A lignin peroxidase mutant and its gene, engineering bacteria, preparation method and application

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to a lignin peroxidase mutant and its gene, engineering bacteria, preparation method and application. The lignin peroxidase mutant is obtained by T158V / A161P mutation on the basis of the wild-type lignin peroxidase shown in SEQ ID NO.1. Compared with the wild-type, the mutant has a certain degree of improvement in enzyme activity and stability at 40°C, which is conducive to expanding the application in industrial fields such as lignin degradation and pulp bleaching.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a lignin peroxidase mutant and its gene, engineering bacteria, preparation method and application. Background Art

[0002] The components of lignocellulosic straw are mainly composed of cellulose, hemicellulose and lignin. The covalent bonding of lignin with hemicellulose and cellulose hinders the effective degradation of straw components. At present, there are many problems in the utilization of straw. On the one hand, the direct return of straw to the field degrades slowly in the natural environment, and the added value of the fertilizer route is low, resulting in reduced grain production and poor economic benefits of the simple pressing process. On the other hand, the use of straw as an industrial raw material has environmental pollution problems. In addition, the industrial field mainly uses high-temperature strong alkali pulping technology to remove lignin and high-temperature strong acid xylose technology to remove hemicellulose. These processes all have the problem of black liquor pollution, which prohibits or restricts development.

[0003] 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 chemical bonds between lignocellulose components in straw, there are huge obstacles to using a single enzyme with specific properties to degrade lignin. The use of enzymes with different functions to degrade lignin by combined action of complex enzymes has great development prospects. 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 lignin peroxidase (LIP), have natural enzymatic properties that cannot meet the requirements for functioning under specific application conditions (such as temperature, pH, enzyme activity, etc.) in complex enzymes, and this goal needs to be achieved through gene mutation.

[0005] Lignin peroxidase, a member of the heme peroxidase superfamily, is the main lignin-degrading enzyme in the biodegradation of lignin. There are two calcium ion binding sites on the surface of lignin peroxidase, which can maintain the protein structure and enzyme activity. The active center of lignin peroxidase contains heme and trivalent iron ion binding sites, so lignin peroxidase mostly acts in the form of isozymes. Among the lignin-degrading enzymes, lignin peroxidase has a higher redox potential and can degrade compounds with a redox potential greater than 1.4V. It has the ability to catalyze the depolymerization of non-phenolic lignin model compounds (such as diarylpropane) and a variety of phenolic compounds (such as guaiacol, vanillyl alcohol, catechol, syringic acid, acetosyringone, etc.).

[0006] The catalytic mechanism of lignin peroxidase is similar to that of other peroxidases, and the catalysis of lignin peroxidase also depends on H2O2. It has a ligand channel that allows small molecule substrates to directly react with heme through the channel. The size and physicochemical properties of the substrate jointly determine whether it can react with heme through the ligand channel. Large molecules such as lignin cannot react with heme through the channel, but ultimately react with heme through the amino acid residues on the enzyme surface via a long-range electron transfer mechanism.

[0007] Lignin peroxidase is mainly derived from fungi and is suitable for functioning under mild conditions. It is limited in the application scenarios of complex enzyme catalytic systems in the industrial field. Improving the activity and stability of lignin peroxidase is an important condition for entering the field of industrial applications.

[0008] Irrational molecular design of proteins is to make a large number of gene mutations under certain conditions without knowing the three-dimensional structure information and mechanism of action of proteins, and then select the desired characteristic mutants through multiple rounds of high-throughput screening methods. Rational molecular design is to carry out targeted mutations on a gene sequence that is most likely to affect the function and properties of proteins on the basis of known three-dimensional structure and function of proteins, and purposefully change one or two amino acid residues or modules of proteins to construct new protein molecules. Compared with irrational design, rational design has the advantages of less workload and easier to obtain effective mutants.

[0009] Therefore, in the present invention, by Phanerodontia chrysosporium ) were molecularly modified to obtain a lignin peroxidase with improved stability. Subsequently, a lignin peroxidase mutant with improved stability was expressed and prepared using the Pichia pastoris expression system. Summary of the invention

[0010] The object of the present invention is to provide a lignin peroxidase mutant with higher stability. lip ) were expressed in Pichia pastoris; overlapping PCR was used to lip The gene is mutated and the mutants are screened using ABTS to obtain mutants with improved stability.

[0011] One of the technical solutions provided by the present invention is a lignin peroxidase mutant, which is obtained by causing T158V and A161P mutations on the basis of the wild-type lignin peroxidase shown in SEQ ID NO.1, and the mutant is named T158V / A161P;

[0012] Furthermore, the amino acid sequence of the lignin peroxidase T158V / A161P mutant is shown in SEQ ID NO.3;

[0013] Furthermore, the nucleotide sequence of the gene encoding the T158V / A161P mutant is shown in SEQ ID NO.4.

[0014] The second technical solution provided by the present invention is a recombinant plasmid or recombinant strain comprising the above mutant encoding gene.

[0015] Furthermore, the expression vector used was pPIC9K, and the expression host was P. pastoris GS115.

[0016] The third technical solution provided by the present invention is the use of the above-mentioned recombinant plasmid or recombinant strain in producing the lignin peroxidase mutant described in one of the technical solutions.

[0017] The fourth technical solution provided by the present invention is the use of the lignin peroxidase mutant described in the first technical solution in degrading lignin.

[0018] The fifth technical solution provided by the present invention is the use of the lignin peroxidase mutant described in the first technical solution in degrading straw.

[0019] The following definitions are used in the present invention:

[0020] Nomenclature of amino acids and DNA sequences: Amino acid residues are named using the generally accepted IUPAC nomenclature, in the form of single-letter or three-letter codes. DNA sequences are named using the generally accepted IUPAC nomenclature.

[0021] The identification of the lignin peroxidase mutant uses "original amino acid + position + replaced amino acid" to indicate the mutated amino acid in the LIP mutant. For example, T158V means that the amino acid at position 158 is replaced by Val from Thr of wild-type LIP; A161P means that the amino acid at position 161 is replaced by Pro from Ala of wild-type LIP. The position number corresponds to the amino acid sequence number of the wild-type LIP mature peptide in SEQ ID NO.1.

[0022] In the present invention, LIP represents wild-type lignin peroxidase, T158V / A161P represents lignin peroxidase mutant, and lowercase italics lip Represents the gene encoding the wild-type lignin peroxidase LIP, in lowercase italics lipm Represents the gene encoding the mutant T158V / A161P.

[0023]

[0024] Beneficial Effects

[0025] The present invention performs molecular modification on the wild-type LIP and obtains a mutant T158V / A161P having improved activity and stability at 40°C compared with the wild-type. The activity of the mutant T158V / A161P is improved to 130.5% of that of the wild-type LIP, and the half-life at 40°C is improved to 186.1% of that of the wild-type LIP. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The electrophoresis diagram of PCR amplification of wild-type lignin peroxidase

[0027] Among them: Lane 1 is the lignin peroxidase gene lip , Lane 2 is DNA Marker.

[0028] Figure 2 This is the enzyme activity change curve of wild-type lignin peroxidase LIP and mutant T158V / A161P at 40°C. DETAILED DESCRIPTION

[0029] The technical content of the present invention is further described below in conjunction with examples, 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.

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

[0031] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride (add 15 g / L agar powder to solid medium), autoclave at 121 °C for 20 min.

[0032] YPD medium: 10 g / L yeast powder, 20 g / L peptone, 20 g / L glucose (add 15 g / L agar powder to solid medium), autoclave at 115 ℃ for 20 min.

[0033] 10×YNB (containing ammonium sulfate): Dissolve 13.4 g of YNB in ​​100 mL of sterile water, sterilize by filtration, and store at 4°C.

[0034] 500× Biotin: Dissolve 20 mg of biotin in 100 mL of sterile water, sterilize by filtration, and store at 4°C.

[0035] 1 mol / L, pH 6.0 potassium phosphate buffer: Prepare 1 mol / L dipotassium hydrogen phosphate and 1 mol / L potassium dihydrogen phosphate and mix to a pH of 6.0.

[0036] MD medium: 2 g glucose and 1.5 g agar powder are dissolved in 90 mL sterile water and sterilized by autoclaving at 115 °C for 20 min. Before use, the temperature of the culture medium is cooled to about 50 °C after dissolution, and 10 mL of the above-mentioned sterilized 10×YNB and 200 μL of 500×biotin are added on the clean bench.

[0037] BMGY medium: 1 g tryptone, 0.5 g yeast powder and 1 mL glycerol were dissolved in 40 mL sterile water and sterilized at 121 °C for 20 min. Before use, 5 mL of the above-mentioned sterilized 10×YNB, 100 μL of 500× biotin, and 5 mL of 1 mol / L, pH 6.0 potassium phosphate buffer were added in the clean bench.

[0038] BMMY medium: 1 g of tryptone and 0.5 g of yeast powder were dissolved in 40 mL of sterile water and sterilized at 121 °C for 20 min. Before use, 5 mL of the above-mentioned sterilized 10×YNB, 100 μL of 500×biotin, 5 mL of 1 mol / L, pH 6.0 potassium phosphate buffer, and 250 μL of methanol were added to the clean bench.

[0039] Britton-Robinson buffer: Add 25 mL of 0.2 mol / L NaOH to 100 mL of a 0.04 mol / L mixture of boric acid, phosphoric acid, and acetic acid.

[0040] Solution A: 0.02 mol / L Tris-HCl, 0.5 mol / L NaCl, 0.02 mol / L imidazole, 1mmol / L DTT.

[0041] Solution B: 0.02 mol / LTris-HCl, 0.5 mol / L NaCl, 0.03 mol / Limidazole, 1mmol / L DTT.

[0042] Solution C: 0.02 mol / L Tris-HCl, 0.5 mol / L NaCl, 0.5 mol / L imidazole, 1mmol / L DTT.

[0043] In the present invention, the wild-type lignin peroxidase LIP sequence is shown in SEQ ID NO.1:

[0044] MAFKQLFAASLALLLSAANAAAAVIEKRATCSNGKTVGDASCCAWFDVLDDIQQNLFHGGQCGAEAHESIRLVFHDSIAISPAMEAQGKFGGGGADGSIMIFDDIETAFHPNIGLDEIVKLQKPFVQKHGVTPGDFIAFAGRVALSNCPGAPQMNFFTGRAPATQPAPDGLVPEPFHTVDQIINRV NDAGEFDELELVWMLSAHSVAAVNDVDPTVQGLPFDSTPGIFDSQFFVETQLRGTAFPGSGGNQGEVESPLPGEIRIQSDHTIARDSRTACEWQSFVNNQSKLVDDFQFIFLALTQLGQDPNAMTDCSDVIPQSKPIPGNLPFSFFPAGKTIKDVEQACAETPFPTLTTLPGPETSVQRIPPPPGA

[0045] In the present invention, the sequence of the T158V / A161P mutant is shown in SEQ ID NO.3:

[0046] MAFKQLFAASLALLLSAANAAAAVIEKRATCSNGKTVGDASCCAWFDVLDDIQQNLFHGGQCGAEAHESIRLVFHDSIAISPAMEAQGKFGGGGADGSIMIFDDIETAFHPNIGLDEIVKLQKPFVQKHGVTPGDFIAFAGRVALSNCPGAPQMNFFVGRPPATQPAPDGLVPEPFHTVDQIINRV NDAGEFDELELVWMLSAHSVAAVNDVDPTVQGLPFDSTPGIFDSQFFVETQLRGTAFPGSGGNQGEVESPLPGEIRIQSDHTIARDSRTACEWQSFVNNQSKLVDDFQFIFLALTQLGQDPNAMTDCSDVIPQSKPIPGNLPFSFFPAGKTIKDVEQACAETPFPTLTTLPGPETSVQRIPPPPGA

[0047] The present invention will be further explained below through specific examples.

[0048] Example 1: Obtaining wild-type lignin peroxidase gene

[0049] The LIP gene sequence from Trichoderma chrysosporium was optimized according to the codon preference of Pichia pastoris GS115 host and submitted to Genewise for synthesis (shown in SEQ ID NO.2). Eco RI and Not I two restriction sites and plasmid pPIC9K connected. The ligation product is first transferred into the cloning host E. coli JM109, coated on LB solid medium, extract the plasmid from the transformant for PCR and double enzyme digestion verification, and sequence verification for successful verification. Restriction endonuclease was used to verify the correct plasmid Sal I was linearized, purified, and then electrotransferred into P. pastoris GS115 was integrated into the GS115 genome (0.2 mm electroporation cuvette, 1500 V, 5 ms), and plated on MD plates. After 2-3 days, the transformants were picked and plated on G418-resistant YPD solid medium. The transformants were sequenced and pPIC9K- lip Plasmid.

[0050] Example 2 Construction of lignin peroxidase mutants

[0051] The T158V / A161P mutation was determined based on the results of bioinformatics analysis, and the mutant T158V / A161P was obtained by constructing it through overlapping PCR.

[0052] 1. Based on overlapping PCR technology, site-directed mutagenesis of T158 and A161 was performed to construct the lignin peroxidase mutant T158V / A161P. The designed mutation primers (upstream primer F, downstream primer R) are as follows:

[0053] F1: CCG GAATTC ATGGCTTTTAAACAATTGTTTGCT (shown in SEQ ID NO.5);

[0054] R1: TGTTGGTAGACCGCCTGC (shown in SEQ ID NO. 6);

[0055] F2: GCAGGCGGTC TACCAACA (shown in SEQ ID NO.7);

[0056] R2: CATG CCATGG AGCACCAGGT GGAGGTGG (shown in SEQ ID NO.8);

[0057] In the first step of overlapping PCR reaction system, F1 and R1 were used as upstream primers, and F2 and R2 were used as downstream primers. lipAs a template, PCR1 reaction was carried out to obtain the upstream fragment and the downstream fragment respectively.

[0058] The reaction system for upstream fragment amplification is:

[0059]

[0060] The reaction system for downstream fragment amplification is:

[0061]

[0062] The amplification program was as follows: pre-denaturation at 98°C for 30 min; denaturation at 98°C for 10 s, annealing at 54°C for 20 s, and extension at 72°C for 5 s, for 30 cycles; and extension at 72°C for 10 min.

[0063] After gel excision and recovery of upstream and downstream fragments, PCR 2 was performed. The reaction system was:

[0064]

[0065] The amplification program was as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 54°C for 20 s, and extension at 72°C for 8 s, for 5 cycles; and extension at 72°C for 10 min.

[0066] After PCR 2, 2 μL of primers F1 and R2 were added to the system, and the PCR 3 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 10 s, 30 cycles; 72°C extension for 10 min. The PCR amplification products were subjected to 1.2% agarose gel electrophoresis, and the PCR products were recovered using a small amount of DNA recovery kit.

[0067] Gene Eco RI and Not I two restriction sites and plasmid pPIC9K connected. The ligation product is first transferred into the cloning host E. coli JM109 was plated on LB solid medium, and plasmids were extracted from transformants for PCR and double enzyme digestion verification. Successful plasmids were sequenced and pPIC9K- T158V / A161P Plasmid. T158V / A161P and plasmid pPIC9K- lip Use restriction enzyme Sal I was linearized, purified and recovered, and then electrotransferred into P. pastoris GS115, integrated into the GS115 genome (0.2 mm electroporation cuvette, 1500 V, 5 ms), and plated on MD plates. After 2-3 days, the transformants were picked and plated on G418-resistant YPD solid medium. The positive transformants were named P. pastorisGS115 / pPIC9K- T158V / A161P and P. pastoris GS115 / pPIC9K- lip .

[0068] Example 3 Expression, purification and enzyme activity determination of lignin peroxidase

[0069] 1. Expression of lignin peroxidase

[0070] Pick P. pastoris GS115 / pPIC9K- T158V / A161P and P. pastoris GS115 / pPIC9K- lip The transformants were incubated in 200 mL BMGY medium containing glycerol for 16-18 h at 30 °C and 200 r / min. Then all the transformants were inoculated into 200 mL BMMY medium and methanol (0.5% v / v) was added for induction culture. 2+ LIP, BMMY medium was supplemented with Cu at a final concentration of 1 mmol / L 2+ . Fermentation was carried out at 30 ℃ and 200 r / min for 6 days, and 0.5% v / v methanol was added every day. After the fermentation, the fermentation broth was centrifuged at 4 ℃ and 12,000 r / min for 30 min, and the supernatant was filtered through a 0.45-µm filter to obtain the crude enzyme solution of T158V / A161P and LIP. The activity of LIP and T158V / A161P was determined by the ABTS method. The principle is that in the presence of hydrogen peroxide, lignin peroxidase can oxidize ABTS to free radical cation ABTS + , ABTS + There is a maximum absorbance at 420nm. As the concentration of ABTS free radicals increases, the absorbance value increases. The time it takes for the absorbance value to change within a certain specified range is expressed as the activity of lignin peroxidase.

[0071] The steps for measuring enzyme activity by ABTS method are as follows: (1) Take 200 μL of Britton-Robinson buffer in a 48-well plate and keep it in a 40℃ water bath for 1 min. (2) Add 500 μL of LIP or T158V / A161P crude enzyme solution diluted to an appropriate concentration and mix well, and then put it in a 40℃ water bath and keep it in a 40℃ water bath for 1 min; (3) Add 25 μL of H2O2 (50 mM) and mix well by pipetting, and react in a 40℃ constant temperature water bath for 10 min. At the beginning and end of the reaction, record the OD value at 420 nm at the beginning and end of the reaction.

[0072] Unit enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 µmol of oxidation product per minute under certain conditions (temperature is 40 °C and pH is 4 unless otherwise specified).

[0073] The enzyme activity calculation formula is: .

[0074] Where D represents the dilution multiple of the enzyme, Vt represents the volume of the total system for enzyme activity determination (mL), △OD represents the absorbance change within 1 min, Vs represents the enzyme solution volume of the reaction system (mL), and E represents the molar absorption coefficient of the product formed by ABTS catalyzed by lignin peroxidase, which is 36000 M -1 cm -1 , d represents the inner diameter / optical path thickness of the 48-well ELISA plate (cm).

[0075] The results showed that the fermentation activity of wild-type LIP was 122.4 U / mL, and that of mutant T158V / A161P was 169.2 U / mL.

[0076] 2. Purification of recombinant protein

[0077] Before the Ni-NTA affinity column purification process, add an appropriate amount of ddH2O to the column and add two times the column volume of solution A to balance the resin; mix the balanced resin and crude enzyme solution, place in a magnetic stirrer, bind at a speed of 100-120 r / min for 1 h, and keep low temperature (4 °C) throughout the process. Add the binding solution to the purification column in 2-3 times in the chromatography cabinet; after the binding solution is completely filtered out, add 10 mL of solution B to elute the impurities bound to the resin; finally, add 10 mL of pre-cooled solution C to the purification column to elute all the target proteins bound to the resin, and collect the filtrate; the eluted protein is dialyzed with 20 mmol / L, pH 4.0 phosphate buffer at 4 °C. The purified LIP or T158V / A161P protein is obtained.

[0078] The enzyme activity of the purified protein was measured according to the aforementioned ABTS method, and the specific activity was calculated. The specific activity of the enzyme (U / mg) = enzyme activity / protein concentration.

[0079] The final calculated activity ratio of wild-type LIP and mutant is as follows:

[0080]

[0081] Example 4 Determination of half-life of LIP or T158V / A161P

[0082] The half-life of purified LIP or T158V / A161P at 40°C (pH 4) was determined by placing purified LIP or T158V / A161P in a 40°C water bath for a certain period of time and then determining the residual enzyme activity. The enzyme activity detection method and reaction system were the same as those for the crude enzyme solution in Example 3. The mutant T158V / A161P was found to be more stable than the wild-type LIP at 40°C. The enzyme activity change curves of the wild-type LIP and the mutant T158V / A161P are shown in FIG. Figure 2 The final calculated half-life of wild-type LIP and mutant T158V / A161P at 40°C is as follows:

[0083]

[0084] Example 5 Application of lignin peroxidase mutants in degrading lignin

[0085] The ability of LIP and the T158V / A161P mutant to degrade lignin was tested using lignin extracted from straw as a substrate. The lignin extraction method was as follows: first, lignin was pretreated with ultrasound in a 2 M NaOH aqueous solution (1 g straw: 20 mL 2 M NaOH solution) at 90 °C. Then, the mixture was stirred at 90 °C and 150 rpm for 1.5 h. The mixture was washed with 0.1 M NaOH to separate the lignin on the cellulose surface. After filtering on nylon cloth, the cellulose-rich residue was removed. Then, hemicellulose was precipitated with 95% ethanol at pH 5.5. The precipitate was filtered and removed. Finally, after ethanol evaporation, alkali-soluble lignin was obtained by precipitation with hydrochloric acid to adjust the pH to 1.5. The lignin-rich solid was then washed with an acid solution at pH 2.0 and freeze-dried. Finally, pure lignin was obtained.

[0086] Take the above extracted lignin and use 0.1 M NaOH solution to prepare lignin solutions with concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mg / L, respectively. Determine the absorbance at 280 nm. According to the absorbance and the corresponding concentration of lignin, draw a standard curve of lignin concentration as shown below:

[0087] y=0.06373+0.01208x (R 2 =0.9995)

[0088] 10 mg of extracted straw lignin was completely dissolved with 0.1 M NaOH solution, and NaOH solution was added to prepare a lignin mother solution with a concentration of 5 mg / mL. The reaction system for lignin degradation was 10 mL, 100 mM sodium acetate buffer at pH 4.0, containing 2 mL of lignin solution, 10 mM H2O2, and 25 mg of purified LIP or T158V / A161P. After incubation at 40°C and 220 rpm for 72 h, the absorbance of the supernatant of the reaction system was measured at 280 nm, and the lignin concentration before and after degradation was calculated according to the standard curve, and the lignin degradation rate was further calculated.

[0089] It was determined that the degradation rate of extracted straw lignin by the mutant T158V / A161P was 1.22 times that of the wild-type LIP.

[0090]

[0091] Example 6 Application of lignin peroxidase mutants to degrade corn stalks

[0092] A certain amount of corn stalks were washed with deionized water to remove surface dirt, and 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 grinder and filtered with a 40-mesh sieve to ensure that they became uniform powders. Finally, they were placed at room temperature for subsequent use. The treated corn stalks were used as substrates to detect the ability of LIP and its mutants to degrade stalks.

[0093] The reaction system was 10 mL: 100 mM sodium acetate buffer at pH 4.0 containing 0.5 g treated corn stover, 10 mM H2O2, and 25 mg purified LIP or T158V / A161P. After incubation at 40 °C and 220 rpm for 72 h, the mixture was centrifuged to determine the lignin content in the stover before and after the reaction. The degradation rate of lignin in the stover was calculated based on this.

[0094] Lignin in straw includes acid-soluble lignin and acid-insoluble lignin. The determination method is to centrifuge the mixture before and after the reaction, extract the solid with anhydrous ethanol for 8 h, add 3 mL of 72% sulfuric acid after complete drying, and stir until the raw materials are fully mixed. After mixing, place it in a 50 ℃ water bath for 60 min; take it out and add 84 mL of deionized water to dilute it to 4%, put it in a 121 ℃ sterilizer, take it out after 45 min, use a G3 (weighing) sand core funnel for vacuum filtration, take 50 mL of 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; wash the filtered residue with hot deionized water to neutrality, dry it in an oven at 105 ℃, and weigh and record it. After drying, transfer it to a resistance furnace and burn it at 575 ℃ for 4 h. Take it out and cool it to room temperature, then weigh and record it. The difference is the acid-insoluble lignin content.

[0095] It was determined that the degradation rate of lignin in straw by the mutant T158V / A161P was 1.31 times that of the wild-type LIP.

[0096]

[0097] 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 lignin peroxidase mutant, characterized in that: The lignin peroxidase mutant is obtained by causing T158V and A161P mutations to the wild-type lignin peroxidase shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

3.

2. A recombinant plasmid or recombinant strain comprising a gene encoding the lignin peroxidase mutant according to claim 1.

3. Use of the recombinant plasmid or recombinant strain according to claim 2 in producing the lignin peroxidase mutant according to claim 1.

4. Use of the lignin peroxidase mutant according to claim 1 in degrading lignin.

5. Use of the lignin peroxidase mutant according to claim 1 in degrading straw.

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