A lytic polysaccharide monooxygenase mutant and use thereof

Abstract

The application discloses a lytic polysaccharide monooxygenase mutant and application thereof, and belongs to the technical field of enzyme engineering and microbial engineering. The enzyme activity of the mutant is more than 88% higher than that of a wild type after molecular modification, and the thermal stability is also significantly improved compared with the wild type. The highest synergistic degree of the lytic polysaccharide monooxygenase mutant of the application in hydrolyzing phosphoric acid swollen cellulose and microcrystalline cellulose reaches 155% and 133% (the wild type is 127% and 120%) respectively. The glucose yield can be significantly improved by using the lytic polysaccharide monooxygenase mutant of the application in synergistically hydrolyzing microcrystalline cellulose with cellulase Celluclast 1.5L and Ctec 2 and in hydrolyzing bagasse pretreated by glycerol under the catalysis of acid or alkali. The application has high practicability and can be widely applied to various agricultural and forestry biomass raw materials to improve the enzymatic hydrolysis efficiency of cellulose substrates.

Description

[0001] This application, with application number 2025102859268, filed on March 11, 2025, is entitled "Construction and Application of a Highly Active and Thermally Stable Cleavage Polysaccharide Monooxygenase Mutant". Technical Field

[0002] This invention relates to a cleavable polysaccharide monooxygenase mutant and its applications, belonging to the fields of enzyme engineering and technology. Background Technology

[0003] Cellulose is a renewable, non-fossil carbon source abundant on Earth and a major component of lignocellulosic biomass, typically found in plant cell walls. As a linear polymer composed of d-glucose units linked by β-glycosidic bonds, cellulose holds significant biological importance. However, cellulose is the most recalcitrant of all plant cell wall polysaccharides, making its degradation a highly challenging task. The conversion of cellulose into monosaccharides, particularly in bioethanol production, has attracted widespread attention because it offers an environmentally friendly and sustainable solution to the growing global energy demand driven by industrial development and urbanization.

[0004] The enzymatic hydrolysis of cellulose involves three key cellulases: endoglucosidase, cellobiase / exoglucosidase, and β-glucosidase. These three enzymes work synergistically to break down cellulose. Endoglucosidase rapidly shortens the polymer length by randomly cleaving the internal glycosidic bonds of cellulose; exoglucosidase releases reducing sugars by hydrolyzing the reducing or non-reducing ends of the cellulose chain. Intermediates such as cellobiose and oligosaccharides are further converted into glucose by β-glucosidase. However, due to the very robust crystalline regions in the cellulose structure, ordinary cellulases have limited effectiveness against them, thus hindering the enzymatic hydrolysis process.

[0005] Since the discovery of lytic polysaccharide monooxygenases (LPMOs) in 2010, this limitation has been effectively mitigated, particularly by AA9 family LPMOs. AA9 LPMOs do not directly hydrolyze polysaccharides; instead, they disrupt the crystalline structure of cellulose by oxidatively cleaving resistant regions within the cellulose, creating pores on its crystalline surface. This structural change allows other glycoside hydrolases (such as endoglucanases and exoglucanases) to more easily access the polysaccharide, thus enabling them to synergistically work with cellulases for more efficient cellulose hydrolysis.

[0006] In industrial applications, enzyme stability and catalytic activity are the most critical enzymatic parameters, directly determining the economic feasibility of the process. Currently, most AA9 LPMOs are derived from wild-type enzymes, whose thermal stability and catalytic activity have not yet reached their full potential. Therefore, to meet the needs of industrial applications, they typically require optimization through protein engineering and other methods. Computer-aided design-based rational design methods have been widely used to identify key amino acids and perform directed evolution, thereby improving the thermal stability and catalytic activity of AA9 LPMOs.

[0007] Modifying AA9 LPMOs to enhance their thermal stability and catalytic activity can not only strengthen their synergistic hydrolysis with cellulase but also make cellulase more efficient in degrading lignocellulose, reducing enzyme usage and thus lowering costs, thereby improving economic efficiency in industrial applications. Therefore, there is an urgent need to develop cleavable polysaccharide monooxygenase mutants with higher activity and better thermal stability to enhance enzyme activity and synergistic effects, thereby increasing the enzymatic hydrolysis rate of different cellulose substrates. Summary of the Invention

[0008] To address the issues of low activity of cleaving polysaccharide monooxygenase and low hydrolysis rate in synergistic cellulase hydrolysis, this invention provides a mutant of cleaving polysaccharide monooxygenase. Compared with wild-type cleaving polysaccharide monooxygenase, the mutant exhibits increased enzyme activity and improved hydrolysis rate in synergistic hydrolysis of phosphate-swelled cellulose, microcrystalline cellulose, and pretreated bagasse.

[0009] The first objective of this invention is to provide a cleaving polysaccharide monooxygenase mutant, characterized in that the cleaving 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 shown in SEQ ID NO.1.

[0010] In one embodiment, the cleaving polysaccharide monooxygenase mutant is a cleaving polysaccharide monooxygenase derived from *Thermophilus pyriformis*. Mt LPMO9V (GenBank:AEO55082.1).

[0011] In one embodiment, the cleaving polysaccharide monooxygenase Mt The amino acid sequence of LPMO9V is shown in SEQ ID NO.7, wherein the first 21 amino acids are the signal peptide, and the sequence from the 22nd amino acid to the end is the sequence of the mature enzyme.

[0012] In one embodiment, a cleaving polysaccharide monooxygenase is used. Mt The 22nd amino acid of LPMO9V is the first amino acid of the wild-type (WT) cleavable polysaccharide monooxygenase, and its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.2.

[0013] In one embodiment, the cleaving polysaccharide monooxygenase mutant corresponds to the amino acid sequence shown in SEQ ID NO.1. The glycine at position 32 is mutated to serine and named G32S; The threonine at position 56 is mutated to cysteine ​​and named T56C; The aspartic acid at position 60 is mutated to tyrosine and named D60Y; The aspartic acid at position 63 is mutated to leucine and named D63L; The proline at position 72 is mutated to valine and named P72V; The threonine at position 79 is mutated to valine and named T79V; The serine at position 80 is mutated to isoleucine and named S80I; The serine at position 84 is mutated to valine and named S84V; The valine at position 90 is mutated to cysteine ​​and named V90C; The alanine at position 98 is mutated to proline and named A98P; The aspartic acid at position 104 is mutated to methionine and named D104M; The proline at position 107 is mutated to leucine and named P107L; The glutamine at position 120 is mutated to tyrosine and named Q120Y; The arginine at position 121 is mutated to valine and named R121V; The aspartic acid at position 128 is mutated to proline and named D128P. The glutamine at position 137 is mutated to isoleucine and named Q137I; The proline at position 143 is mutated to asparagine or alanine, named P143N and P143A respectively. The alanine at position 154 is mutated to proline and named A154P. The aspartic acid at position 160 is mutated to glycine and named D160G; The alanine at position 170 is mutated to cysteine ​​and named A170C; The alanine at position 175 is mutated to cysteine ​​and named A175C.

[0014] In one embodiment, the cleaving polysaccharide monooxygenase mutant simultaneously exhibits the following mutations: The alanine at position 170 is mutated to cysteine, and the alanine at position 175 is mutated to cysteine, named A170C / A175C. 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. 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. 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. 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. 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. 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.

[0015] 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.

[0016] A second object of the present invention is to provide a polynucleotide of any of the above-described cleavable polysaccharide monooxygenase mutants.

[0017] A third objective of this invention is to provide a carrier for the aforementioned polynucleotides.

[0018] In one embodiment, the carrier includes pPIC series carriers, pPICZ series carriers, pPICZα, and pET series carriers; Optionally, the pPIC series vectors include pPIC9, pPIC9K, and pPIC3.5K, etc.; the pPICZ series vectors include pPICZA, pPICZB, and pPICZC, etc.; the pPICZα series vectors include pPICZαA, pPICZαB, and pPICZαC, etc.; and the pET series vectors include pET-22b.

[0019] A fourth object of the present invention is to provide cells expressing any of the above-described cleaving polysaccharide monooxygenase mutants; In one embodiment, the cells include Pichia pastoris, Escherichia coli, Saccharomyces cerevisiae, and filamentous fungi; Alternatively, filamentous fungi include Aspergillus niger, Aspergillus oryzae, etc.

[0020] A fifth object of the present invention is to provide the use of any of the above-described cleavable polysaccharide monooxygenase mutants, or the above-described polynucleotides, or the above-described polynucleotide vectors, or the above-described cells in the hydrolysis of cellulose.

[0021] A sixth object of the present invention is to provide a method for hydrolyzing cellulose, using any of the above-described cleaving polysaccharide monooxygenase mutants and cellulase for synergistic enzymatic hydrolysis; Optionally, the cellulase includes thermophilic endoglucanase or cellulase.

[0022] In one embodiment, the thermophilic endoglucanase is Dt CelA, the amino acid sequence of which is shown in SEQ ID NO.5.

[0023] In one embodiment, the cellulase includes commercially available cellulases, such as Celluclast or Ctec2.

[0024] In one embodiment, the cellulose comprises pretreated bagasse, phosphate-swollen cellulose, and microcrystalline cellulose.

[0025] In one embodiment, the pretreated bagasse is either alkali-treated bagasse or acid-treated bagasse, and the preparation method includes: Dry sugarcane, glycerol, and NaOH (or sulfuric acid) are mixed, heated and stirred, filtered and washed to prepare alkali-treated bagasse (acid-treated bagasse).

[0026] In one embodiment, the amount of the cleaving polysaccharide monooxygenase mutant added is 0.2~2 mg / g dry substrate; the amount of cellulase (total amount of cleaving polysaccharide monooxygenase and commercial cellulase) added is 4~10 mg / g dry substrate.

[0027] In one embodiment, the amount of cellulose added is 0.5~2% w / v.

[0028] In one embodiment, the enzymatic hydrolysis conditions are 45-50°C for 12-96 h.

[0029] In one embodiment, using 2% w / v MCC (or acid-catalyzed glycerol pretreated substrate) as the substrate, 1 mM gallic acid (not added when the substrate is MCC), 0.4 mg / g of the cleaving polysaccharide monooxygenase mutant, and 1.5 L of a 9.6 mg / g dry matrix enzyme mixture of cellulase, the glucose yield reached 77% after 96 h of reaction (74% when the substrate is acid-catalyzed glycerol pretreated). When using 2% w / v MCC (or acid-catalyzed glycerol pretreated substrate) as substrate, 1 mM gallic acid (not added when substrate is MCC), 0.4 mg / g of cleaving polysaccharide monooxygenase mutant, and 4.27 mg / g of cellulase in Ctec2 dry matrix enzyme mixture, the glucose yield reached 81% after 72 h of reaction (74% when the substrate is acid-catalyzed glycerol pretreated).

[0030] In one embodiment, when the substrate was pretreated with 2% w / v base-catalyzed glycerol, 1 mM gallic acid, 0.2 mg / g of the cleaving polysaccharide monooxygenase mutant, and 9.8 mg / g of cellulase in 1.5 L of a dry matrix enzyme mixture of Celluclast were added, and the reaction was carried out for 96 h, resulting in a glucose yield of 86%. When the substrate was pretreated with 2% w / v base-catalyzed glycerol, and the amount of 1 mM gallic acid, 0.2 mg / g of the cleaving polysaccharide monooxygenase mutant, and 4.47 mg / g of cellulase in the dry matrix enzyme mixture of Ctec2 was added, the glucose yield reached 77% after 72 h of reaction.

[0031] Beneficial effects This invention uses cleaving polysaccharide monooxygenase Mt LPMO9V is wild-type and is produced by cleaving polysaccharide monooxygenases. Mt Point mutations were performed at different sites in LPMO9V, which increased enzyme activity by more than 88% and improved the synergistic effect of cleaving polysaccharide monooxygenase and cellulase on different cellulose substrates.

[0032] The mutant A170C / A175C / Q120Y showed the best results, as detailed below: (1) The cleavage polysaccharide monooxygenase mutant A170C / A175C / Q120Y (M3) of the present invention has an enzyme activity that is 88% higher than that of the wild type under standard conditions, reaching 63.4 U / g; (2) The cleaving polysaccharide monooxygenase mutant A170C / A175C / Q120Y (M3) of the present invention, in conjunction with thermophilic endoglucanase Dt When CelA is used to hydrolyze swollen cellulose and microcrystalline cellulose, it exhibits better synergistic effects compared to the wild type, with the highest synergistic degree reaching 155% and 133% (the wild type is 127% and 120%, respectively). (3) The cleavable polysaccharide monooxygenase mutant A170C / A175C / Q120Y (M3) of the present invention showed a higher synergistic effect when synergistically hydrolyzing microcrystalline cellulose with cellulase Celluclast 1.5L and pretreating sugarcane bagasse substrate with acid / base catalysis. The glucose yield at 96h increased from 58%, 56% and 61% to 77%, 74% and 86% respectively (70%, 66% and 78% for wild type). It also showed a better synergistic effect when synergistically hydrolyzing microcrystalline cellulose with Ctec 2 and pretreating sugarcane bagasse substrate with acid / base catalysis. The glucose yield at 72h increased from 60%, 61% and 66% to 81%, 74% and 86% respectively (75%, 65% and 77% for wild type). Attached Figure Description

[0033] Figure 1 The relative enzyme activities of different polysaccharide monooxygenase mutants relative to the wild type; Figure 2 For mutant M3 and Dt CelA co-hydrolyzes PASC and MCC; where a and b are wild-type. Mt LPMO9V and Dt CelA co-hydrolyzes PASC and MCC; c and d are mutants M3 and Dt CelA synergistically hydrolyzes PASC and MCC; Figure 3 The mutant M3 and Celluclast 1.5L were used for the synergistic hydrolysis of MCC and pretreatment of substrates; where a, b, and c were the synergistic hydrolysis of MCC by mutant M3 and Celluclast 1.5L, acid-treated bagasse (ac-AGO), and alkali-treated bagasse (al-AGO), respectively. Figure 4 The mutant M3 and Ctec2 co-hydrolyze MCC and pre-treated substrates; where a, b, and c are respectively the mutant M3 and Ctec2 co-hydrolyze MCC, acid-treated bagasse (ac-AGO), and alkali-treated bagasse (al-AGO). Detailed Implementation

[0034] The technical solutions described below will be clearly and completely described with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0035] Experimental materials E. coli strains ( E. coli DH5α was preserved in our laboratory; expression strain: Pichia pastoris GS115 was preserved in our laboratory; plasmid pPIC9K- Mt LPMO9V is preserved in our laboratory; thermophilic endoglucanase Dt CelA was previously constructed in the laboratory from E. coli. E. coli The thermophilic endoglucanase expressed by the BL21(DE3) preserved strain Dt The amino acid sequence of CelA is shown in SEQ ID NO.5. (Thermophilic endoglucanase) Dt CelA can be directly passed through E. coli E. coli BL21 (DE3) expression was obtained by isolation and purification.

[0036] The enzyme activity and molecular weight used in this invention are respectively Mt LPMO9V wild type (WT): 34.2 U / g (30℃, pH 7.5, i.e., standard conditions for enzyme activity detection), molecular weight approximately 24.4 kDa; Dt CelA: 63 U / g, molecular weight 37.1 kDa; in the embodiments of this invention, the enzyme addition amount is expressed in mg / g dry substrate.

[0037] Unless otherwise specified, w / v in this article refers to g / mL.

[0038] Detection methods 1. Determination of reducing sugar concentration (DNS method): During hydrolysis, 0.4 mL of the hydrolysate was taken and the supernatant was obtained by centrifugation at 8000 rpm for 5 min. The supernatant was diluted to an appropriate ratio, and the reducing sugar content was determined using DNS (distillation reagent), thereby calculating the concentration of reducing sugars.

[0039] Synergy (DS) calculation formula: .

[0040] Among them, R DtCelA for Dt CelA reducing sugar concentration; R LPMO R represents the reducing sugar concentration of LPMO.DtCelA+LPMO This represents the concentration of reducing sugars produced by the two enzymes in the system.

[0041] 2. Glucose yield: At specified time intervals, 0.4 mL of sample was extracted and heated at 100 °C for 5 min to inactivate the enzyme. The sample was then centrifuged at 8000 rpm for 5 min, and the supernatant was filtered through a 0.22 µm filter and appropriately diluted. Glucose concentration was determined using high-performance liquid chromatography (HPLC, Chomaster CM5110, Hitachi, Japan), equipped with a refractometer and a packed column (Bio-Rad Aminex HPX-87H, 9 µm, 7.8 × 300 mm). The mobile phase was 5 mM dilute H₂SO₄, the flow rate was 0.6 mL / min, isocratic elution was performed, and the column temperature was 60 °C.

[0042] The formula for calculating glucose yield is: .

[0043] 3. Preparation of phosphoric acid swollen cellulose (PASC): The specific steps are as follows: (1) Add 0.2 g MCC (microcrystalline cellulose) to a 50 mL centrifuge tube and moisten with 0.6 mL ddH2O to form a suspension; (2) Slowly add 10 mL of pre-cooled 86.2% phosphoric acid and stir thoroughly until the final concentration is 83.2%. Before adding the last 2 mL of phosphoric acid, the cellulose suspension should be mixed evenly. (3) Place on ice for 1 hour, stirring every 10 minutes; (4) Centrifuge at 4 ℃ and 5000 rpm for 20 min, suspend the particles in cold water, add 10 mL of cold water each time, for a total of 40 mL, and stir thoroughly after each addition. Finally, a white turbid precipitate is produced. Centrifuge at 4 ℃ and 6500 rpm for 25 min. (5) Wash the precipitate with 50 mL of cold water, repeating four times; (6) Add 0.5 mL of 2 mol / L Na2CO3 solution to neutralize phosphoric acid, add 45 mL of cold water to resuspend, and centrifuge at 4 ℃ and 6500 rpm for 25 min; (7) Add 50 mL of cold water to resuspend and centrifuge twice to make the pH about 5 to 7; (8) Store in a refrigerator at 4 ℃ or -20 ℃.

[0044] 4. The preparation method of acid / base catalytic glycerol pretreatment substrate (i.e., glycerol organic solvent pretreatment of bagasse under acid / base catalysis) is as follows: (1) Grind the sugarcane bagasse into fine particles using a grinder, and then sieve it through a 60-mesh sieve and dry it in a 60℃ oven until it reaches a constant weight; (2) Place 10 g of dried sugarcane bagasse in a three-necked flask, add 140 g of glycerol and 4% NaOH (0.4 g based on bagasse) or 6% sulfuric acid (0.6 g based on bagasse). (3) Place the three-necked flask in a heating mantle and heat it to 195°C (alkali treatment) or 200°C (acid treatment). At the same time, stir with a stir paddle to make it mix and heat evenly, and react for 45 min (alkali treatment) or 15 min (acid treatment). (4) Start timing when the specified temperature is reached. When the reaction ends, add 100 mL of hot water to stop the reaction. Filter the mixture through a G1 sintered glass funnel. Wash the filter cake with hot water until it is neutral and remove the glycerol.

[0045] (5) Place the processed sugarcane bagasse in a 60 ℃ oven and dry it to constant weight. Collect it, dry it, and store it for later use.

[0046] 5. Pichia pastoris electroconversion The preparation and transformation (electroporation method) of Pichia pastoris GS115 competent cells are as follows: (1) Pichia pastoris GS115 was streaked on YPD plates and cultured at 30℃ for 2 days to isolate single colonies. Single colonies were isolated and purified by spot inoculation with YNB basic medium and supplemented medium containing His. Single colonies that grew on supplemented medium but not on basic medium were picked and streaked on YPD plates and stored at 4℃. (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. Incubate overnight at 30 °C and 250-300 rpm / min. (3) Take 20 µL of the culture and inoculate it into a 50 mL Erlenmeyer flask containing 20 mL of fresh culture medium. Incubate overnight at 28–30 °C and 250–300 rpm / min until OD reaches 200 °C. 600 Reaching 1.3~1.5; (4) Dispense the cell culture into multiple 1.5mL EP tubes, centrifuge at 1500g for 30s at 4℃, and resuspend the bacterial pellet in 1mL of ice-cold sterile water. (5) Centrifuge according to step (4), and resuspend the bacterial precipitate with 1 mL of ice-cooled sterile water; (6) Centrifuge according to step (4), and resuspend the bacterial precipitate with 1 mL of ice-cold 1M sorbitol solution; (7) Centrifuge according to step (4), resuspend the bacterial precipitate with 60µL of ice-cold 1M sorbitol solution, and immediately use it for electroporation transformation; (8) Dissolve 5~20µg of linearized DNA in 5~10µL of TE solution, mix it with 80µL of the bacterial cells obtained in step (7) above, and transfer it to an ice-cold electroconversion cup at 0.2cm. (9) Place the electroconversion cup in an ice bath for 5 minutes; (10) Voltage 3 kV, electric shock time 6 ms; (11) After the electroporation is complete, immediately add 1 mL of 1 mol / L ice-cold sorbitol solution to the electroporation cup, mix the cells well, and transfer the contents of the electroporation cup to a new 1.5 mL centrifuge tube; (12) Spread the bacterial suspension onto MD plates, one plate for every 200~600µL; (13) Place the plate at 30°C and incubate until a single colony appears.

[0047] 6. Pichia pastoris protein induction expression and purification (1) Induced expression Single colonies grown in MD medium were selected by picking them with toothpicks and transferring them to YPD medium containing 1 g / L, 3 g / L, and 5 g / L of genimycin for screening. Recombinant bacteria growing at high antibiotic concentrations were inoculated into BMGY medium and cultured overnight until OD. 600 The concentration was 2-6, and then the cells were transferred to BMMY medium until the OD value of the bacteria reached 2-6. 600 The mixture was cultured in a shaker at 30 °C and 260 rpm for oscillation fermentation. Methanol solution with a final concentration of 1% was added every 24 h to induce enzyme production for 7 consecutive days. After fermentation, the fermentation broth was centrifuged at 4 °C and 10,000 rpm for 20 min and the supernatant was collected. (2) Protein purification The collected supernatant was precipitated by adding ammonium sulfate to a final concentration of 70%. Excess salts were then removed using a dialysis bag, and Ni was used as the final solution. + Proteins were purified using NTA nickel column affinity chromatography, following the manufacturer's instructions. The purified proteins were then stored at 4 °C.

[0048] 7. Enzyme activity detection Determination using 2,6-DMP and H2O2 as co-substrate Mt Enzyme activity, optimal temperature, and thermal stability of LPMO9V wild-type (WT) and mutant: (1) Prepare 116 mM phosphate buffer (pH 7.5); 10 mM 2,6-DMP solution; and 5 mM H2O2 solution. All solutions should be used within 12 h after preparation.

[0049] (2) Add 860 μL of phosphate buffer, 100 μL of 2,6-DMP solution and 20 μL of H2O2 solution to a centrifuge tube and mix thoroughly. Incubate at the appropriate temperature for 15 min.

[0050] (3) After adding LPMO protein sample to a final concentration of 1 μM and mixing well, the increase in absorbance within 5 min at a wavelength of 469 nm was measured at different temperatures (ε469 = 53200 L / mol / cm) to calculate the activity of LPMO. The residual activity was also determined after incubation at different temperatures for different times.

[0051] One unit of enzyme activity is defined as the conversion of 2 μmol of 2,6-DMP per minute under standard reaction conditions. The relative enzyme activity of the mutant is calculated with the enzyme activity of WT as 100%.

[0052] Example 1: Preparation of mutants of polysaccharide monooxygenase with different mutation sites 1. Constructing recombinant mutant vectors Lysogenic polysaccharide monooxygenase derived from thermophilic pyridamole Mt The complete gene sequence of LPMO9V (GenBank: AEO55082.1) was obtained from the NCBI and CAZy databases. Its amino acid sequence is shown in SEQ ID NO.6 and its nucleotide sequence is shown in SEQ ID NO.7.

[0053] The nucleotide sequence shown in SEQ ID NO.7 was inserted into the pPIC9K vector after the AOXⅠ promoter. Bam HⅠ restriction site and Not Between the restriction enzyme sites, a His tag was introduced into the C segment of the gene for subsequent purification, and the recombinant plasmid pPIC9K- was constructed. Mt LPMO9V, this plasmid was synthesized by Tianlin Biotechnology Wuxi Co., Ltd.

[0054] Mt LPMO9V (amino acid sequence shown in SEQ ID NO. 6) consists of a signal peptide sequence and an enzyme sequence; the first 21 amino acids constitute the signal peptide (i.e., MRYYFLQLAAAAAFAVNSAAG), and the sequence from amino acid position 22 to the end constitutes the mature enzyme sequence. Therefore, position 22 is taken as the first amino acid of the wild-type (WT) cleaving polysaccharide monooxygenase, and its amino acid sequence is shown in SEQ ID NO. 1, while its nucleotide sequence is shown in SEQ ID NO. 2.

[0055] Site-directed mutagenesis was achieved by designing primers for PCR, using plasmid pPIC9K- MtUsing LPMO9V as a template and primers as shown in Table 6, mutants were constructed primarily using PCR site-directed mutagenesis. The resulting recombinant mutant vectors contained multiple mutants with corresponding mutations at the amino acid sequence (SEQ ID NO.1) and nucleotide sequence (SEQ ID NO.2). The mutants were named according to the sequence "pre-mutation amino acid + mutation site + post-mutation amino acid," as shown in Table 1. For example, G32S represents a mutation of glycine (G) at position 32 to serine (S) based on SEQ ID NO.1; A170C / A175C represents a mutation of alanine (A) at position 170 to cysteine ​​(C) and alanine (A) at position 175 to cysteine ​​(C) based on SEQ ID NO.1.

[0056] Table 1. Mutant Primers

[0057] 2. Linearization and transformation of recombinant mutant vectors The above-mentioned recombinant mutant vectors carrying different mutants were used Sac I. Restriction fast cutases were used to cut the protein at its optimal temperature for 1 h, and the protein was purified using a product recovery kit. The protein was transformed, expressed, and purified using Pichia pastoris GS115 competent cells to obtain mutants G32S, D60Y, D63L, P72V, T79V, S80I, S84V, A98P, D104M, P107L, Q120Y, R121V, D128P, Q137I, P143N, P143A, A154P, D160G, T56C / V90C, and A170C / A175C.

[0058] Example 2: Determination of enzyme activity, optimal temperature, and thermal stability of mutants Wild-type and mutant enzymes prepared in Example 1 were analyzed using a rapid enzyme activity assay with 2,6-DMP, using 2,6-DMP and H2O2 as co-substrate. Mt Enzyme activity, optimal temperature, and thermal stability of LPMO9V wild-type (WT) and mutant.

[0059] By constructing disulfide bond mutants T56C / V90C and A170C / A175C, it was found that A170C / A175C had higher activity than T56C / V90C, and there was no significant difference in thermal stability between the two. Therefore, A170C / A175C was selected for further research. Simultaneously, site-directed mutagenesis was used to mutate other sites. Sites with activity increased by more than 55% were iteratively mutated based on the A170C / A175C (named M1) mutant. Primers are shown in Table 1, resulting in mutants A170C / A175C / G32S, A170C / A175C / P107L, A170C / A175C / Q120Y, A170C / A175C / Q137I, A170C / A175C / A154P, and A170C / A175C / D160G.

[0060] The enzyme activity and relative enzyme activity of the above mutants were detected, and the results are as follows: Figure 1 As shown in Table 2, mutant A170C / A175C / Q120Y exhibited the highest activity among all iterative mutants, showing an increase of over 80% compared to the wild-type (WT, 34.2 U / g) enzyme activity, and a significant improvement over WT. Therefore, mutant A170C / A175C / Q120Y (named M3) was selected for further research.

[0061] Table 2 Enzyme activity data

[0062] Example 3 Application of mutant synergistic cellulase in hydrolyzing cellulose substrates The mutant M3 prepared in Example 2 was used to test its synergistic effect with cellulase and thermophilic endoglucanase. Dt CelA preserves E. coli in the laboratory. E. coli After BL21(DE3) expression, the crude enzyme was obtained by sonication and then processed using Ni + Proteins were purified using NTA nickel column affinity chromatography. Dt The amino acid sequence of CelA is shown in SEQ ID NO.5.

[0063] (1) Synergistic thermophilic endoglucanase Dt CelA hydrolyzed phosphoric acid swells cellulose and microcrystalline cellulose Wild type tested separately Mt The high-temperature synergistic effect of LPMO9V (WT) and M3 with cellulase enables WT and M3 to interact with thermophilic endoglucanase. DtCelA was used to synergistically hydrolyze phosphate-swellable cellulose (PASC) and microcrystalline cellulose (MCC) at 70 °C for a total reaction time of 96 h. Samples were taken at regular intervals to determine the concentration of reducing sugars in the hydrolysate. The specific method is as follows: The mutant M3 was combined with a thermostable endoglucanase. Dt CelA was used to synergistically hydrolyze cellulose (PASC or MCC) matrices to detect the synergistic effect between the two, using wild-type... Mt LPMO9V (WT) was used as a control.

[0064] The enzyme reaction was carried out in 5 mL acetate buffer (50 mM, pH 5) containing 0.5% w / v PASC (2% w / v when the substrate was MCC) and 10 mg / g dry matrix. Dt CelA, 1 mM ascorbic acid (AscA), and M3 at 5 mg / g dry substrate (when the substrate is MCC, the amount of M3 added is 1 mg / g dry substrate). The reaction solution was placed in a shaker at 70 °C and 180 rpm for 96 h. At regular intervals, 0.4 mL of sample was taken and boiled at 100 °C for 10 min to terminate the reaction. The supernatant was collected by centrifugation at 10,000 rpm for 10 min. The reducing sugar content in the supernatant was measured by the DNS method.

[0065] The results are as follows Figure 2 As shown, mutant M3 in synergistic... Dt The maximum cooperativity of CelA hydrolysis of PASC is 155% (WT is 127%), while the maximum cooperativity of CelA hydrolysis of MCC is 132% (WT is 120%).

[0066] (2) Hydrolyze MCC and pretreated bagasse substrate with synergistic cellulase Celluclast 1.5L or Ctec 2. Hydrolysis of bagasse substrate pretreated with glycerol under acid / al-catalyzed conditions at a concentration of 2% w / v using M3 in conjunction with Celluclast 1.5L or Ctec 2 was performed, and the glucose yield was determined. The specific steps are as follows: M3 was used in conjunction with Celluclast 1.5L or Ctec 2 to hydrolyze fibrous substrates. The hydrolysis reaction was carried out at 50 °C for 96 h on substrates pretreated with 2% w / v MCC or acid / base catalytic glycerol, yielding wild-type products. Mt LPMO9V (WT) was used as a control.

[0067] The enzyme reaction was carried out in 50 mM, pH 5 acetate buffer. 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 M3 (when the substrate was base-catalyzed glycerol pretreated substrate, the amount of M3 added was 0.2 mg / g dry substrate), and a total concentration of 10 mg / g dry matrix (i.e., the total concentration of Celluclast and M3 was 10 mg / g dry matrix) in 1.5 L of Celluclast (or a Ctec2 enzyme mixture with a total concentration of 4.67 mg / g dry matrix M3). The reaction was carried out at 50 °C and 180 rpm for 96 h (Celluclast 1.5 L) or 72 h (Ctec2).

[0068] Results of synergistic use with Celluclast 1.5L are as follows: Figure 3 As shown, M3 can assist Celluclast 1.5L in the hydrolysis of cellulose substrates. Compared with the absence of M3, the glucose yield of hydrolyzed MCC substrates was 58% at 96 h, and the glucose yield with M3 was 77% (WT was 71%). For the ac-AGO and al-AGO substrates, the glucose yields without LPMO were 56% and 61%, respectively, and the glucose yields with M3 were 74% and 86%, respectively (WT were 66% and 78%).

[0069] The results of the synergistic Ctec 2 are as follows Figure 4 As shown, M3 also significantly improved glucose yield when synergistically hydrolyzing cellulose substrates with Ctec2. Compared to the absence of M3, the glucose yields at 72 h for hydrolyzing MCC, ac-AGO, and al-AGO substrates were 60%, 61%, and 66%, respectively, while the glucose yields with M3 were 81%, 74%, and 86% (WTs of 75%, 65%, and 77%, respectively).

[0070] The results in summary indicate that mutant M3 can efficiently synergistically work with thermophilic endoglucanase and cellulase.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0072] The sequence used in this invention Mt LPMO9V (signal peptide removed) amino acid sequence SEQ ID NO.1: HYIFQQFATGGTKYPPWKYIRRNTNPDWLQNGPVTDLSSTDLRCNVGGQVSNGTETITLDAGDEFSFILDTPVYHAGPTSLYMSKAPGAVADYDGGGAWFKIYDWGPSGTSWTLSGTYTQRIPKCIPDGEYLLRIQQIGLHNPGAAPQFYISCAQVKVVDGGSTNPTPTAQIPGAFHSNDPGLTVNIYNDPLTNYVVPGPRVFSC Mt LPMO9V (signal peptide removed) nucleotide sequence SEQ ID NO.2: CATTACATTTTTCAGCAGTTCGCAACAGGAGGTACTAAATATCCACCCTGGAAGTATATTCGTCGAAATACGAATCCTGACTGGTTGCAAAACGGCCCTGTTACCGACCTTAGTTCCACGGACCTACGATGCAATGTTGGAGGTCAAGTCTCAAATGGTACAGAGACGATCACTTTAGATGCAGGTGACGAGTTTAGTTTCATCCTAGATACACCAGTTTACCACGCTGGTCCAACAAGTTTGTACATGTCAAAAGCCCCTGGAGCTGTTGCTGACTATGATGGTGGAGGAGCATGGTTCAAGATTTACGATTGGGGACCTTCAGGAACTTCTTGGACCTTGAGTGGTACCTACACCCAGAGAATCCCCAAGTGCATTCCAGACGGTGAGTATCTGTTGAGAATACAACAAATTGGTTTGCATAACCCCGGTGCTGCTCCTCAGTTTTACATTTCCTGTGCCCAGGTTAAGGTCGTGGACGGTGGCTCAACTAACCCTACACCAACTGCTCAAATTCCTGGTGCATTTCATTCCAATGATCCCGGTCTGACCGTTAACATTTACAACGATCCCCTAACTAACTACGTGGTACCAGGTCCTAGAGTTTTCAGTTGC Mutant A170C / A175C / Q120Y sequence SEQ ID NO.3: HYIFQQFATGGTKYPPWKYIRRNTNPDWLQNGPVTDLSSTDLRCNVGGQVSNGTETITLDAGDEFSFILDTPVYHAGPTSLYMSKAPGAVADYDGGGAWFKIYDWGPSGTSWTLSGTYTYRIPKCIPDGEYLLRIQQIGLHNPGAAPQFYISCAQVKVVDGGSTNPTPTCQIPGCFHSNDPGLTVNIYNDPLTNYVVPGPRVFSC Mutant A170C / A175C / Q120Y nucleotide sequence SEQ ID NO.4: CATTACATTTTTCAGCAGTTCGCAACAGGAGGTACTAAATATCCACCCTGGAAGTATATTCGTCGAAATACGAATCCTGACTGGTTGCAAAACGGCCCTGTTACCGACCTTAGTTCCACGGACCTACGATGCAATGTTGGAGGTCAAGTCTCAAATGGTACAGAGACGATCACTTTAGATGCAGGTGACGAGTTTAGTTTCATCCTAGATACACCAGTTTACCACGCTGGTCCAACAAGTTTGTACATGTCAAAAGCCCCTGGAGCTGTTGCTGACTATGATGGTGGAGGAGCATGGTTCAAGATTTACGATTGGGGACCTTCAGGAACTTCTTGGACCTTGAGTGGTACCTACACCTATAGAATCCCCAAGTGCATTCCAGACGGTGAGTATCTGTTGAGAATACAACAAATTGGTTTGCATAACCCCGGTGCTGCTCCTCAGTTTTACATTTCCTGTGCCCAGGTTAAGGTCGTGGACGGTGGCTCAACTAACCCTACACCAACTTGCCAAATTCCTGGTTGCTTTCATTCCAATGATCCCGGTCTGACCGTTAACATTTACAACGATCCCCTAACTAACTACGTGGTACCAGGTCCTAGAGTTTTCAGTTGC Dt CelA amino acid sequence SEQ ID NO.5: MRLVVSFLLVVSAFLFSAEVVLTDIGATDITFKGFPVTMELNFWNVKSYEGETWLKFDGEKVQFYADIYNIVLQNPDSWVHGYPEIYYGYKPWAAHNSGTEILPVKVKDLPDFYVTLDYSIWYENDLPINLAMETWITRKPDQTSVSSGDVEIMVWFYNNILMPGGQKVDEFTTTIEINGSPVETKWDVYFAPWGWDYLAFRLTTPMKDGRVKFNVKDFVEKAAEVIKKHSTRVENFDEMYFCVWEIGTEFGDPNTTAAKFGWTFKDFSVEIGE Mt Amino acid sequence of LPMO9V with signal peptide SEQ ID NO.6: MRYYFLQLAAAAAFAVNSAAGHYIFQQFATGGTKYPPWKYIRRNTNPDWLQNGPVTDLSSTDLRCNVGGQVSNGTETITLDAGDEFSFILDTPVYHAGPTSLYMSKAPGAVADYDGGGAWFKIYDWGPSGTSWTLSGTYTQRIPKCIPDGEYLLRIQQIGLHNPGAAPQFYISCAQVKVVDGGSTNPTPTAQIPGAFHSNDPGLTVNIYNDPLTNYVVPGPRVFSC Mt Nucleotide sequence of LPMO9V with signal peptide SEQ ID NO.7: ATGAGGTACTACTTCTTGCAATTGGCTGCTGCAGCTGCTTTTGCTGTTAACTCTGCTGCAGGTCATTACATTTTTCAGCAGTTCGCAACAGGAGGTACTAAATATCCACCCTGGAAGTATATTCGTCGAAATACGAATCCTGACTGGTTGCAAAACGGCCCTGTTACCGACCTTAGTTCCACGGACCTACGATGCAATGTTGGAGGTCAAGTCTCAAATGGTACAGAGACGATCACTTTAGATGCAGGTGACGAGTTTAGTTTCATCCTAGATACACCAGTTTACCACGCTGGTCCAACAAGTTTGTACATGTCAAAAGCCCCTGGAGCTGTTGCTGACTATGATGGTGGAGGAGCATGGTTCAAGATTTACGATTGGGGACCTTCAGGAACTTCTTGGACCTTGAGTGGTACCTACACCCAGAGAATCCCCAAGTGCATTCCAGACGGTGAGTATCTGTTGAGAATACAACAAATTGGTTTGCATAACCCCGGTGCTGCTCCTCAGTTTTACATTTCCTGTGCCCAGGTTAAGGTCGTGGACGGTGGCTCAACTAACCCTACACCAACTGCTCAAATTCCTGGTGCATTTCATTCCAATGATCCCGGTCTGACCGTTAACATTTACAACGATCCCCTAACTAACTACGTGGTACCAGGTCCTAGAGTTTTCAGTTGC

Claims

1. A cleavable polysaccharide monooxygenase mutant, characterized in that, The cleaving polysaccharide monooxygenase mutant is based on the amino acid sequence shown in SEQ ID NO.1, with glutamine at position 137 mutated to isoleucine; Alternatively, alanine at position 170 is mutated to cysteine, alanine at position 175 is mutated to cysteine, and glutamine at position 137 is mutated to isoleucine.

2. A polynucleotide encoding the cleavable polysaccharide monooxygenase mutant of claim 1.

3. A vector carrying the polynucleotide of claim 2.

4. The carrier according to claim 3, characterized in that, The vectors include pPIC series vectors, pPICZ series vectors, pPICZα and pET series vectors.

5. Cells expressing the cleavage polysaccharide monooxygenase mutant of claim 1.

6. The cell according to claim 5, characterized in that, The cells include Pichia pastoris, Escherichia coli, Saccharomyces cerevisiae, Aspergillus niger, and Aspergillus oryzae.

7. The use of the cleavage polysaccharide monooxygenase mutant of claim 1, or the polynucleotide of claim 2, or the carrier of the polynucleotide of claims 3-4, or the cell of claims 5-6 in the hydrolysis of cellulose.

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