High-temperature-resistant laccase and preparation and application thereof

Abstract

The present application belongs to the technical field of genetic engineering of enzymes, and particularly relates to a high-temperature-resistant laccase as well as preparation and application thereof. A laccase gene (lac) from Bacillus safensis is expressed in Escherichia coli BL21; the gene lac is mutated by using error-prone PCR technology, and the mutant is screened by using ABTS, so that the mutant L48R / N453P with improved thermal stability is selected, and the preparation is realized by expression in Bacillus subtilis WB600, Bacillus amyloliquefaciens CGMCC No.11218 and Pichia pastoris GS115.

Description

Technical Field

[0001] This invention belongs to the field of enzyme genetic engineering technology, specifically relating to a high-temperature resistant laccase and its preparation and application. Background Technology

[0002] Agricultural wastes such as corn stalks contain abundant lignocellulose, with lignin, one of its main components, being plentiful and considered an important direction for biomass resource development. However, lignin has a complex structure and high stability, making it difficult to effectively convert and utilize. Traditional methods for lignin degradation require conditions such as high temperature, high pressure, strong acid, or strong alkali, which are not only energy-intensive and environmentally unfriendly but also cause non-selective degradation of lignin, increasing costs.

[0003] Enzymatic catalysis is a green and efficient method for lignin degradation, with mild reaction conditions that avoid the drawbacks of traditional degradation methods. Among lignin-degrading enzymes, laccase is an important polyphenol oxidase that catalyzes the oxidation of phenolic structural units in lignin, cleaving C-C / C==C bonds and side-chain linkages of the benzene ring in the lignin molecule, thereby reducing the molecular weight and degree of polymerization of lignin and increasing its solubility and reactivity. However, due to the complexity of the chemical bonds between the components of lignocellulose straw, there are significant obstacles to degrading lignin using a single enzyme with specific properties. It is possible to use a combination of enzymes with different functions as a composite enzyme to work synergistically in the same scenario to degrade lignin. This technology has already entered the industrialization stage, for example, patent CN 116479670 B. Lignin-degrading enzymes with specific functional properties, such as laccase, cannot meet the specific application conditions (e.g., temperature, pH, enzyme activity) required for their function as a composite enzyme in a specific scenario. Therefore, it is necessary to achieve this goal through laccase mutants and their genes, engineered bacteria, and other methods.

[0004] Laccase (EC1.10.3.2) is a widely distributed polycopper monomeric glycoprotein, also known as polyphenol oxidase, belonging to the polycopper oxidase family. Most laccases are monomeric enzymes, with molecular weights ranging from 50-130 kDa. Laccase generally consists of three parts: a polypeptide chain, copper atoms, and a polysaccharide molecule. The copper atom is located at the active site of the laccase. Based on differences in magnetic and spectral properties, the four copper atoms in the laccase molecule can be divided into three types: Type I Cu... 2+ (T1Cu) contains one copper atom and has a distinct absorption peak near 610 nm, which is related to the blue color of the enzyme. Type II Cu 2+ (T₂Cu) contains one copper atom and shows no absorption in the visible spectrum, but EPR can be detected. Type III Cu... 2+(T3Cu) contains two copper atoms and is an antiferromagnetically coupled ion, exhibiting electron adsorption at around 330 nm. Laccase can serve as an environmentally friendly and versatile "green catalyst" both in vivo and in vitro. Therefore, laccase has wide applications in industrial fields.

[0005] Current research on laccases primarily focuses on plants, insects, fungi, and bacteria. Studies have shown that plant laccases participate in the lignification of plant cell walls, maintaining cell wall structure and mechanical rigidity; they can also provide defense mechanisms against environmental stresses and promote wound healing; and they can convert Fe(II) to Fe(III). In the animal kingdom, research on insect laccases is the most extensive. Laccase activity has been detected in mosquitoes, bees, fruit flies, pine caterpillars, and swallowtail butterflies. Laccases oxidize toxic compounds ingested by insects, thus protecting the insect gut. They produce melanin in the midgut, serving as a primitive immune response against parasite invasion, and also participate in the tanning (coloring and hardening) of the insect cuticle.

[0006] Fungal laccases have long been a focus of research both domestically and internationally, and currently, all commercially available laccases are fungal in origin. Most fungal laccases operate under relatively mild conditions, with optimal temperatures typically between 30-60°C and optimal pH levels between 4.0-6.0. However, they become unstable under high temperature, high alkalinity, and high salinity conditions. The application of fungal laccases is somewhat limited by their applicability to temperature and pH conditions. Some industrial operations require extreme conditions, namely higher temperatures, pH values, and salt concentrations. Bacterial laccases generally have an optimal temperature of 50°C and exhibit good thermal stability within the 0-70°C range. Among bacterial laccases, Bacillus-derived laccases have better temperature and pH stability, leading to more extensive research. High temperatures are often required in the industrial degradation of straw. Therefore, further improving the stability of wild-type Bacillus laccases would be beneficial for their industrial applications.

[0007] Irrational molecular design of proteins involves inducing a large number of mutations in genes under certain conditions without knowing the three-dimensional structural information and mechanism of action of the protein, and then using multiple rounds of high-throughput screening methods to select mutants with the desired characteristics.

[0008] 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.

[0009] In addition to the advantages of the aforementioned Bacillus expression systems, the Pichia pastoris expression system also offers advantages related to eukaryotic expression: 1. It more closely resembles mammalian protein folding and modification, enhancing protein activity. 2. It offers high yield, making it suitable for projects with high production requirements. 3. It is particularly suitable for proteins requiring complex post-translational modifications.

[0010] Therefore, in this invention, a laccase mutant with improved thermostability was obtained by mutating the laccase gene derived from Bacillus safensis. Subsequently, a heat-resistant mutant laccase was prepared by expressing it using Bacillus and Pichia pastoris expression systems. Summary of the Invention

[0011] 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. Error-prone 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, *Bacillus amyloliquefaciens* CGMCC No. 11218, and *Pichia pastoris* GS115.

[0012] The technical approach to achieving the objective of this invention is summarized as follows:

[0013] The laccase gene *lac* from *Bacillus safensis* was mutated, and the mutant L48R / N453P and its encoding gene *lacm1* were screened using an *E. coli* expression system. The mutant's half-life at 80°C was increased to 176% of that of wild-type laccase (LAC). The optimal mutant laccase L48R / N453P was efficiently prepared using *Bacillus subtilis* WB600, *Bacillus amyloliquefaciens* CGMCC No. 11218, and *Pichia pastoris* GS115.

[0014] One of the technical solutions provided by the present invention is a laccase mutant, which is obtained by a mutation containing L48R and N453P on the basis of the wild-type laccase shown in SEQ ID NO.1. The laccase mutant is named L48R / N453P mutant.

[0015] Furthermore, the amino acid sequence of the L48R / N453P mutant is shown in SEQ ID NO.3;

[0016] Furthermore, the nucleotide sequence of the gene lacm1 encoding the L48R / N453P mutant is shown in SEQ ID NO.4.

[0017] The second technical solution provided by the present invention is a recombinant plasmid or recombinant strain containing the above-mentioned mutant encoding gene;

[0018] Furthermore, the expression vector used was pET-28a(+), and the host was Escherichia coli, Bacillus subtilis, Bacillus amyloliquefaciens, or Pichia pastoris;

[0019] Furthermore, the host cell is Escherichia coli BL21;

[0020] Furthermore, the host cell is Bacillus subtilis WB600, or Bacillus amyloliquefaciens CGMCC No.11218, or Pichia pastoris GS115;

[0021] 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.

[0022] 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.

[0023] The fourth technical solution provided by this invention is the application of the laccase mutant described in technical solution one, particularly its application in the degradation of lignocellulose;

[0024] Furthermore, it is used in the degradation of straw or bagasse;

[0025] Furthermore, it can be applied to the degradation of corn stalks, rice stalks, or wheat stalks.

[0026] In this invention, the following definitions are used:

[0027] 1. Nomenclature of amino acids and DNA nucleic acid sequences

[0028] 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.

[0029] 2. Identification of laccase mutants

[0030] The mutated amino acid in the LAC mutant is represented by "original amino acid + position + substituted amino acid". For example, L48R indicates that the amino acid at position 48 is replaced by Arg from the wild-type LAC, and the position number corresponds to the amino acid sequence number of the mature peptide of the wild-type LAC in SEQ ID NO.1.

[0031] In this invention, LAC represents wild-type laccase, L48R / N453P represents the laccase mutant, lowercase italic lac represents the coding gene for wild-type laccase LAC, and lowercase italic lacm1 represents the coding gene for the mutant L48R / N453P.

[0032] The specific information is shown in the table below.

[0033]

[0034] Beneficial effects

[0035] This invention utilizes error-prone PCR technology to mutate wild-type LAC, resulting in a mutant L48R / N453P with improved thermal stability at 80℃ compared to the wild-type, and an increased half-life at 80℃ of 176% of that of wild-type laccase (LAC). Attached Figure Description

[0036] Figure 1 Electrophoresis diagram of PCR amplification of wild-type laccase

[0037] Where M is the DNA Marker and lane 1 is the laccase gene lac.

[0038] Figure 2 Image for pET-lac plasmid digestion verification

[0039] Where: M is DNA Marker, and 1 is pET-lac digestion map after double digestion with NcoI and XhoI.

[0040] Figure 3 The curves show the changes in enzyme activity over time for wild-type and mutant L48R / N453P laccase at 80℃. Detailed Implementation

[0041] 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.

[0042] The solutions and culture media used in the embodiments of this invention are as follows:

[0043] Lysis buffer (mM): Tris 20, NaCl 500, dithiothreitol 1, imidazole 20.

[0044] Wash buffer (mM): Tris 20, NaCl 500, dithiothreitol 1, imidazole 100.

[0045] Elution Buffer (mM): Tris 20, NaCl 500, dithiothreitol 1, imidazole 500.

[0046] LB medium (g / L): yeast extract 5.0, tryptone 10.0, NaCl 10.0, the remainder being water. Solid medium supplemented with 2% agar.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] In the present invention, the mature peptide sequence of wild-type laccase LAC is shown in SEQ ID NO.1: MNLEKFVDELPIPEVAEPVKKNPRQTYYEIAMEEVFLKVHRDLPPTKLWTYNGSLPGPTIKANRNEKVKVKWMNKLPLKHFLPVDHTIHSSHHDEPEVKTVVHLHGGVTPASSDGYPEAWFSRDFEATGPFFERETYVYPNHQQACTLWYHDHAMALTRLNVYAGLAGFYLISDAFEKSLELPKDDYDIPLMIMDRTFQEDGSLFYPSRPNDTPEDSDIPDPSIVPFFCGETILVNGKVWPYLEVEPRKYRFRILNASNTRTYELHLDNDATILQIGSDGGFLPRPVRHQSFSIAPAERFDVIIDFSAYENKTITLKNTAGCGQDVNPETDANIMQFKVTRPLKGRVPKTLRPIFKPLPPLRPSRADRERKLTLTGTQDKYGRPILLLDNHFWNDPVTENPRLGSLEVWSIVNPTRGTHPIHLHLVQFRVLDRRPFDTEVYQSTGEIVYTGPNEAPPLHEQGYKDTIQAHAGEVIRIVARFVPYSGRYVWHCHILEHEDYDMMRPMDIIQ。

[0051] In this invention, the mature peptide sequence of the L48R / N453P mutant is shown in SEQ ID NO.3: MNLEKFVDELPIPEVAEPVKKNPRQTYYEIAMEEVFLKVHRDLPPTKRWTYNGSLPGPTIKANRNEKVKVKWMNKLPLKHFLPVDHTIHSSHHDEPEVKTVVHLHGGVTPASSDGYPEAWFSRDFEATGPFFERETYVYPNHQQACTLWYHDHAMALTRLNVYAGLAGFYLISDAFEKSLELPKDDYDIPLMIMDRTFQEDGSLFYPSRPNDTPEDSDIPDPSIVPFFCGETILVNGKVWPYLEVEPRKYRF RILNASNTRTYELHLDNDATILQIGSDGGFLPRPVRHQSFSIAPAERFDVIIDFSAYENKTITLKNTAGCGQDVNPETDANIMQFKVTRPLKGRVPKTLRPIFKPLPPLRPSRADRERKLTLTGTQDKY GRPILLLDNHFWNDPVTENPRLGSLEVWSIVNPTRGTHPIHLHLVQFRVLDRRPFDTEVYQSTGEIVYTGPPEAPPLHEQGYKDTIQAHAGEVIRIVARFVPYSGRYVWHCHILEHEDYDMMRPMDIIQ.

[0052] The present invention will be further explained and illustrated below through specific embodiments.

[0053] Example 1: Obtaining the wild-type laccase gene

[0054] 1. Genomic DNA was extracted from Bacillus safensis TCCC 111022 using the OMEGA Bacterial DNA Kit. The extraction steps are as follows:

[0055] (1) Inoculate the strain onto LB solid plates with an inoculation loop and incubate overnight at 37°C.

[0056] (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.

[0057] (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.

[0058] (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.

[0059] (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.

[0060] (6) Add 5 μL of RNase, invert and mix several times, and incubate at room temperature for 10 min.

[0061] (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.

[0062] (8) Add 220 μL of anhydrous ethanol and mix by blowing and sucking.

[0063] (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.

[0064] (10) Add 500 μL HBC buffer, centrifuge at 12000 r / min for 1 min, and discard the filtrate.

[0065] (11) Add 700 μL DNA wash buffer, let stand for 1 min, centrifuge at 12000 r / min for 1 min, and discard the filtrate.

[0066] (12) Add 500 μL DNA wash buffer, let stand for 1 min, centrifuge at 12000 r / min for 1 min, and discard the filtrate.

[0067] (13) 12000r / min for 2min, discard the waste liquid tube, and put the recovery column into a new EP tube.

[0068] (14) Place in a 55℃ metal bath to dry for 10 minutes.

[0069] (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.

[0070] 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:

[0071] Upstream primer P1:

[0072] 5'-GAAGGAGATATACCATGGGCATGAACCTAGAAAAATTTGTTGAC-3'

[0073] Downstream primer P2:

[0074] 5'-TGGTGGTGGTGGTGCTCGAGCTGAATGATATCCATCGGTCT-3'

[0075] Using P1 and P2 as upstream and downstream primers, amplification was performed using the laccase genome of Bacillus salsa as a template.

[0076] The amplification reaction system is as follows:

[0077] upstream primer P1 2.0μL Downstream primer P2 2.0μL Genomic DNA as template 2.0μL Primer Star Max Enzyme 25μL <![CDATA[ddH2O]]> 19μL

[0078] 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.

[0079] Example 2: Determination of LAC activity and temperature stability

[0080] 1. ABTS assay for LAC enzyme activity

[0081] Measurement method:

[0082] (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.

[0083] (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.

[0084] (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.

[0085] Calculate enzyme activity:

[0086]

[0087] In the formula: △OD = OD_end - OD_initial;

[0088] V1 represents the total volume of the reaction system (μL);

[0089] △t represents the reaction time (min);

[0090] V2 is added to the enzyme solution in μL;

[0091] ε represents the molar absorptivity of the product at 420 nm, which is 36 mM. -1 cm -1 ;

[0092] d represents the inner diameter / optical path thickness (cm) of the 96-well microplate.

[0093] Enzyme specific activity (U / mg) = enzyme activity / protein concentration.

[0094] 2. Temperature stability determination of LAC

[0095] LAC 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.

[0096] Example 3: Construction and Screening of LAC Mutants

[0097] 1. Constructing mutant libraries

[0098] Error-prone PCR: Error-prone PCR was performed using the wild-type coding gene lac as a template. The reaction system is as follows:

[0099] <![CDATA[ddH2O]]> 10μL Recombinant plasmid pET28a-lac (5 ng / μL) 2μL Upstream primer P1 (10 μmol / L) 2μL Downstream primer P2 (10 μmol / L) 2μL Taq DNA polymerase 0.5μL 10×Taq buffer 5μL dATP (10 mmol / L) 1μL dGTP (10 mmol / L) 1μL dTTP (10 mmol / L) 5μL dCTP (10 mmol / L) 5μL <![CDATA[MgCl2(25mmol / L)]]> 14μL <![CDATA[MnCl2(10mmol / L)]]> 2.5μL

[0100] Note: The reagents required above are from Takara Bio Inc.

[0101] After the system is complete, perform the error-prone PCR reaction. The program settings are as follows:

[0102] a. Pre-denaturation: 95℃ for 5 min;

[0103] b. Denaturation: 95℃ for 30 seconds;

[0104] c. Annealing: 56℃ for 45 seconds;

[0105] d. Extension: 72℃ for 50 seconds;

[0106] eb-d reaction for 35 cycles;

[0107] f. Extension: 72℃ for 10 minutes.

[0108] After the PCR reaction, the PCR amplification products were subjected to 0.8% agarose gel electrophoresis, and the PCR products were recovered using a small-volume DNA recovery kit to obtain different laccase mutant genes lacm. Recombinant plasmid pET-lacm was constructed by enzyme digestion and ligation, and then transformed into Escherichia coli BL21 to obtain the recombinant strain BL21 / pET-lacm.

[0109] 2. Mutant screening

[0110] (1) Take 2 mL of sterilized LB medium and add it to a 96-well plate. Add 2 μL of Kan resistance (concentration 50 μg / mL), add different mutant single colonies, and incubate overnight in a shaker at 37℃. The control is wild type.

[0111] (2) Transfer the bacterial culture to a new 24-well plate at an inoculation rate of 2%, add 1 mL of sterile LB (containing Kan resistance) to each well, and incubate at 37°C and 600 r / min for 3 h (incubate until OD600 is 0.6-0.8). Add 0.5 mM IPTG to each well and incubate overnight at 16°C in a shaker.

[0112] (3) Collect bacterial cells using a plate centrifuge at 5000 r / min for 15 min. Discard the supernatant, mix the bacterial cells with pH 7.0 buffer solution, and collect the bacterial solutions into 2 mL centrifuge tubes. Add 80 μL of lysozyme to lyse the cells. After lysis, centrifuge and collect the supernatant. Divide the supernatant into two parts. Incubate one part in an 80°C water bath according to step 1 of Example 2 to determine the enzyme activity of different mutants. Incubate the other part at 80°C for 30 min, and then incubate it according to step 2 of Example 2 to determine the thermal stability of different mutants.

[0113] (4) Transformers in which the supernatant of the mutant catalyzed ABTS at 420 nm showed higher absorbance than the wild type and which, after incubation at 80℃ for 30 min, still showed higher absorbance than the wild type at 420 nm were sequenced to obtain the mutant gene lacm1 (SEQ ID NO.4). Finally, the mutant L48R / N453P (SEQ ID NO.3) with higher activity and stability than the wild type was obtained through initial screening. The transformant containing this mutant was named recombinant strain BL21 / pET-lacm1.

[0114] Example 4: Expression, purification, and enzyme activity assay of WT and L48R / N453P

[0115] 1. Induced expression of recombinant strains

[0116] (1) On LB plates, pick one single colony of BL21 / pET-lacm1 and BL21 / pET-lac respectively, inoculate them into 5mL LB tubes (containing 50μg / mL Kan), and place them in a shaker at 37℃ for 10h.

[0117] (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.

[0118] (3) Add 125 μL of IPTG (final concentration 0.5 mmol / L) and incubate in a shaker at 16℃ for 16-20 h;

[0119] (4) The fermentation broth was purified to prepare wild-type laccase and laccase mutant.

[0120] 2. Ni column purification of recombinant proteins

[0121] (1) Broken bacterial cells

[0122] 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.

[0123] 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.

[0124] (2) WT and L48R / N453P bonded with nickel pillar

[0125] 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.

[0126] 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.

[0127] (3) Protein purification

[0128] a. Add the binding buffer to the purification column in 2-3 fractions in the chromatography cabinet;

[0129] b. After the binding solution has been completely filtered out, add 10 mL of wash buffer to elute the impurities bound to the resin;

[0130] 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;

[0131] d. Transfer all eluent to an ultrafiltration centrifuge tube. Centrifuge and ultrafilter until 1 mL of solution remains. Add pre-cooled 50 mM Tris-HCl buffer (pH 7.0) and repeat the replacement twice. The purified WT and L48R / N453P proteins are then obtained.

[0132] Following step 1 of Example 2, the specific activity was determined, and the final calculated specific activities of wild-type LAC and mutant LACM are as follows:

[0133] Laccase Enzyme activity (U / mg) WT 6.6 L48R / N453P 8.6

[0134] Temperature stability was determined according to step 2 of Example 2, and the thermal stability at 80°C was obtained (i.e., the enzyme activity of WT and L48R / N453P mutants after incubation at 80°C for different times was determined according to step 2 of Example 2, and enzyme activity change curves were plotted, as shown in Figure 2). Figure 3 As shown in the figure, the final calculated half-lives of wild-type LAC and each mutant are as follows:

[0135] Laccase Half-life (min) WT 42.7 L48R / N453P 75.2

[0136] Example 5: Expression and preparation of laccase mutant in Bacillus subtilis

[0137] The laccase mutant L48R / N453P encoding gene lacm1 and the wild-type laccase encoding gene lac were respectively ligated with expression plasmid pLY-3 to obtain new recombinant plasmids pLY-3-lacm1 and pLY-3-lac.

[0138] The recombinant plasmids were transformed into Bacillus subtilis WB600, and after screening for kanamycin (Kan) resistance and enzyme digestion verification, the mutant recombinant strain WB600 / pLY-3-lacm1 and the wild-type laccase recombinant strain WB600 / pLY-3-lac were obtained.

[0139] Recombinant strains WB600 / pLY-3-lacm1 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).

[0140] The ATBS method described in Example 2 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 33.4 U / mL, and the fermentation enzyme activity of L48R / N453P was 44.9 U / mL.

[0141] Example 6: Expression and preparation of laccase mutants in recombinant Bacillus amyloliquefaciens strains

[0142] The laccase mutant L48R / N453P encoding gene lacm1 and the wild-type laccase encoding gene lac were respectively ligated with expression plasmid pLY-3 to obtain new recombinant plasmids pLY-3-lacm1 and pLY-3-lac.

[0143] 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-lacm1 and the wild-type laccase recombinant strain CGMCC No.11218 / pLY-3-lac were obtained.

[0144] Recombinant strains CGMCC No.11218 / pLY-3-lacm1 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, 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).

[0145] The ATBS method described in Example 2 was used to determine the laccase activity obtained from Bacillus amyloliquefaciens fermentation (the supernatant was collected after centrifugation of the fermentation broth for enzyme activity determination). The enzyme activity of wild-type Bacillus amyloliquefaciens was 67.2 U / mL, and that of L48R / N453P fermentation was 88.3 U / mL.

[0146] Example 7: Expression and preparation of laccase mutant in Pichia pastoris GS115 recombinant strain

[0147] The laccase mutant L48R / N453P encoding gene lacm1 and the wild-type laccase encoding gene lac were ligated with expression plasmid pPIC9K to obtain new recombinant plasmids pPIC9K-lacm1 and pPIC9K-lac, respectively.

[0148] The recombinant plasmids pPIC9K-lacm1 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 strain GS115 / pPIC9K-lacm1 and the wild-type laccase recombinant strain GS115 / pPIC9K-lac were obtained by enzyme digestion verification.

[0149] (1) Pick single colonies of recombinant expression strains GS115 / pPIC9K-lacm1 and GS115 / pPIC9K-lac, inoculate them into 5mL YPD test tubes containing 50μg / mL Kan, and incubate at 30℃ and 200r / min for 24h.

[0150] (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;

[0151] (3) Collect the bacterial culture in BMGY medium using a 50mL centrifuge tube, and collect the bacterial cells by centrifugation; add 20mL of BMMY medium to resuspend the bacterial cells, centrifuge to collect the bacteria, and repeat once; add 10mL of BMMY medium to resuspend the bacterial cells, transfer the bacterial culture to BMMY medium using a pipette, add methanol to a final concentration of 0.5% every 12h, and ferment for 6d.

[0152] The ATBS method described in Example 2 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). The laccase fermentation activity of wild-type Pichia pastoris GS115 was 47.51 U / mL, and that of L48R / N453P was 64.2 U / mL.

[0153] Example 8: Application of laccase mutant in the degradation of corn stalks and sugarcane bagasse

[0154] 1. Pre-treatment of corn stalks and sugarcane bagasse

[0155] 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.

[0156] 2. Laccase degradation of lignin

[0157] The treated corn stalks and sugarcane bagasse were used as samples to be degraded. Wild-type laccase and mutant laccase L48R / N453P of this invention were added to each sample in a 20 mL system: 1 g of the sample to be degraded was placed in a 50 mL centrifuge tube, 50 U of WT / mutant laccase was added, and then pH 5.0 solution containing 5 mM Cu was added. 2+ Add the citrate-disodium hydrogen phosphate buffer to a final volume of 20 mL and react in a water bath at 50 °C and 150 rpm for 6 h.

[0158] The lignin degradation rate of laccase wild-type and mutant L48R / N453P was determined. Details are as follows:

[0159] (1) The treated corn stalks and sugarcane bagasse were extracted with benzene / alcohol for 8 hours and then dried to dryness for use.

[0160] (2) Weigh 0.3g and add 3mL of 72% sulfuric acid, stirring until the raw materials are fully mixed. After mixing, place in a 30℃ water bath and keep warm for 60min;

[0161] (3) Add 84 mL of deionized water to dilute to 4%, put it into a sterilizer at 121℃, take it out after 45 min, use a G3 (weigh, recorded as m0) sand core funnel for vacuum filtration, take 50 mL of filtrate to measure the absorbance of the acid hydrolysate at 205 nm, calculate the acid-soluble lignin content, and the determination method is GB / 10337-89 Determination of Acid-Soluble Lignin in Papermaking Raw Materials and Pulp;

[0162] The residue after filtration was washed with hot deionized water until neutral, dried in an oven at 105°C, and the weight was recorded (m1). After drying, it was transferred to an electric resistance furnace and calcined at 575°C for 4 hours. After cooling to room temperature, it was weighed and recorded (m2), and the content of acid-insoluble lignin was calculated. The final calculated lignin degradation rate is as follows:

[0163] 1. Degradation results of lignin in corn stalks

[0164] The lignin degradation rate was 23.6% after treatment with mutant L48R / N453P, and 16.9% after treatment with WT.

[0165] Group Lignin degradation rate (%) Corn stalks + WT 16.9 Corn stalks + L48R / N453P 23.6

[0166] 2. Degradation results of lignin in sugarcane bagasse

[0167] The lignin degradation rate was 23.2% after treatment with mutant L48R / N453P, and 15.8% after treatment with WT.

[0168] Group Lignin degradation rate (%) sugarcane bagasse + WT 15.8 sugarcane bagasse + L48R / N453P 23.2

[0169] The above results indicate that, after laccase treatment of straw and sugarcane bagasse, the mutant exhibits a higher lignin degradation rate for both corn straw and sugarcane bagasse compared to the WT mutant.

[0170] The embodiments described above 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 was obtained by L48R and N453P mutations on the wild-type laccase shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.

3.

2. The encoding gene of the laccase mutant according to claim 1.

3. The encoding gene as described in claim 2, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

4.

4. A recombinant plasmid or recombinant strain containing the mutant encoding gene of claim 2.

5. The use of the recombinant plasmid or recombinant strain of claim 4 in the production of the laccase mutant of claim 1.

6. The application of the laccase mutant according to claim 1, characterized in that, It is used in the degradation of lignocellulose.

7. The application as described in claim 6, characterized in that, The lignocellulose includes: straw and sugarcane bagasse.

8. The application as described in claim 7, characterized in that, The straw includes: corn straw, rice straw and wheat straw.

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