A high temperature resistant laccase mutant and its engineering bacteria, preparation and application

Abstract

The present invention belongs to the field of enzyme genetic engineering technology, and specifically relates to a thermostable laccase mutant and its engineering bacteria, preparation and application. Bacillus pumilus )-derived laccase gene ( lac ) were expressed in Escherichia coli BL21; the gene was PCR-PCR-enhanced lac The mutants were mutated and screened by ABTS, and the mutant V347D / Y463W with improved thermal stability was selected, and expressed in Bacillus subtilis WB600, Bacillus amyloliquefaciens CGMCC No.11218, and Pichia pastoris GS115.

Description

Technical Field

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

[0002] Agricultural industrial wastes, such as rice bran, wheat bran, bagasse, corn stover, and peanut shell waste, are rich in lignocellulose. Currently, most of the waste is dumped or incinerated, which pollutes the ecosystem or slows down the decomposition rate. Therefore, it is crucial to convert these lignocelluloses into commodities that can be effectively consumed. As a renewable matrix, lignocellulose has been widely studied for the production of reducing sugars and biofuels, mainly including hemicellulose, cellulose, and lignin. In order to release cellulose and hemicellulose, preliminary lignin removal pretreatment is required. The current industrial processes for removing lignin are strong alkali high temperature pulping technology for cellulose utilization and strong acid high temperature xylose (or furfural) technology for hemicellulose utilization. These processes all have problems of prohibition or restriction of development caused by environmental pollution.

[0003] The bio-enzyme method is the most effective way to solve pollution and increase benefits by replacing strong acid and strong alkali processes to degrade lignin. However, due to the complexity of the chemical bonds between the components of lignocellulose straw, there are huge obstacles to using a single enzyme with specific properties to degrade lignin. The use of enzymes with different functions to combine into a composite enzyme in the same scenario makes it possible to degrade lignin. At present, this technical solution has entered the industrialization stage, for example: Patent CN 116479670 B, etc.

[0004] Lignin-degrading enzymes with specific functional properties, such as laccase, do not have natural enzymatic properties that allow them to function under the conditions required for specific application scenarios in complex enzymes (such as temperature, pH, enzyme activity, etc.). This goal needs to be achieved through laccase mutants and their genes, engineered bacteria, and preparation.

[0005] Laccase (EC1.10.3.2), also known as phenolase, polyphenol oxidase, lacquer phenol oxidase, etc., was first discovered in the sap of Rhus verniciflua. It is characterized by a metal-containing oxidase and is one of the oldest enzymes ever. There are more than 200 substrates that can be catalyzed by laccase. In the process of laccase-catalyzed oxidation, T1Cu is the main electron acceptor. It transfers electrons to a trinuclear copper ion cluster (TNC) containing T2Cu and T3Cu atoms through a highly conserved His-Cys-His tripeptide, and then reduces O2 to H2O. It can catalyze the single-electron oxidation reaction of electron-rich substrates with O2, accompanied by the reduction of oxygen to completely non-toxic water, which is not only green and environmentally friendly, but also has a wide range of substrate specificity. Compared with the traditional method of lignin degradation, laccase opens the phenolic ring in the lignin molecule through the reaction of oxidizing phenolic compounds, thereby leading to the chain breakage of lignin. This process makes the structure of the lignin molecule more open and active, improving the degradability and availability of lignin. Therefore, the study of laccase has attracted widespread attention.

[0006] The sources of laccase are very wide, and they are divided into plant laccase, insect laccase, fungal laccase and bacterial laccase. Laccases from different sources play different roles. At present, most of the commercialized, more and more in-depth researched laccases have been identified to be derived from fungi, among which white rot fungal laccases account for a large proportion. Fungal laccases play an important role in lignin degradation, pigmentation, spore formation, stress defense, and fungus-plant-pathogen / host interactions. Studies have found that plant-derived laccases can promote plant wound healing and participate in

[0007] The metabolism of flavonoids, maintenance of the structural integrity of the cell wall, response to salt stress and degradation of environmental pollutants, etc. Compared with plant laccases, there are fewer reports on insect-derived laccases. The physiological processes of insect laccases in the tanning, hardening and pigmentation of their cuticles are essential for insect growth, development and survival. Bacterial laccases have a very wide range of substrate oxidation and perform different functions in organisms.

[0008] At present, the research on fungal laccases at home and abroad is relatively extensive. Studies have found that most fungal laccases can work better under mild environmental conditions, with the optimum temperature usually between 30-55 °C and the optimum reaction pH between 4-6. Only a few are neutral and resistant to high temperatures. Fungal laccases are usually unstable under high temperature and alkaline conditions, which limits their application. Compared with fungal laccases, bacterial laccases have better thermal stability and pH stability. Among them, laccases derived from Bacillus have been widely studied because of their relatively good heat resistance and alkaline stability. In the industrial degradation process of straw, high temperature is often required to assist in accelerating the decomposition and conversion of organic matter in the straw to generate usable fertilizer or energy. Therefore, further improving the thermal stability of wild-type Bacillus laccase is conducive to its industrial application.

[0009] Protein engineering refers to the modification or creation of proteins with desired functions or properties, such as catalytic activity, binding affinity, stability, solubility, specificity or novelty. The two main approaches to protein engineering are rational design and irrational design (or directed evolution). Irrational design (or directed evolution) refers to the generation of a large library of protein mutants by introducing random mutations and recombination, and then searching for proteins with desired properties through screening or selection.

[0010] The Bacillus expression system has the following advantages: 1. It can efficiently secrete various proteins; 2. Many Bacillus have been used in the fermentation industry for a long time, are non-pathogenic, and do not produce any endotoxins; 3. The genetic background of Bacillus microorganisms has been very clear, and they grow rapidly and have no special requirements for nutrients; 4. The codon preference is not obvious; 5. The fermentation process is simple. Bacillus is an aerobic bacteria and does not require anaerobic fermentation equipment. After the fermentation is completed, the fermentation liquid and bacterial cells can be simply separated to enter the separation, purification and recovery stage of the target protein; 6. It has stress resistance and can produce a variety of heat-resistant enzyme preparations.

[0011] In addition to the advantages of the Bacillus expression system mentioned above, the Pichia pastoris expression system also has the advantages of eukaryotic expression: 1. It is closer to the protein folding and modification of mammals, which improves protein activity. 2. It has high yield and is suitable for projects with high yield requirements. 3. It is particularly suitable for proteins that require complex post-translational modifications.

[0012] Therefore, in the present invention, by Bacillus pumilus ) laccase gene was mutated to screen out laccase mutants with improved thermal stability. Subsequently, the thermostable mutant laccase was expressed and prepared using Bacillus and Pichia pastoris expression systems. Summary of the invention

[0013] Based on the problem of high temperature application of laccase, in order to obtain laccase with improved thermal stability, its existing properties need to be further modified. The purpose of the present invention is to provide a high temperature resistant laccase mutant. Bacillus pumilus )-derived laccase gene ( lac ) were expressed in Escherichia coli BL21; the gene was PCR-PCR-enhanced lac Mutations were generated and the mutants were screened using ABTS to select mutants with improved thermal stability. The mutants were expressed and prepared in Bacillus subtilis WB600, Bacillus amyloliquefaciens CGMCC No.11218, and Pichia pastoris GS115.

[0014] The technical route for achieving the purpose of the present invention is summarized as follows:

[0015] From Bacillus pumilus ( Bacillus pumilus ) was mutated, and the mutant V347D / Y463W and the encoding gene were screened using the E. coli expression system. lacm1 The half-life of the enzyme at 80 ℃ was increased to 151% of that of the wild-type laccase (LAC). The optimal mutant laccase V347D / Y463W was efficiently prepared using Bacillus subtilis WB600 and Pichia pastoris GS115.

[0016] One of the technical solutions provided by the present invention is a laccase mutant, which is obtained by causing mutations including V347D and Y463W on the basis of the wild-type laccase shown in SEQ ID NO.1, and the laccase mutant is named V347D / Y463W mutant;

[0017] Furthermore, the amino acid sequence of the V347D / Y463W mutant is shown in SEQ ID NO.3;

[0018] Furthermore, the coding gene of the V347D / Y463W mutant lacm1 , the nucleotide sequence is shown in SEQ ID NO.4.

[0019] The second technical solution provided by the present invention is a recombinant plasmid or recombinant strain containing the above mutant encoding gene;

[0020] Furthermore, the expression vector used is pET-28a(+), and the host is Escherichia coli, Bacillus subtilis, Bacillus amyloliquefaciens, or Pichia pastoris;

[0021] Furthermore, the host cell is Escherichia coli BL21;

[0022] Furthermore, the host cell is Bacillus subtilis WB600, or Bacillus amyloliquefaciens CGMCC No.11218, or the host cell is Pichia pastoris GS115;

[0023] Preferably, the recombinant strain is obtained by connecting the mutant encoding gene to the expression vector pET-28a(+) and expressing it in the host Escherichia coli.

[0024] The third technical solution provided by the present invention is the use of the above-mentioned recombinant plasmid or recombinant strain in producing the laccase mutant described in the first technical solution.

[0025] The fourth technical solution provided by the present invention is the application of the laccase mutant described in the first technical solution, especially in the degradation of lignocellulose; further, in the degradation of straw or bagasse;

[0026] Furthermore, the straw includes but is not limited to: corn straw, rice straw or wheat straw.

[0027] The following definitions are used in the present invention:

[0028] 1. Nomenclature of amino acid and DNA sequences

[0029] The generally accepted IUPAC nomenclature for amino acid residues is used, either as a single letter or three letter code. The generally accepted IUPAC nomenclature for DNA nucleic acid sequences is used.

[0030] 2. Identification of Laccase Mutants

[0031] The mutated amino acid in the LAC mutant is represented by "original amino acid + position + replaced amino acid". For example, V347D means that the amino acid at position 347 is replaced by Asp from Val of wild-type LAC, and the position number corresponds to the amino acid sequence number of the wild-type LAC mature peptide in SEQ ID NO.1.

[0032] In the present invention, LAC represents wild-type laccase, V347D / Y463W represents laccase mutants, and lowercase italics lac Indicates the gene encoding the wild-type laccase LAC, in lowercase italics lacm1 The gene encoding the mutant V347D / Y463W is indicated.

[0033] The specific information is shown in the following table.

[0034]

[0035] Beneficial Effects

[0036] The invention utilizes error-prone PCR technology to mutate the wild-type LAC to obtain a mutant V347D / Y463W having improved thermal stability at 80°C relative to the wild-type, and a half-life at 80°C increased to 151% of the wild-type laccase (LAC). BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The electrophoresis diagram of PCR amplification of wild-type laccase

[0038] Among them: M is DNA Marker, lane 1 is laccase gene lac .

[0039] Figure 2 For pET- lac Plasmid restriction enzyme cutting verification diagram

[0040] Among them: M is DNA Marker, 1 is pET- lac through Nco I and XOt I double enzyme digestion map.

[0041] Figure 3 The curves showing the changes in enzyme activity of wild-type and mutant V347D / Y463W laccase at 80°C over time. DETAILED DESCRIPTION

[0042] The technical content of the present invention is further described below in conjunction with the embodiments, but the present invention is not limited to these embodiments, and the protection scope of the present invention cannot be limited by the following embodiments.

[0043] Some solutions and culture media used in the embodiments of the present invention are as follows:

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

[0045] Wash buffer (mM): Tris 20, NaCl 500, dithiothreitol 1, imidazole 100.

[0046] Elution Buffer (mM): Tris 20, NaCl 500, dithiothreitol 1, imidazole 500.

[0047] LB medium (g / L): 5.0% yeast extract, 10.0% tryptone, 10.0% NaCl, and the rest is water. For solid medium, add 2% agar.

[0048] 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.

[0049] 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.

[0050] Fermentation medium (g / L): 64 corn flour, 40 soybean meal, add 2.7 amylase, 4 Na2HPO4, 0.3 KH2PO4, and the rest is water; keep warm at 90℃ for 30min and then sterilize at 121℃ for 20min.

[0051] In the present invention, the mature peptide sequence of the wild-type laccase LAC is shown in SEQ ID NO.1: MNLEKFVDELPIPEVAKPVKKNPRQTYYEIAMEEVFLKVHRDLPPTKLWTYNGSLPGPTIQANRNEKVKVKWMNKLPLKHFLPVDHTIHEGHHDEPEVKTVVHLHGGVTPASSDGYPEAWFSRDFEATGPFFEREVYVYPNHQQACTLWYHDHAMALTRLNVYAGLAGFYLISDAFEKSLELPKDDYDIPLMIMDRTFQEDGSLFYPSRPNDTPEDSDLPDPSIVPFFCGETILVNGKVWPYLEVEPRKYRF RILNASNTRTYELHLDNDATILQIGSDGGFLPRPVRHQSFSIAPAERFDVIIDFSAYENKTITLKNTAGCGQDVNPETDANIMQFKVTRPLKGRVPKTLRLIFKPLPTLRPSQADRERTLTLSGTQDKY GRPILLLDNQFWNDPVTENPRLGSLEVWSIVNPTRGTHPIHLHLVQFRVLDRRPFDTEVYQSTGEIVYTGPNEAPPLHEQGYKDTIQAHAGEVIRIVARFVPYSGRYVWHCHILEHEDYDMMRPMDIIQ.

[0052] In the present invention, the mature peptide sequence of the V347D / Y463W mutant is shown in SEQ ID NO.3:

[0053] MNLEKFVDELPIPEVAKPVKKNPRQTYYEIAMEEVFLKVHRDLPPTKLWTYNGSLPGPTIQANRNEKVKVKWMNKLPLKHFLPVDHTIHEGHHDEPEVKTVVHLHGGVTPASSDGYPEAWFSRDFEA TGPFFEREVYVYPNHQQACTLWYHDHAMALTRLNVYAGLAGFYLISDAFEKSLELPKDDYDIPLMIMDRTFQEDGSLFYPSRPNDTPEDSDLPDPSIVPFFCGETILVNGKVWPYLEVEPRKYRFRIL NASNTRTYELHLDNDATILQIGSDGGFLPRPVRHQSFSIAPAERFDVIIDFSAYENKTITLKNTAGCGQDVNPETDANIMQFKVTRPLKGRDPKTLRLIFKPLPTLRPSQADRERTLTLSGTQDKYGR PILLLDNQFWNDPVTENPRLGSLEVWSIVNPTRGTHPIHLHLVQFRVLDRRPFDTEVYQSTGEIVYTGPNEAPPLHEQGWKDTIQAHAGEVIRIVARFVPYSGRYVWHCHILEHEDYDMMRPMDIIQ.

[0054] The present invention will be further explained below through specific examples.

[0055] Example 1 Obtaining wild-type laccase gene

[0056] 1. Use the kit (OMEGA: Bacterial DNA Kit) to extract Bacillus pumilus ( Bacillus pumilus ) The genomic DNA of TCCC 11568 was extracted as follows:

[0057] (1) Use an inoculation loop to inoculate the strain onto a LB solid plate and culture at 37 °C overnight.

[0058] (2) Pick a single colony from the culture plate and inoculate it into liquid test tube culture medium. Incubate overnight at 37 °C and 220 rpm with shaking.

[0059] (3) Place 3 mL-5 mL of bacterial solution in a sterilized EP tube, centrifuge at 12,000 rpm for 2 min, and discard the supernatant.

[0060] (4) Add 200 μL of sterile water to the EP tube to resuspend the bacteria, then add 50 μL of lysozyme, pipette to mix, and keep at 37 °C for 20 min.

[0061] (5) Add 100 μL BTL buffer and 20 μL proteinase K to the EP tube, vortex to mix, and incubate at 55 °C for 40 min, vortexing to mix every 20 min.

[0062] (6) Add 5 μL of RNase, invert several times to mix, and leave at room temperature for 10 min.

[0063] (7) Centrifuge at 12,000 r / min for 2 min to remove the undigested portion. Transfer the supernatant to a new EP tube, add 220 μL of BDL buffer, and incubate at 65 °C in a water bath for 15 min.

[0064] (8) Add 220 μL of anhydrous ethanol and mix by pipetting.

[0065] (9) Transfer the liquid in the EP tube into the recovery column and let it stand for 1 min. Centrifuge at 12,000 r / min for 1 min. Pour the filtrate back into the recovery column. Repeat twice and discard the waste liquid.

[0066] (10) Add 500 μL HBC buffer, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0067] (11) Add 700 μL DNA wash buffer, let stand for 1 min, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0068] (12) Add 500 μL DNA wash buffer, let stand for 1 min, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0069] (13) Incubate at 12,000 r / min for 2 min, discard the waste liquid tube, and place the recovery column in a new EP tube.

[0070] (14) Dry in a metal bath at 55 °C for 10 min.

[0071] (15) Add 50 μL of 55°C sterile water, let stand at room temperature for 5 min, centrifuge at 12,000 rpm for 2 min, discard the recovery column, and the liquid in the EP tube is the genome.

[0072] 2. Using the extracted genome of Bacillus pumilus as a template, a pair of primers were designed upstream and downstream of the ORF frame to introduce restriction enzyme sites respectively. Nco I. XOt I. Laccase gene of the present invention lac The amplification primers are as follows:

[0073] Upstream primer P1:

[0074] 5'-CATGCCATGGGCATGAACCTAGAAAAATTTGTTGACG-3'

[0075] Downstream primer P2:

[0076] 5'-CCCTCGAGCTGGATGATATCCATCGGCC-3'

[0077] P1 and P2 were used as upstream and downstream primers, and the laccase genome of Bacillus pumilus was used as a template for amplification.

[0078] The amplification reaction system is:

[0079]

[0080] The amplification program was as follows: 98°C pre-denaturation for 30 s; 98°C denaturation for 10 s, 54°C annealing for 20 s, 72°C extension for 8 s, 30 cycles; 72°C extension for 10 min. The PCR amplification product was subjected to 0.8% agarose gel electrophoresis, and a 1530 bp band was obtained ( Figure 1 ), using a small amount of DNA recovery kit to recover the PCR product, and obtain the wild-type laccase gene of the present invention. lac (SEQ ID NO.2), lac pET-28a(+) plasmid were cleaved with restriction endonucleases Nco I and XOt I was double-digested, and the gel-recovered lac Connect with the vector pET vector to obtain the recombinant plasmid pET- lac , enzyme digestion verification Figure 2 As shown, it was transformed into Escherichia coli BL21 and named as the recombinant strain BL21 / pET- lac .

[0081] Example 2 LAC activity and temperature stability determination

[0082] ABTS assay method for LAC enzyme activity:

[0083] (1) Take 200 µL of pH 5.5 citric acid-sodium hydrogen phosphate buffer (50 mM, containing 5 mM Cu 2+ ) in a 96-well ELISA plate and incubate in a water bath at 80 °C for 1 min;

[0084] (2) Add 10 μL of LAC / LACM diluted to an appropriate concentration, mix well, and place in an 80 °C water bath for 1 min;

[0085] (3) Add 30 µL of ABTS (50 mM) and mix by pipetting. Incubate the mixture in a constant temperature water bath at 80 °C for 10 min. Record the initial and final OD values ​​at 420 nm.

[0086] Calculation of enzyme activity:

[0087]

[0088] Where: △OD = OD end - OD start;

[0089] V1 represents the total volume of the reaction system (µL);

[0090] △t represents the reaction time (min);

[0091] V2: volume of enzyme solution added (µL);

[0092] ε represents the molar absorption coefficient of the product at 420 nm, 36 mM -1 cm -1 ;

[0093] d represents the inner diameter / optical path thickness of the 96-well ELISA plate (cm).

[0094] Enzyme specific activity (U / mg) = enzyme activity / protein concentration.

[0095] Temperature stability of LAC was determined by placing LAC in a pH 7.0 buffer and keeping it at 80 °C for different time periods. Samples were taken at regular intervals and the residual enzyme activity was measured at pH 5.0 and 80 °C after the insulation. The enzyme activity without insulation treatment was taken as 100% to calculate the residual enzyme activity, draw the enzyme activity change curve, and further calculate the half-life.

[0096] Example 3 Construction and screening of LAC mutants

[0097] 1. Construction of mutant library

[0098] Error-prone PCR: wild-type coding gene lac Error-prone PCR was performed with 1% β-glutamic acid as the template, and the reaction system was as follows:

[0099]

[0100] Note: The above reagents are from Takara Biotech Co., Ltd.

[0101] After the system is completed, the error-prone PCR reaction is performed, and the program settings are as follows:

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

[0103] b. Denaturation: 95 ℃ 30 s;

[0104] c. Annealing: 56 ℃ 45 s;

[0105] d. Extension: 72 °C 50 s;

[0106] EB-D reaction for 35 cycles;

[0107] f. Extension: 72 ℃ 10 min.

[0108] After the PCR reaction was completed, the PCR amplification products were subjected to 0.8% agarose gel electrophoresis, and the PCR products were recovered using a small amount of DNA recovery kit to obtain different laccase mutant genes. lacm . Recombinant plasmid pET- lacm , and then the recombinant plasmid pET- lacm 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 (effective concentration 50 µg / mL) and different mutant colonies. Incubate overnight in a shaker at 37 °C. The wild type is used as the control.

[0111] (2) Transfer the bacterial solution to a new 24-well plate at a 2% inoculum volume, add 1 mL of sterilized LB (containing Kan resistance) to each well, and culture at 37 °C, 600 r / min for 3 h (cultivate until OD600 is 0.6-0.8), add 0.5 mM IPTG to each well, and induce overnight culture at 16 °C in a shaker;

[0112] (3) Collect the cells using a plate centrifuge, centrifuge at 5000 r / min for 15 min, discard the supernatant, mix the cells by pipetting with a pH 7.0 buffer, collect the cell suspension into 2 mL centrifuge tubes, and add 80 μL of lysozyme to disrupt the cells. After the disruption is completed, collect the supernatant by centrifugation, and divide the supernatant into two parts. One part is placed in an 80 ℃ water bath according to step 1 of Example 2 to determine the enzyme activity of different mutants; the other part is kept at 80 ℃ for 30 min, and the residual enzyme activity is determined according to step 2 of Example 2;

[0113] (4) Select the mutant transformants whose supernatant catalyzed ABTS absorbance at 420 nm is higher than that of the wild type and whose absorbance at 420 nm catalyzed ABTS is still higher than that of the wild type after being incubated at 80°C for 30 min for sequencing to obtain the mutant gene lacm1 (shown in SEQ ID NO.4), and finally the mutant V347D / Y463W (shown in SEQ ID NO.3) with higher activity and stability than the wild type was obtained in the initial screening. The transformant containing the mutant was named the recombinant strain BL21 / pET- lacm1 .

[0114] Example 4 Expression, purification and enzyme activity determination of WT and V347D / Y463W

[0115] 1. Inducible expression of recombinant strains

[0116] (1) On the LB plate, pick BL21 / pET- lacm1 and BL21 / pET- lac A single colony was inoculated into a 5 mL LB tube (containing 50 µg / mL Kan) and placed in a shaker at 37 °C for 10 h.

[0117] (2) Transfer the recombinant bacterial suspension into 250 mL LB medium (final concentration of 50 µg / mL Kan) and culture in a shaker at 37 °C for 2-2.5 h.

[0118] (3) Add 125 μL of IPTG (final concentration 0.5 mmol / L) and induce the culture in a shaking incubator at 16 °C for 16-20 h;

[0119] (4) Purifying the fermentation broth to prepare wild-type laccase and laccase mutants.

[0120] 2. Ni column purification of recombinant protein

[0121] (1) Disruption of bacterial cells

[0122] The fermentation broth was collected using a centrifuge cup and centrifuged at 10,000 rpm for 15 min. The supernatant was discarded and 20 mL of Lysis buffer was added to blow the bacteria away. Ultrasonic waves were used to break the bacteria, destroy the cell wall and release the protein inside the bacteria.

[0123] After the disruption was completed, the bacterial solution was poured into a 50 mL centrifuge tube and centrifuged at 12000 rpm for 30 min at 4 °C to collect the supernatant.

[0124] (2) WT and V347D / Y463W binding to nickel column

[0125] a. Before the nickel column purification process, add an appropriate amount of ddH2O to the purification column and add two column volumes of Lysis buffer to balance the resin;

[0126] b. Mix the equilibrated resin and bacterial supernatant, place in a magnetic stirrer, and stir at 80-100 r / min for 1 h, while keeping the temperature low (4 °C) throughout the process.

[0127] (3) Protein purification

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

[0129] b. After the binding solution is 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 the eluate to an ultrafiltration centrifuge tube, centrifuge and ultrafilter until 1 mL of the solution remains, add pre-cooled 50 mM, pH 7.0 Tris-HCl buffer, and repeat the replacement twice to obtain the purified WT and V347D / Y463W proteins.

[0132] The specific activity was determined according to step 1 of Example 2, and the specific activity of the wild-type LAC and the mutant V347D / Y463W was finally calculated as follows:

[0133]

[0134] The temperature stability was determined according to step 2 of Example 2, and the thermal stability at 80°C was obtained (i.e., the enzyme activities of WT and V347D / Y463W mutants after being incubated at 80°C for different times were determined according to the method of step 2 of Example 2, and the enzyme activity change curve was plotted. The enzyme activity change curve is shown in FIG. Figure 3As shown), the half-lives of wild-type LAC and mutants were finally calculated as follows:

[0135]

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

[0137] Laccase mutant V347D / Y463W encoding gene lacm1 and wild-type laccase encoding gene lac The new recombinant plasmid pLY-3- lacm1 and pLY-3- lac ;

[0138] The recombinant plasmids were transferred into Bacillus subtilis WB600, and the mutant recombinant bacteria WB600 / pLY-3- lacm1 and wild-type laccase recombinant strain WB600 / pLY-3- lac .

[0139] The recombinant strain WB600 / pLY-3- lacm1, WB600 / pLY-3- lac The samples were inoculated into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL) respectively, cultured at 37°C, 220 r / min overnight, transferred to 50 mL of fresh fermentation medium (containing kanamycin, 50 µg / mL) at a 2% inoculum amount, and cultured at 37°C, 220 r / min for 48 h (fermentation medium (g / L): corn flour 64, soybean cake powder 40, 2.7 amylase, Na2HPO44, KH2PO40.3, and the rest was water; kept at 90°C for 30 min and sterilized at 121°C for 20 min).

[0140] The ATBS method in Example 2 was used to determine the activity of the laccase obtained by fermentation of Bacillus subtilis (the fermentation broth was centrifuged and the supernatant was taken to determine the activity). In Bacillus subtilis, the activity of the wild type enzyme was 955.6 U / mL, and the activity of the fermentation enzyme of V347D / Y463W was 1287.2 U / mL.

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

[0142] Laccase mutant V347D / Y463W encoding gene lacm1 and wild-type laccase encoding gene lac The new recombinant plasmid pLY-3- lacm1 and pLY-3- lac ;

[0143] The recombinant plasmids were transferred into Bacillus amyloliquefaciens CGMCC No.11218, and the mutant recombinant bacteria CGMCC No.11218 / pLY-3- lacm1 and wild-type laccase recombinant strain CGMCC No.11218 / pLY-3- lac .

[0144] The recombinant strain CGMCC No.11218 / pLY-3- lacm1 、CGMCC No.11218 / pLY-3- lac The samples were inoculated into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL) respectively, cultured at 37°C, 220 r / min overnight, transferred to 50 mL of fresh fermentation medium (containing kanamycin, 50 µg / mL) at a 2% inoculum amount, and cultured at 37°C, 220 r / min for 48 h (fermentation medium (g / L): corn flour 64, soybean cake powder 40, 2.7 amylase, Na2HPO44, KH2PO40.3, and the rest water; kept at 90°C for 30 min and sterilized at 121°C for 20 min).

[0145] The ATBS method in Example 2 was used to determine the activity of the laccase obtained by fermentation of Bacillus amyloliquefaciens (the fermentation broth was centrifuged and the supernatant was taken to determine the enzyme activity). The enzyme activity of the wild type of Bacillus amyloliquefaciens was 1918.5 U / mL, and the fermentation enzyme activity of V347D / Y463W was 2583.3 U / mL.

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

[0147] Laccase mutant V347D / Y463W encoding gene lacm1 and wild-type laccase encoding gene lac The new recombinant plasmid pPIC9K- lacm1 and pPIC9K- lac ;

[0148] Use restriction enzyme SalI The recombinant plasmid pPIC9K- lacm1 and pPIC9K- lac Linearization, the linearized recombinant plasmids were electroporated into Pichia pastoris GS115, and the mutant recombinant bacteria GS115 / pPIC9K- lacm1 and wild-type laccase recombinant bacteria GS115 / pPIC9K- lac .

[0149] (1) Select the recombinant expression strain GS115 / pPIC9K- lacm1 and GS115 / pPIC9K- lac A single colony was inoculated into a 5 mL YPD tube containing 50 μg / mL Kan and cultured at 30 °C, 200 rpm for 24 h;

[0150] (2) Pipette 1 mL of bacterial solution and inoculate it into BMGY enrichment medium. Cultivate at 30 °C and 220 rpm for 16-19 h.

[0151] (3) Collect the bacterial liquid in the BMGY medium in a 50-mL centrifuge tube and collect the bacteria by centrifugation; add 20 mL of BMMY medium to resuspend the bacteria and collect the bacteria by centrifugation, and repeat once; add 10 mL of BMMY medium to resuspend the bacteria and transfer the bacterial liquid to the BMMY medium with a pipette. Add methanol at a final concentration of 0.5% every 12 hours and ferment for 6 days.

[0152] The ATBS method in Example 2 was used to determine the activity of the laccase obtained from the fermentation of Pichia pastoris (the fermentation broth was centrifuged and the supernatant was taken to determine the enzyme activity). In the fermentation activity of laccase in Pichia pastoris GS115, the wild-type enzyme activity was 1337.9 U / mL, and the fermentation enzyme activity of V347D / Y463W was 1798.6 U / mL.

[0153] Example 8 Application of Laccase Mutants in Degrading Corn Straw and Bagasse

[0154] Preliminary treatment of corn stalks and sugarcane bagasse Corn stalks and sugarcane bagasse were sourced from local enterprises in Jiangsu, China. They were first washed with deionized water to remove surface dirt, then placed in a constant temperature drying oven at 60 °C for drying to reduce the moisture content to less than 2%. The dried raw materials were crushed with a pulverizer and filtered with a 40-mesh sieve to ensure that they became uniform powders. Finally, they were placed at room temperature for subsequent use.

[0155] 2. Laccase degradation of lignin

[0156] The treated corn stalks and sugarcane bagasse were used as samples to be degraded, and the wild-type laccase and mutant laccase V347D / Y463W of the present invention were added respectively. A 20 mL system was used: 1 g of the sample to be degraded was taken in a 50 mL centrifuge tube, 50 U of WT / mutant laccase was added, and then 5 mM Cu was added at pH 5.0. 2+ The volume was filled to 20 mL with citric acid-sodium hydrogen phosphate buffer and shaken in a water bath at 50 °C and 150 r / min for 6 h.

[0157] The lignin degradation rate after treatment with the wild-type laccase and the mutant V347D / Y463W was determined as follows:

[0158] (1) The treated corn stalks and sugarcane bagasse were extracted with benzene / alcohol for 8 h and dried to absolute dryness for later use;

[0159] (2) Weigh 0.3 g and add 3 mL of 72% sulfuric acid, stirring until the raw materials are fully mixed. After mixing, place in a 30°C water bath and keep warm for 60 min;

[0160] (3) Add 84 mL of deionized water to dilute to 4%, put it into a 121 °C autoclave, take it out after 45 min, use a G3 (weighed, recorded as m0) sand core funnel for vacuum filtration, take 50 mL of the filtrate to measure the absorbance of the acid hydrolyzate at 205 nm, and calculate the acid-soluble lignin content. The determination method is GB / 10337-89 Determination of acid-soluble lignin in papermaking raw materials and pulp;

[0161] Use hot deionized water to wash the filtered residue until it is neutral, dry it in an oven at 105°C, and weigh and record (m1). After drying, transfer it to a resistance furnace and burn it at 575°C for 4 hours. Take it out and cool it to room temperature, then weigh and record (m2), and calculate the content of acid-insoluble lignin. The final calculated lignin degradation rate is as follows:

[0162] 1. Degradation results of lignin in corn straw

[0163] The lignin degradation rate was 29.1% after treatment with mutant V347D / Y463W, and 21.3% after treatment with WT.

[0164]

[0165] 2. Degradation results of lignin in bagasse

[0166] The lignin degradation rate was 26.5% after treatment with mutant V347D / Y463W, and 19.6% after treatment with WT.

[0167]

[0168] The above results showed that the mutant had higher lignin degradation rates on corn straw and sugarcane bagasse than WT after laccase treatment of corn straw and sugarcane bagasse.

[0169] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A laccase mutant, characterized in that: The laccase mutant is obtained by causing mutations of V347D and Y463W on the wild-type laccase shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

3.

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

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

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

5. Use of the laccase mutant according to claim 1.

6. The use according to claim 5, characterized in that It is used in the degradation of lignocellulose.

7. The use according to claim 6, characterized in that The lignocellulose includes but is not limited to straw and bagasse.

8. The use according to claim 7, characterized in that The straw includes but is not limited to corn straw, rice straw or wheat straw.

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