Construction and application of high-activity and thermal-stability cracking polysaccharide monooxygenase mutant

Abstract

The invention discloses construction and application of a high-activity and thermal-stability cracking polysaccharide monooxygenase mutant, and belongs to the technical field of enzyme engineering and microbial engineering. After the wild-type enzyme is subjected to molecular modification, the enzyme activity is improved by 88% or above compared with that of the wild-type enzyme, and the thermal stability is also remarkably improved compared with that of the wild-type enzyme. According to the present invention, the highest synergy degrees of the cleavable polysaccharide monooxygenase mutant and the thermophilic endoglucanase for enzymolysis of the swelling phosphate cellulose and the microcrystalline cellulose respectively achieve 155% and 133% (the wild type is 127% and 120%); the glucose yield can be remarkably increased by cooperating with 1.5 L of cellulase Celluclast and Ctec 2 to hydrolyze microcrystalline cellulose and pretreating bagasse with glycerol under the catalysis of acid / base. The method is high in practicability and can be widely applied to agriculture and forestry biomass raw materials with various sources, and the enzymolysis efficiency of cellulose substrates is improved.

Description

Technical Field

[0001] The present invention relates to the construction and application of a highly active and thermally stable lytic polysaccharide monooxygenase mutant, belonging to the fields of enzyme engineering and technology. Background Art

[0002] Cellulose is a renewable non-fossil carbon source that is abundantly present on Earth and is one of the main components of lignocellulosic biomass, usually present in plant cell walls. As a linear polymer composed of d-glucose units linked by β-glycosidic bonds, cellulose has important biological significance. However, cellulose is the most difficult to degrade among all plant cell wall polysaccharides, making its degradation process a highly challenging task. The process of converting cellulose into monosaccharides, especially its application in bioethanol production, has attracted extensive attention because it provides an environmentally friendly and sustainable solution to meet the growing global energy demand driven by industrial development and urbanization.

[0003] The enzymatic hydrolysis process of cellulose involves three key cellulases: endoglucanase, cellobiohydrolase / exoglucanase, and β-glucosidase. These three types of enzymes work together to decompose cellulose. Endoglucanase randomly cleaves the internal glycosidic bonds of cellulose, rapidly shortening the polymer length; exoglucanase releases reducing sugars by hydrolyzing the reducing or non-reducing ends of the cellulose chain. Intermediate products such as cellobiose and oligosaccharides are further converted into glucose by β-glucosidase. However, due to the very strong crystalline regions in the cellulose structure, ordinary cellulases have limited effects on it, resulting in hindrance to the enzymatic hydrolysis process.

[0004] Since the discovery of lytic polysaccharide monooxygenases (LPMOs) in 2010, this limitation has been effectively improved, especially for LPMOs of the AA9 family. AA9 LPMOs do not directly hydrolyze polysaccharides, but rather oxidatively cleave the resistant regions in cellulose, forming pores on its crystalline surface, thereby disrupting the crystalline structure of cellulose. This structural change makes other glycoside hydrolases (such as endoglucanase and exoglucanase) more accessible to the polysaccharide, thus working synergistically with cellulases to achieve more efficient cellulose hydrolysis.

[0005] In industrial applications, the stability and catalytic activity of enzymes are the most critical enzymatic parameters, which directly determine the economic feasibility of processes. Currently, most AA9 LPMOs are derived from wild-type enzymes, and the thermal stability and catalytic activity of these enzymes have not fully realized their potential. Therefore, in order to meet the requirements of industrial applications, it is usually necessary to optimize them through means such as protein engineering. Rational design methods based on computer-aided design have been widely used to identify key amino acids and conduct directed evolution, so as to improve the thermal stability and catalytic activity of AA9 LPMOs.

[0006] By modifying AA9 LPMOs to enhance their thermal stability and catalytic activity, not only can the synergistic hydrolysis with cellulases be strengthened, but also cellulases can be made more efficient in degrading lignocellulose, and the amount of enzyme used can be reduced, thereby reducing costs and further improving the economic benefits in industrial applications. Therefore, there is an urgent need to develop lytic polysaccharide monooxygenase mutants with higher activity and better thermal stability to enhance enzyme activity and synergy, and then improve the enzymatic hydrolysis rate of different cellulose substrates. Summary of the Invention

[0007] To solve the problems of low enzyme activity of lytic polysaccharide monooxygenase and low enzymatic hydrolysis rate in synergy with cellulase, the present invention provides a lytic polysaccharide monooxygenase mutant. Compared with the wild-type lytic polysaccharide monooxygenase, the enzyme activity of the mutant is improved; when synergistically enzymatically hydrolyzing phosphoric acid swollen cellulose, microcrystalline cellulose and pretreated sugarcane bagasse, the enzymatic hydrolysis rate is increased.

[0008] The first object of the present invention is to provide a lytic polysaccharide monooxygenase mutant, characterized in that the lytic polysaccharide monooxygenase mutant has one or more amino acid mutations at the 32nd, 56th, 60th, 63rd, 72nd, 79th, 80th, 84th, 90th, 98th, 104th, 107th, 120th, 121st, 128th, 137th, 143rd, 154th, 160th, 170th or 175th positions based on the amino acid sequence shown in SEQ ID NO.1.

[0009] In one embodiment, the lytic polysaccharide monooxygenase mutant is derived from the lytic polysaccharide monooxygenase MtLPMO9V (GenBank: AEO55082.1) of Myceliophthora thermophila.

[0010] In one embodiment, the amino acid sequence of the lytic polysaccharide monooxygenase MtLPMO9V is as shown in SEQ ID NO.7, wherein the first 21 amino acids are the signal peptide, and the amino acids from the 22nd position to the end are the mature enzyme sequence.

[0011] In one embodiment, the 22nd amino acid of the amino acids of the lytic polysaccharide monooxygenase MtLPMO9V is used as the first amino acid of the wild-type (WT) lytic polysaccharide monooxygenase, and its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0012] In one embodiment, the lytic polysaccharide monooxygenase mutant corresponds to the amino acid sequence shown in SEQ ID NO.1,

[0013] The glycine at the 32nd position is mutated to serine and named G32S;

[0014] The threonine at the 56th position is mutated to cysteine and named T56C;

[0015] The aspartic acid at the 60th position is mutated to tyrosine and named D60Y;

[0016] The aspartic acid at the 63rd position is mutated to leucine and named D63L;

[0017] The proline at the 72nd position is mutated to valine and named P72V;

[0018] The threonine at the 79th position is mutated to valine and named T79V;

[0019] The serine at the 80th position is mutated to isoleucine and named S80I;

[0020] The serine at the 84th position is mutated to valine and named S84V;

[0021] The valine at the 90th position is mutated to cysteine and named V90C;

[0022] The alanine at the 98th position is mutated to proline and named A98P;

[0023] The aspartic acid at the 104th position is mutated to methionine and named D104M;

[0024] The proline at the 107th position is mutated to leucine and named P107L;

[0025] The glutamine at the 120th position is mutated to tyrosine and named Q120Y;

[0026] The arginine at the 121st position is mutated to valine and named R121V;

[0027] The aspartic acid at the 128th position is mutated to proline and named D128P;

[0028] The glutamine at position 137 is mutated to isoleucine, named Q137I;

[0029] The proline at position 143 is mutated to asparagine or alanine, named P143N and P143A respectively;

[0030] The alanine at position 154 is mutated to proline, named A154P;

[0031] The aspartic acid at position 160 is mutated to glycine, named D160G;

[0032] The alanine at position 170 is mutated to cysteine, named A170C;

[0033] The alanine at position 175 is mutated to cysteine, named A175C.

[0034] In one embodiment, the lytic polysaccharide monooxygenase mutant has the following mutations simultaneously:

[0035] The alanine at position 170 is mutated to cysteine and the alanine at position 175 is mutated to cysteine, named A170C / A175C;

[0036] Preferably, the alanine at position 170 is mutated to cysteine, the alanine at position 175 is mutated to cysteine, and the glycine at position 32 is mutated to serine, named A170C / A175C / G32S;

[0037] Preferably, the alanine at position 170 is mutated to cysteine, the alanine at position 175 is mutated to cysteine, and the proline at position 107 is mutated to leucine, named A170C / A175C / P107L;

[0038] Preferably, the alanine at position 170 is mutated to cysteine, the alanine at position 175 is mutated to cysteine, and the glutamine at position 120 is mutated to tyrosine, named A170C / A175C / Q120Y;

[0039] Preferably, the alanine at position 170 is mutated to cysteine, the alanine at position 175 is mutated to cysteine, and the glutamine at position 137 is mutated to isoleucine, named A170C / A175C / Q137I;

[0040] Preferably, the alanine at position 170 is mutated to cysteine, the alanine at position 175 is mutated to cysteine, and the alanine at position 154 is mutated to proline, named A170C / A175C / A154P;

[0041] Preferably, the alanine at position 170 is mutated to cysteine, the alanine at position 175 is mutated to cysteine, and the aspartic acid at position 160 is mutated to glycine, named A170C / A175C / D160G.

[0042] In one embodiment, the amino acid sequence of the mutant A170C / A175C / Q120Y is shown in SEQ ID NO.3; the nucleotide sequence is shown in SEQ ID NO.4.

[0043] The second object of the present invention is to provide a polynucleotide of any of the above lytic polysaccharide monooxygenase mutants.

[0044] The third object of the present invention is to provide a vector of the above polynucleotide.

[0045] In one embodiment, the vector includes pPIC series vectors, pPICZ series vectors, pPICZα, pET series vectors;

[0046] Optionally, the pPIC series vectors include pPIC9, pPIC9K and pPIC3.5K, etc.; the pPICZ series vectors include pPICZA, pPICZB, pPICZC, etc.; the pPICZα series vectors include pPICZαA, pPICZαB, pPICZαC, etc.; the pET series vectors include pET-22b.

[0047] The fourth object of the present invention is to provide a cell expressing any of the above lytic polysaccharide monooxygenase mutants;

[0048] In one embodiment, the cell includes Pichia pastoris, Escherichia coli, Saccharomyces cerevisiae and filamentous fungi;

[0049] Optionally, the filamentous fungi include Aspergillus niger, Aspergillus oryzae, etc.

[0050] The fifth object of the present invention is to provide the application of any of the above lytic polysaccharide monooxygenase mutants or the above polynucleotide or the vector of the above polynucleotide or the above cell in hydrolyzing cellulose.

[0051] The sixth object of the present invention is to provide a method for hydrolyzing cellulose, using any of the above lytic polysaccharide monooxygenase mutants and cellulase for synergistic enzymatic hydrolysis;

[0052] Optionally, the cellulase includes a thermophilic endoglucanase or a cellulase.

[0053] In one embodiment, the thermophilic endoglucanase is DtCelA, and its amino acid sequence is as shown in SEQ ID NO.5.

[0054] In one embodiment, the cellulase includes a commercial cellulase, for example, Celluclast or Ctec2.

[0055] In one embodiment, the cellulose includes pretreated bagasse, phosphoric acid swollen cellulose, and microcrystalline cellulose.

[0056] In one embodiment, the pretreated bagasse is alkali-treated bagasse and acid-treated bagasse, and the preparation method includes:

[0057] Mix dried sugarcane, glycerol, and NaOH (or sulfuric acid), heat and stir, filter and wash to obtain alkali-treated bagasse (acid-treated bagasse).

[0058] In one embodiment, the addition amount of the lytic polysaccharide monooxygenase mutant is 0.2 - 2 mg / g of dry substrate; the addition amount of the cellulase (the total amount of the lytic polysaccharide monooxygenase and the commercial cellulase) is 4 - 10 mg / g of dry substrate.

[0059] In one embodiment, the addition amount of the cellulose is 0.5 - 2% w / v.

[0060] In one embodiment, the enzymatic hydrolysis condition is enzymatic hydrolysis at 45 - 50 °C for 12 - 96 h.

[0061] In one embodiment, when using 2% w / v of MCC (or acid-catalyzed glycerol pretreated substrate) as the substrate, 1 mM gallic acid (not added when the substrate is MCC), the addition amount of the lytic polysaccharide monooxygenase mutant is 0.4 mg / g, and the cellulase is Celluclast 1.5L with 9.6 mg / g of dry substrate enzyme mixture, reacting for 96 h, and the glucose yield reaches 77% (when the substrate is acid-catalyzed glycerol pretreated substrate, the glucose yield reaches 74%);

[0062] When using 2% w / v of MCC (or acid-catalyzed glycerol pretreated substrate) as the substrate, 1 mM gallic acid (not added when the substrate is MCC), the addition amount of the lytic polysaccharide monooxygenase mutant is 0.4 mg / g, and the cellulase is Ctec2 with 4.27 mg / g of dry substrate enzyme mixture, reacting for 72 h, and the glucose yield reaches 81% (when the substrate is acid-catalyzed glycerol pretreated substrate, the glucose yield reaches 74%).

[0063] In one embodiment, when the substrate pretreated with 2% w / v alkali-catalyzed glycerol is used as the substrate, with 1 mM gallic acid, the addition amount of the lytic polysaccharide monooxygenase mutant is 0.2 mg / g, and the cellulase is Celluclast 1.5L at 9.8 mg / g dry substrate enzyme mixture. After reacting for 96 h, the glucose yield reaches 86%;

[0064] When the substrate pretreated with 2% w / v alkali-catalyzed glycerol is used as the substrate, with 1 mM gallic acid, the addition amount of the lytic polysaccharide monooxygenase mutant is 0.2 mg / g, and the cellulase is Ctec2 at 4.47 mg / g dry substrate enzyme mixture. After reacting for 72 h, the glucose yield reaches 77%.

[0065] Beneficial effects

[0066] In the present invention, the lytic polysaccharide monooxygenase MtLPMO9V is used as the wild type. By performing point mutations at different sites of the lytic polysaccharide monooxygenase MtLPMO9V, the enzyme activity after mutation is increased by more than 88%, and the synergistic enzymatic hydrolysis effect of the lytic polysaccharide monooxygenase and cellulase on different cellulose substrates is improved.

[0067] The mutant A170C / A175C / Q120Y has the best effect, which is specifically as follows:

[0068] (1) The enzyme activity of the lytic polysaccharide monooxygenase mutant A170C / A175C / Q120Y (M3) of the present invention is increased by 88% compared with the wild type under standard conditions, and the enzyme activity reaches 63.4 U / g;

[0069] (2) When the lytic polysaccharide monooxygenase mutant A170C / A175C / Q120Y (M3) of the present invention synergistically hydrolyzes phosphoric acid swollen cellulose and microcrystalline cellulose with the thermophilic endoglucanase DtCelA, compared with the wild type, it has a better synergistic effect, and the highest synergy degrees can reach 155% and 133% (the wild type is 127% and 120% respectively);

[0070] (3) The lytic polysaccharide monooxygenase mutant A170C / A175C / Q120Y (M3) of the present invention shows a higher synergistic effect when synergistically hydrolyzing microcrystalline cellulose and the substrate of sugarcane bagasse pretreated with glycerol under acid / alkali catalysis with cellulase Celluclast1.5L. The glucose yields at 96 h are increased from 58%, 56% and 61% to 77%, 74% and 86% respectively (the wild type is 70%, 66% and 78%); when synergistically hydrolyzing microcrystalline cellulose and the substrate of sugarcane bagasse pretreated with glycerol under acid / alkali catalysis with Ctec 2, it also shows a better synergistic effect. The glucose yields at 72 h are increased from 60%, 61% and 66% to 81%, 74% and 86% respectively (the wild type is 75%, 65% and 77%). Description of the drawings

[0071] Figure 1 represents the relative enzyme activity of different lytic polysaccharide monooxygenase mutants to the wild type;

[0072] Figure 2 represents the synergistic hydrolysis of PASC and MCC by mutant M3 and DtCelA; wherein, a and b are the synergistic hydrolysis of PASC and MCC by wild-type MtLPMO9V and DtCelA; c and d are the synergistic hydrolysis of PASC and MCC by mutant M3 and DtCelA;

[0073] Figure 3 represents the synergistic hydrolysis of MCC and pretreated substrates by mutant M3 and Celluclast 1.5L; wherein, a, b, and c are the synergistic hydrolysis of MCC, acid-pretreated sugarcane bagasse (ac-AGO), and alkali-pretreated sugarcane bagasse (al-AGO) by mutant M3 and Celluclast 1.5L, respectively;

[0074] Figure 4 represents the synergistic hydrolysis of MCC and pretreated substrates by mutant M3 and Ctec2; wherein, a, b, and c are the synergistic hydrolysis of MCC, acid-pretreated sugarcane bagasse (ac-AGO), and alkali-pretreated sugarcane bagasse (al-AGO) by mutant M3 and Ctec2, respectively. Detailed implementation manners

[0075] The technical solutions described in the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0076] Experimental materials

[0077] The Escherichia coli strain (E. coli DH5α) is preserved by this laboratory; the expression strain: Pichia pastoris GS115 is preserved by this laboratory; the plasmid pPIC9K-MtLPMO9V is preserved by this laboratory; the thermophilic endoglucanase DtCelA is expressed by the strain reserved in the previous construction of Escherichia coli E. coli BL21(DE3) in the laboratory. The amino acid sequence of the thermophilic endoglucanase DtCelA is shown in SEQ ID NO.5, and the thermophilic endoglucanase DtCelA can be directly obtained by expression and purification through Escherichia coli E. coli BL21(DE3).

[0078] In the present invention, the enzyme activities and molecular weights of the enzymes used are as follows: MtLPMO9V wild type (WT): 34.2 U / g (at 30 °C and pH 7.5, which are the standard conditions for detecting enzyme activity), and the molecular weight is approximately 24.4 kDa; DtCelA: 63 U / g, and the molecular weight is 37.1 kDa. In the examples of the present invention, the enzyme addition amounts are all added in the unit of mg / g dry substrate.

[0079] Unless otherwise specified, w / v in the text is g / mL.

[0080] Detection method

[0081] 1. Determination of reducing sugar concentration (DNS method):

[0082] During the hydrolysis process, 0.4 mL of the hydrolysis solution was taken, and the supernatant was obtained by centrifugation. The centrifugation conditions were: rotation speed 8000 rpm, time 5 min. The supernatant was diluted to an appropriate multiple, and the reducing sugar content was determined using DNS, and the reducing sugar concentration was calculated therefrom.

[0083] Calculation formula for synergy degree (DS):

[0084]

[0085] Among them, R DtCelA is the reducing sugar concentration of DtCelA; R LPMO is the reducing sugar concentration of LPMO; R DtCelA+LPMO is the reducing sugar concentration produced by the two enzymes in the system.

[0086] 2. Glucose yield:

[0087] At regular time intervals, 0.4 mL of the sample was taken and heated at 100 °C for 5 minutes to inactivate the enzyme. Then the sample was centrifuged at 8000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane and appropriately diluted. The glucose concentration was detected by high performance liquid chromatography (HPLC, Chomaster CM5110, Hitachi, Japan), equipped with a refractive index detector and a packed column (Bio-Rad Aminex HPX-87H, 9 μm, 7.8×300 mm). The mobile phase was 5 mM dilute H 2 SO 4 , the flow rate was 0.6 mL / min, isocratic elution, and the column temperature was 60 °C.

[0088] The calculation formula for glucose yield is:

[0089]

[0090] 3. Preparation of phosphoric acid swollen cellulose (PASC), the specific steps are as follows:

[0091] (1) Add 0.2 g of MCC (microcrystalline cellulose) to a 50 mL centrifuge tube, and add 0.6 mL of ddH 2 O to wet it and form a suspension;

[0092] (2) Slowly add 10 mL of pre-cooled 86.2% phosphoric acid, stir well, and finally it becomes 83.2%. Before adding the last 2 mL of phosphoric acid, the cellulose suspension should be mixed evenly;

[0093] (3) Place it on ice for 1 h and stir every 10 min;

[0094] (4) Centrifuge at 4 °C and 5000 rpm for 20 min. Suspend the particles with cold water, add 10 mL of cold water each time, for a total of 40 mL. Stir well after each addition. Finally, a white turbid precipitate is produced. Centrifuge at 4 °C and 6500 rpm for 25 min;

[0095] (5) Wash the precipitate with 50 mL of cold water and repeat four times;

[0096] (6) Add 0.5 mL of 2 mol / L Na 2 CO 3 solution to neutralize the phosphoric acid, resuspend with 45 mL of cold water, and centrifuge at 4 °C and 6500 rpm for 25 min;

[0097] (7) Resuspend with 50 mL of cold water and centrifuge twice to make the pH about 5 - 7;

[0098] (8) Store in a refrigerator at 4 °C or -20 °C.

[0099] 4. Preparation method of acid / base-catalyzed glycerol pretreated substrate (i.e., acid / base-catalyzed glycerol organic solvent pretreated bagasse), the specific steps are as follows:

[0100] (1) Grind the bagasse with a grinder into fine particles, sieve it with a 60-mesh sieve, and place it in an oven at 60 °C until it reaches a constant weight;

[0101] (2) Place 10 g of dried bagasse in a three-necked flask, add 140 g of glycerol and 4% NaOH (based on bagasse, 0.4 g) or 6% sulfuric acid (based on bagasse, 0.6 g);

[0102] (3) Place the three-necked flask in a heating mantle and heat to 195 °C (alkali treatment) or 200 °C (acid treatment), and at the same time stir with a stirrer paddle to make it mix and heat evenly. React for 45 min (alkali treatment) or 15 min (acid treatment);

[0103] (4) Start timing when the specified temperature is reached. Add 100 mL of hot water to end the reaction when the reaction is over. Filter the mixture through a G1 sintered glass funnel. Wash the filter cake with hot water until neutral and wash away glycerol.

[0104] (5) Place the treated bagasse in an oven at 60 °C and dry it to a constant weight. Collect and store it for future use after drying.

[0105] 5. Pichia pastoris electrotransformation

[0106] Use the preparation and transformation (electroporation method) of Pichia pastoris GS115 competent cells. The steps are as follows:

[0107] (1) Streak Pichia pastoris GS115 on a YPD plate and culture it at 30 °C for 2 days to isolate single colonies. Use YNB minimal medium and supplemented medium containing His for spot inoculation and purification. Pick the single colonies that grow on the supplemented medium but not on the minimal medium and streak them on a YPD plate, and store them at 4 °C;

[0108] (2) Pick a single colony of Pichia pastoris GS115 from the YPD plate and inoculate it into a 50 mL Erlenmeyer flask containing 5 mL of YPD medium. Culture it overnight at 30 °C, 250 - 300 rpm / min;

[0109] (3) Take 20 μL of the culture and inoculate it into a 50 mL Erlenmeyer flask containing 20 mL of fresh medium. Culture it overnight at 28 - 30 °C, 250 - 300 rpm / min until the OD 600 reaches 1.3 - 1.5;

[0110] (4) Aliquot the cell culture into multiple 1.5 mL EP tubes, centrifuge at 4 °C, 1500 g for 30 s, and resuspend the cell pellet with 1 mL of ice-cold sterile water;

[0111] (5) Centrifuge according to step (4) and resuspend the cell pellet with 1 mL of ice-cold sterile water;

[0112] (6) Centrifuge according to step (4) and resuspend the cell pellet with 1 mL of ice-cold 1 M sorbitol solution;

[0113] (7) Centrifuge according to step (4) and resuspend the cell pellet with 60 μL of ice-cold 1 M sorbitol solution, and immediately use it for electroporation;

[0114] (8) Dissolve 5 - 20 μg of linearized DNA in 5 - 10 μL of TE solution, mix it with 80 μL of the cell suspension obtained in step (7) above, and transfer it to a 0.2 cm ice-cold electroporation cuvette;

[0115] (9) Incubate the electroporation cuvette in an ice bath for 5 min;

[0116] (10) The voltage is 3 kV and the electric shock time is 6 ms;

[0117] (11) Immediately after the electric shock, add 1 mL of 1 mol / L ice-cold sorbitol solution to the electroporation cuvette, mix the cells, and transfer the contents of the electroporation cuvette to a new 1.5 mL centrifuge tube;

[0118] (12) Spread the cell suspension on the MD plate, 200 - 600 μL per plate;

[0119] (13) Incubate the plate at 30 °C until single colonies appear.

[0120] 6. Pichia pastoris protein induction expression and purification

[0121] (1) Induction expression

[0122] Pick single colonies grown on the MD medium into YPD medium containing 1 g / L, 3 g / L, and 5 g / L geneticin for screening. Inoculate the recombinant bacteria growing in high-concentration antibiotics into BMGY medium and culture overnight until the OD 600 reaches 2 - 6, then transfer to BMMY medium until the cell OD 600 reaches 1, and perform shaking fermentation culture in a shaker at 30 °C and 260 rpm. Add methanol solution with a final concentration of 1% every 24 h for induced fermentation to produce enzymes for 7 consecutive days; after fermentation, centrifuge the fermentation broth at 4 °C and 10,000 rpm for 20 min, and collect the supernatant;

[0123] (2) Protein purification

[0124] Precipitate the collected supernatant by adding ammonium sulfate with a final concentration of 70%. After removing the excess salt using a dialysis bag, use the method of Ni + -NTA nickel column affinity chromatography for protein purification, and purify according to its instruction manual; store the purified protein in a 4 °C refrigerator.

[0125] 7. Enzyme activity detection

[0126] Use 2,6-DMP and H 2 O 2 as co-substrates to determine the enzyme activity, optimal temperature, and thermal stability of MtLPMO9V wild type (WT) and mutants:

[0127] (1) Prepare 116 mM phosphate buffer at pH 7.5; 10 mM 2,6-DMP solution; 5 mM H 2 O 2 solution. All solutions should be used within 12 h after preparation.

[0128] (2) Add 860 μL of phosphate buffer, 100 μL of 2,6-DMP solution, and 20 μL of H 2 O 2 solution into a centrifuge tube, mix well, and incubate at the corresponding temperature for 15 min.

[0129] (3) After adding the LPMO protein sample with a final concentration of 1 μM, mix well. At different temperatures, measure the increase in absorbance at a wavelength of 469 nm within 5 min (ε469 = 53200 L / mol / cm) to calculate the activity of LPMO. And measure its residual activity after incubating at different temperatures for different times.

[0130] One unit of enzyme activity is defined as the conversion of 2 μmol of 2,6-DMP per minute under standard reaction conditions. Taking the enzyme activity of WT as 100%, calculate the relative enzyme activity of the mutant.

[0131] Example 1 Preparation of Lytic Polysaccharide Monooxygenase Mutants with Different Mutation Sites

[0132] 1. Construction of Recombinant Mutation Vectors

[0133] The complete gene sequence of the lytic polysaccharide monooxygenase MtLPMO9V (GenBank: AEO55082.1) from Myceliophthora thermophila was obtained from the NCBI and CAZy databases. Its amino acid sequence is shown in SEQ ID NO.6, and the nucleotide sequence is shown in SEQ ID NO.7.

[0134] Insert the nucleotide sequence shown in SEQ ID NO.7 between the BamHⅠ and NotⅠ restriction enzyme sites behind the AOXⅠ promoter in the pPIC9K vector, and introduce a His tag at the C segment of the gene for subsequent purification to construct the recombinant plasmid pPIC9K-MtLPMO9V. This plasmid was synthesized by Tianlin Biotechnology Co., Ltd. in Wuxi.

[0135] MtLPMO9V (amino acid sequence shown in SEQ ID NO.6) consists of a signal peptide sequence and an enzyme sequence; among them, the first 21 amino acids are the signal peptide (i.e., MRYYFLQLAAAAAFAVNSAAG), and the amino acids from the 22nd position to the end are the mature enzyme sequence. Therefore, the 22nd position is used as the first amino acid of the wild-type (WT) lytic polysaccharide monooxygenase, and its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0136] Site-directed mutagenesis was completed by designing site-directed mutagenesis primers and performing PCR. Using plasmid pPIC9K-MtLPMO9V as a template and the primers shown in Table 6, the construction of mutants was mainly carried out using PCR site-directed mutagenesis technology. Multiple mutants with corresponding position mutations were obtained based on the amino acid sequence (SEQ ID NO.1) and nucleotide sequence (SEQ ID NO.2). According to "pre-mutation amino acid + mutation site + post-mutation amino acid", the mutants were named as described in Table 1. For example, G32S means that on the basis of SEQ ID NO.1, glycine (G) at position 32 was mutated to serine (S); A170C / A175C means that on the basis of SEQ ID NO.1, alanine (A) at position 170 was mutated to cysteine (C), and alanine (A) at position 175 was mutated to cysteine (C).

[0137] Table 1 Mutant Primers

[0138]

[0139]

[0140] 2. Linearization and Transformation of Recombinant Mutant Vectors

[0141] The above recombinant mutant vectors carrying different mutants were cut with SacⅠ restriction endonuclease at its optimal temperature for 1 h and purified using a product recovery kit; Pichia pastoris GS115 competent cells were used for transformation, expression, and purification of proteins to obtain mutants G32S, D60Y, D63L, P72V, T79V, S80I, S84V, A98P, D104M, P107L, Q120Y, R121V, D128P, Q137I, P143N, P143A, A154P, D160G, T56C / V90C, A170C / A175C.

[0142] Example 2 Detection of Enzyme Activity, Optimal Temperature, and Thermal Stability of Mutants

[0143] Take the wild type and its mutants prepared in Example 1, and use the 2,6-DMP rapid enzyme activity assay method to measure the enzyme activity, optimal temperature, and thermal stability of wild-type (WT) and mutant MtLPMO9V with 2,6-DMP and H 2 O 2 as co-substrates.

[0144] By constructing disulfide bond mutants T56C / V90C and A170C / A175C, it was found that the activity of A170C / A175C was higher than that of T56C / V90C, and there was no significant difference in their thermal stabilities. Therefore, A170C / A175C was selected for subsequent research. At the same time, other sites were mutated by site-directed mutagenesis, and the sites with more than 55% increase in activity were iteratively mutated on the basis of the A170C / A175C (named M1) mutant. The primers are shown in Table 1, and mutants A170C / A175C / G32S, A170C / A175C / P107L, A170C / A175C / Q120Y, A170C / A175C / Q137I, A170C / A175C / A154P, and A170C / A175C / D160G were constructed respectively.

[0145] The enzyme activity and relative enzyme activity of the above mutants were detected. The results are shown in Figure 1 and Table 2. The mutants A170C / A175C / Q120Y had the highest activity among all iterative mutants, with an enzyme activity increase of more than 80% compared with the wild type (WT, 34.2 U / g), showing a significant improvement compared with WT. Therefore, the mutant A170C / A175C / Q120Y (named M3) was selected for subsequent research.

[0146] Table 2 Enzyme activity data

[0147]

[0148]

[0149] Example 3 Application of Mutant in Synergistic Hydrolysis of Cellulose Substrate with Cellulase

[0150] Using the mutant M3 prepared in Example 2, its synergistic effect with cellulase was detected. After the thermophilic endoglucanase DtCelA was expressed by the laboratory-preserved Escherichia coli E. coli BL21(DE3) and the crude enzyme was obtained by ultrasonic disruption, it was then purified by Ni + -NTA nickel column affinity chromatography. The amino acid sequence of DtCelA is shown in SEQ ID NO.5.

[0151] (1) Synergistic hydrolysis of phosphoric acid swollen cellulose and microcrystalline cellulose with thermophilic endoglucanase DtCelA

[0152] The high-temperature synergistic effects of wild-type MtLPMO9V (WT) and M3 with cellulase were detected separately. WT and M3 were co-hydrolyzed with the thermophilic endoglucanase DtCelA on phosphoric acid swollen cellulose (PASC) and microcrystalline cellulose (MCC) at 70 °C. The total reaction time was 96 h, and samples were taken at regular intervals to measure the reducing sugar concentration in the hydrolysis solution. The specific method is as follows:

[0153] The mutant M3 was co-hydrolyzed with the heat-resistant endoglucanase DtCelA on cellulose (PASC or MCC) substrates to detect the synergistic effect between the two, with wild-type MtLPMO9V (WT) as a control.

[0154] The enzyme reaction was carried out in 5 mL of acetate buffer (50 mM, pH 5). The reaction solution contained 0.5% w / v PASC (when the substrate was MCC, the concentration was 2% w / v), 10 mg / g dry substrate DtCelA, 1 mM ascorbic acid AscA, and 5 mg / g dry substrate of M3 (when the substrate was MCC, the addition amount of M3 was 1 mg / g dry substrate). The reaction solution was placed in a shaker at 70 °C and 180 rmp for 96 h. At regular intervals, 0.4 mL of the sample was taken and boiled at 100 °C for 10 min to terminate the reaction, and the supernatant was collected by centrifugation at 10000 rpm for 10 min. The amount of reducing sugar in the supernatant was measured by the DNS method.

[0155] The results are as Figure 2 shown. The maximum synergy degree of the mutant M3 in co-hydrolyzing PASC with DtCelA was 155% (127% for WT), and the maximum synergy degree in hydrolyzing MCC was 132% (120% for WT).

[0156] (2) Co-hydrolyzing MCC and pretreated bagasse substrates with cellulase Celluclast 1.5L or Ctec 2

[0157] M3 was used to co-hydrolyze 2% w / v MCC and acid / base-catalyzed glycerol-pretreated bagasse substrates (ac / al-AGO) with Celluclast 1.5L or Ctec 2 to detect their glucose yields. The specific steps are as follows:

[0158] M3 was co-hydrolyzed with Celluclast 1.5L or Ctec 2 on cellulosic substrates. Hydrolysis reactions were carried out on 2% w / v MCC or acid / base-catalyzed glycerol-pretreated substrates at 50 °C for 96 h, with wild-type MtLPMO9V (WT) as a control.

[0159] The enzymatic reaction was carried out in 50 mM acetate buffer at pH 5. The reaction solution included 1 mM gallic acid (not added when the substrate was MCC), 2% w / v MCC or acid-catalyzed glycerol pretreated substrate, 0.4 mg / g dry substrate of M3 (when the substrate was alkali-catalyzed glycerol pretreated substrate, the addition amount of M3 was 0.2 mg / g dry substrate), Celluclast 1.5L with a total concentration of 10 mg / g dry substrate (that is, the total concentration of Celluclast and M3 was 10 mg / g dry substrate) (or Ctec2 with an enzyme mixture of M3 concentration of 4.67 mg / g dry substrate). It was placed in a shaker at 50 °C at 180 rpm for 96 h (for Celluclast 1.5L) or 72 h (for Ctec2).

[0160] The results of the cooperation with Celluclast 1.5L are as Figure 3 shown. M3 can assist Celluclast 1.5L in the hydrolysis of cellulosic substrates. Compared with the case without adding M3, the glucose yield of hydrolyzing MCC substrate was 58% at 96 h, and the glucose yield was 77% (71% for WT) after adding M3. For the substrates ac-AGO and al-AGO without LPMO, the glucose yields were 56% and 61% respectively, and the glucose yields were 74% and 86% respectively (66% and 78% for WT respectively) after adding M3.

[0161] The results of the cooperation with Ctec 2 are as Figure 4 shown. M3 can also significantly improve the glucose yield when cooperating with Ctec2 in the hydrolysis of cellulosic substrates. Compared with the case without adding M3, the glucose yields of hydrolyzing MCC, ac-AGO and al-AGO substrates were 60%, 61% and 66% respectively at 72 h, and the glucose yields were 81%, 74% and 86% respectively (75%, 65% and 77% for WT respectively) after adding M3.

[0162] The above results show that the mutant M3 can efficiently cooperate with endoglucanase and cellulase.

[0163] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0164] The sequences used in the present invention

[0165] The amino acid sequence of MtLPMO9V (signal peptide removed) SEQ ID NO.1:

[0166] HYIFQQFATGGTKYPPWKYIRRNTNPDWLQNGPVTDLSSTDLRCNVGGQVSNGTETITLDAGDEFSFILDTPVYHAGPTSLYMSKAPGAVADYDGGGAWFKIYDWGPSGTSWTLSGTYTQRIPKCIPDGEYLLRIQQIGLHNPGAAPQFYISCAQVKVVDGGSTNPTPTAQIPGAFHSNDPGLTVNIYNDPLTNYVVPGPRVFSC

[0167] Nucleotide sequence of MtLPMO9V (signal peptide removed) SEQ ID NO.2:

[0168] CATTACATTTTTCAGCAGTTCGCAACAGGAGGTACTAAATATCCACCCTGGAAGTATATTCGTCGAAATACGAATCCTGACTGGTTGCAAAACGGCCCTGTTACCGACCTTAGTTCCACGGACCTACGATGCAATGTTGGAGGTCAAGTCTCAAATGGTACAGAGACGATCACTTTAGATGCAGGTGACGAGTTTAGTTTCATCCTAGATACACCAGTTTACCACGCTGGTCCAACAAGTTTGTACATGTCAAAAGCCCCTGGAGCTGTTGCTGACTATGATGGTGGAGGAGCATGGTTCAAGATTTACGATTGGGGACCTTCAGGAACTTCTTGGACCTTGAGTGGTACCTACACCCAGAGAATCCCCAAGTGCATTCCAGACGGTGAGTATCTGTTGAGAATACAACAAATTGGTTTGCATAACCCCGGTGCTGCTCCTCAGTTTTACATTTCCTGTGCCCAGGTTAAGGTCGTGGACGGTGGCTCAACTAACCCTACACCAACTGCTCAAATTCCTGGTGCATTTCATTCCAATGATCCCGGTCTGACCGTTAACATTTACAACGATCCCCTAACTAACTACGTGGTACCAGGTCCTAGAGTTTTCAGTTGC

[0169] Mutant A170C / A175C / Q120Y sequence SEQ ID NO.3:

[0170] HYIFQQFATGGTKYPPWKYIRRNTNPDWLQNGPVTDLSSTDLRCNVGGQVSNGTETITLDAGDEFSFILDTPVYHAGPTSLYMSKAPGAVADYDGGGAWFKIYDWGPSGTSWTLSGTYTYRIPKCIPDGEYLLRIQQIGLHNPGAAPQFYISCAQVKVVDGGSTNPTPTCQIPGCFHSNDPGLTVNIYNDPLTNYVVPGPRVFSC

[0171] Mutant A170C / A175C / Q120Y nucleotide sequence SEQ ID NO.4:

[0172] CATTACATTTTTCAGCAGTTCGCAACAGGAGGTACTAAATATCCACCCTGGAAGTATATTCGTCGAAATACGAATCCTGACTGGTTGCAAAACGGCCCTGTTACCGACCTTAGTTCCACGGACCTACGATGCAATGTTGGAGGTCAAGTCTCAAATGGTACAGAGACGATCACTTTAGATGCAGGTGACGAGTTTAGTTTCATCCTAGATACACCAGTTTACCACGCTGGTCCAACAAGTTTGTACATGTCAAAAGCCCCTGGAGCTGTTGCTGACTATGATGGTGGAGGAGCATGGTTCAAGATTTACGATTGGGGACCTTCAGGAACTTCTTGGACCTTGAGTGGTACCTACACCTATAGAATCCCCAAGTGCATTCCAGACGGTGAGTATCTGTTGAGAATACAACAAATTGGTTTGCATAACCCCGGTGCTGCTCCTCAGTTTTACATTTCCTGTGCCCAGGTTAAGGTCGTGGACGGTGGCTCAACTAACCCTACACCAACTTGCCAAATTCCTGGTTGCTTTCATTCCAATGATCCCGGTCTGACCGTTAACATTTACAACGATCCCCTAACTAACTACGTGGTACCAGGTCCTAGAGTTTTCAGTTGC

[0173] DtCelA amino acid sequence SEQ ID NO.5:

[0174] MRLVVSFLLVVSAFLFSAEVVLTDIGATDITFKGFPVTMELNFWNVKSYEGETWLKFDGEKVQFYADIYNIVLQNPDSWVHGYPEIYYGYKPWAAHNSGTEILPVKVKDLPDFYVTLDYSIWYENDLPINLAMETWITRKPDQTSVSSGDVEIMVWFYNNILMPGGQKVDEFTTTIEINGSPVETKWDVYFAPWGWDYLAFRLTTPMKDGRVKFNVKDFVEKAAEVIKKHSTRVENFDEMYFCVWEIGTEFGDPNTTAAKFGWTFKDFSVEIGE

[0175] The amino acid sequence of MtLPMO9V with a signal peptide SEQ ID NO.6:

[0176] MRYYFLQLAAAAAFAVNSAAGHYIFQQFATGGTKYPPWKYIRRNTNPDWLQNGPVTDLSSTDLRCNVGGQVSNGTETITLDAGDEFSFILDTPVYHAGPTSLYMSKAPGAVADYDGGGAWFKIYDWGPSGTSWTLSGTYTQRIPKCIPDGEYLLRIQQIGLHNPGAAPQFYISCAQVKVVDGGSTNPTPTAQIPGAFHSNDPGLTVNIYNDPLTNYVVPGPRVFSC

[0177] The nucleotide sequence of MtLPMO9V with a signal peptide SEQ ID NO.7:

[0178] ATGAGGTACTACTTCTTGCAATTGGCTGCTGCAGCTGCTTTTGCTGTTAACTCTGCTGCAGGTCATTACATTTTTCAGCAGTTCGCAACAGGAGGTACTAAATATCCACCCTGGAAGTATATTCGTCGAAATACGAATCCTGACTGGTTGCAAAACGGCCCTGTTACCGACCTTAGTTCCACGGACCTACGATGCAATGTTGGAGGTCAAGTCTCAAATGGTACAGAGACGATCACTTTAGATGCAGGTGACGAGTTTAGTTTCATCCTAGATACACCAGTTTACCACGCTGGTCCAACAAGTTTGTACATGTCAAAAGCCCCTGGAGCTGTTGCTGACTATGATGGTGGAGGAGCATGGTTCAAGATTTACGATTGGGGACCTTCAGGAACTTCTTGGACCTTGAGTGGTACCTACACCCAGAGAATCCCCAAGTGCATTCCAGACGGTGAGTATCTGTTGAGAATACAACAAATTGGTTTGCATAACCCCGGTGCTGCTCCTCAGTTTTACATTTCCTGTGCCCAGGTTAAGGTCGTGGACGGTGGCTCAACTAACCCTACACCAACTGCTCAAATTCCTGGTGCATTTCATTCCAATGATCCCGGTCTGACCGTTAACATTTACAACGATCCCCTAACTAACTACGTGGTACCAGGTCCTAGAGTTTTCAGTTGC。

Claims

1. A lytic polysaccharide monooxygenase mutant, characterized in that: The lytic polysaccharide monooxygenase mutant has one or more amino acid mutations at positions 32, 56, 60, 63, 72, 79, 80, 84, 90, 98, 104, 107, 120, 121, 128, 137, 143, 154, 160, 170 or 175 based on the amino acid sequence as shown in SEQ ID NO.

1.

2. The lytic polysaccharide monooxygenase mutant according to claim 1, characterized in that Corresponding to the amino acid sequence shown in SEQ ID NO.1, The glycine at position 32 is mutated to serine; The threonine at position 56 is mutated to cysteine; The aspartic acid at position 60 is mutated to tyrosine; The aspartic acid at position 63 is mutated to leucine; The proline at position 72 is mutated to valine; The threonine at position 79 is mutated to valine; The serine at position 80 is mutated to isoleucine; The serine at position 84 is mutated to valine; The valine at position 90 is mutated to cysteine; The alanine at position 98 is mutated to proline; The aspartic acid at position 104 is mutated to methionine; The proline at position 107 is mutated to leucine; The glutamine at position 120 is mutated to tyrosine; The arginine at position 121 is mutated to valine; The aspartic acid at position 128 is mutated to proline; The glutamine at position 137 is mutated to isoleucine; The proline at position 143 is mutated to asparagine or alanine; The alanine at position 154 is mutated to proline; The aspartic acid at position 160 is mutated to glycine; The alanine at position 170 is mutated to cysteine; The alanine at position 175 is mutated to cysteine.

3. The lytic polysaccharide monooxygenase mutant according to claims 1-2, characterized in that: The following mutations also exist: The alanine at position 170 is mutated to cysteine, and the alanine at position 175 is mutated to cysteine; Preferably, the alanine at position 170 is mutated to cysteine, the alanine at position 175 is mutated to cysteine, and the glycine at position 32 is mutated to serine; Preferably, the alanine at position 170 is mutated to cysteine, the alanine at position 175 is mutated to cysteine, and the proline at position 107 is mutated to leucine; Preferably, the alanine at position 170 is mutated to cysteine, the alanine at position 175 is mutated to cysteine, and the glutamine at position 120 is mutated to tyrosine; Preferably, the alanine at position 170 is mutated to cysteine, the alanine at position 175 is mutated to cysteine, and the glutamine at position 137 is mutated to isoleucine; Preferably, the alanine at position 170 is mutated to cysteine, the alanine at position 175 is mutated to cysteine, and the alanine at position 154 is mutated to proline; Preferably, the alanine at position 170 is mutated to cysteine, the alanine at position 175 is mutated to cysteine, and the aspartic acid at position 160 is mutated to glycine.

4. A polynucleotide encoding the lytic polysaccharide monooxygenase mutant according to any one of claims 1 to 3.

5. A vector carrying the polynucleotide according to claim 5.

6. The carrier according to claim 5, characterized in that The vectors include pPIC series vectors, pPICZ series vectors, pPICZα and pET series vectors.

7. A cell expressing the lytic polysaccharide monooxygenase mutant according to any one of claims 1 to 4.

8. The cell according to claim 7, characterized in that The cells include Pichia pastoris, Escherichia coli, Saccharomyces cerevisiae, Aspergillus niger, and Aspergillus oryzae.

9. Use of the lytic polysaccharide monooxygenase mutant according to any one of claims 1 to 3, the polynucleotide according to claim 4, the vector of the polynucleotide according to claim 5, or the cell according to claim 7 in hydrolyzing cellulose.

10. A method for hydrolyzing cellulose, characterized in that: Using the lytic polysaccharide monooxygenase mutant according to any one of claims 1 to 3 and cellulase for synergistic enzymolysis; Optionally, the cellulase comprises a thermophilic endoglucanase or a cellulase.

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