherein the compound is ###0002### 8. Use of the laccase mutant in the degradation of corn stalks or sugarcane residues in cooperation with mediator 1-hydroxybenzotriazole and cellulase.

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