A lytic polysaccharide monooxygenase mutant and its application

Abstract

The present invention relates to a lytic polysaccharide monooxygenase mutant and its application, belonging to the technical fields of enzyme engineering and microbial engineering. The lytic polysaccharide monooxygenase mutant of the present invention does not change the optimal temperature and pH, and the enzyme activity is increased by 55% compared with the wild type. The enzymatic hydrolysis rate of the lytic polysaccharide monooxygenase mutant of the present invention for phosphorylated swollen cellulose in combination with a thermophilic endoglucanase at high temperature is increased by 17% compared with the wild type; the enzymatic hydrolysis rate for bagasse in combination with a thermophilic endoglucanase and a thermophilic endoxylanase is increased by 29% more than that of the wild type. The present invention has strong practicability and can be widely applied to agricultural and forestry biomass raw materials from various sources to improve the enzymatic hydrolysis efficiency of cellulose substrates.

Description

Technical Field

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

[0002] Cellulose, as one of the most abundant polysaccharides in plant cell walls, has always attracted extensive research interest. The decomposition of cellulose is of great significance for the renewable utilization of biomass resources, the production of biofuels, and the development of sustainable production processes. However, the complex structure and strong crystalline properties of cellulose make it difficult to degrade. Therefore, scientists have been searching for effective methods to improve the hydrolysis process of cellulose to better release the glucose contained therein, which can then be used for the production of biofuels or biochemicals.

[0003] Lytic polysaccharide monooxygenases (LPMOs) are a class of enzymes discovered in recent studies that can disrupt the polymeric structure of cellulose. LPMOs are a class of enzymes with oxidative properties that can introduce oxygen atoms during the cellulose hydrolysis process, thereby improving the degradation of cellulose. The role of LPMOs is to break the structure of crystalline cellulose in the microcrystalline part of cellulose, thereby increasing the accessibility of cellulose fibers and making it easier for other cellulose-hydrolyzing enzymes to degrade cellulose and release glucose. LPMOs can cleave the glycosidic bonds in the crystalline region of cellulose through an oxidation-reduction method, thereby improving the hydrolysis efficiency of cellulase on cellulose.

[0004] The analysis of protein structure often plays an important role in the research of enzymes. Although LPMOs have a wide range of sources, their structures are highly conserved. Researchers have compared through various means (such as crystal structure analysis, nuclear magnetic resonance spectroscopy, etc.) and found that all current LPMOs have a similar overall three-dimensional structure (composed of multiple β-sheets connected by α-helices and loop rings) and a conserved β-sandwich structure core region, where the presence of hydrophobic residues, hydrogen bonds, and disulfide bonds provides stability for this region. Although LPMOs have great potential in cellulose hydrolysis, there are still huge challenges. The most important problem is the enzyme activity of LPMOs. Most LPMOs show low catalytic efficiency in practical applications, which may lead to a decrease in the hydrolysis rate of cellulose. In order to give full play to the role of LPMOs, it is necessary to improve their enzyme activity levels to more effectively degrade cellulose. This will also help solve the problems in the utilization of biomass resources, increase the renewable energy yield of biomass, and promote the development of sustainable production.

[0005] A Site-Directed Mutagenesis-Modified Lytic Polysaccharide Monooxygenase and Its Construction Method and Application (CN110551699B) performs point mutations on lytic polysaccharide monooxygenase, improving the enzyme activity of the mutant and the synergistic hydrolysis of microcrystalline cellulose by the mutant and cellulase. However, the enzyme hydrolysis substrate of this technology is microcrystalline cellulose, and the substrate is relatively demanding. Currently, there are few reports on mutating lytic polysaccharide monooxygenase, and there are various cellulose substrates. In addition to microcrystalline cellulose, phosphorylated swollen cellulose and bagasse are more common. Therefore, it is urgent to develop different mutants of lytic polysaccharide monooxygenase to enhance enzyme activity and synergistic effect and improve the enzyme hydrolysis rate of different cellulose substrates. Summary of the Invention

[0006] To solve the problems of low enzyme activity of lytic polysaccharide monooxygenase and low enzyme hydrolysis rate in synergism with cellulase, the present invention provides a mutant of lytic polysaccharide monooxygenase. Compared with the wild-type lytic polysaccharide monooxygenase, the enzyme activity of the mutant is improved; the mutant enzyme of the present invention can synergistically process a variety of cellulose substrates, such as enzymatically hydrolyzing phosphorylated swollen cellulose and bagasse, and can process high-concentration substrates (such as 15% bagasse), and the enzyme hydrolysis rate is increased.

[0007] The first object of the present invention is to provide a mutant of lytic polysaccharide monooxygenase, which is obtained by mutating any one or more of the 17th, 6th, 89th, or 88th positions of the lytic polysaccharide monooxygenase with the initial amino acid sequence shown in SEQ ID NO.1.

[0008] In one embodiment, the gene of the lytic polysaccharide monooxygenase is derived from the Integrated Microbial Genomes / MetaGenomes database of the Joint Genome Institute (https: / / img.jgi.doe.gov / cgi-bin / m / main.cgi; IMG genome ID 2199352008).

[0009] In one embodiment, the initial nucleotide sequence of the lytic polysaccharide monooxygenase is shown in SEQ ID NO.1.

[0010] In one embodiment, the amino acid sequence of the mutant of lytic polysaccharide monooxygenase is shown in SEQ ID NO.2.

[0011] In one embodiment, the mutant of lytic polysaccharide monooxygenase is obtained by mutating the 17th aspartic acid of the initial amino acid sequence to tryptophan (abbreviated as D17W);

[0012] Or by mutating the 17th aspartic acid to phenylalanine (abbreviated as D17F);

[0013] Or by mutating the 6th valine to arginine (abbreviated as V6R);

[0014] Or obtained by mutating threonine at position 89 to isoleucine (abbreviated as T89I);

[0015] Or obtained by mutating leucine at position 88 to arginine (abbreviated as L88R).

[0016] The second object of the present invention is to provide a gene encoding the above-mentioned lytic polysaccharide monooxygenase mutant.

[0017] The present invention also provides a recombinant plasmid carrying the above-mentioned lytic polysaccharide monooxygenase mutant gene.

[0018] In one embodiment, the vector of the recombinant plasmid is a pET vector.

[0019] In one embodiment, the vector of the recombinant plasmid is pET-20b.

[0020] The present invention also provides a host cell carrying the above-mentioned gene or recombinant plasmid.

[0021] In one embodiment, the host cell is a bacterium or a fungus.

[0022] In one embodiment, the host cell is Escherichia coli.

[0023] The present invention also provides the application of the above-mentioned lytic polysaccharide monooxygenase mutant or the gene encoding the lytic polysaccharide monooxygenase mutant or the recombinant plasmid carrying the above-mentioned lytic polysaccharide monooxygenase mutant gene or the host cell carrying the above-mentioned gene or recombinant plasmid in hydrolyzing cellulose.

[0024] The present invention also provides a method for hydrolyzing cellulose, wherein the above-mentioned lytic polysaccharide monooxygenase mutant or the above-mentioned host cell and other cellulases are simultaneously added to cellulose for enzymatic hydrolysis.

[0025] In one embodiment, the other cellulases are any one or combination of endoglucanase, xylanase or Celluclast.

[0026] In one embodiment, the amino acid sequence of endoglucanase is as shown in SEQ ID NO.8; the amino acid sequence of xylanase is as shown in SEQ ID NO.9.

[0027] Beneficial effects

[0028] The present invention performs point mutations on different sites of lytic polysaccharide monooxygenase, and the obtained mutant has a significantly improved enzyme activity. The optimal temperature of the lytic polysaccharide monooxygenase mutant of the present invention is 70 °C, and it can cooperate with other thermophilic enzymes and cellulases to process various fibrous substrates. It can not only process high-concentration substrates but also significantly improve the enzymatic hydrolysis rate.

[0029] Specifically, the mutant D17W has the best effect, as follows:

[0030] (1) The lytic polysaccharide monooxygenase mutant D17W of the present invention has an enzyme activity 55% higher than that of the wild type without changing the optimum temperature and pH;

[0031] (2) When the lytic polysaccharide monooxygenase mutant D17W of the present invention synergistically enzymatically hydrolyzes phosphoric acid swollen cellulose, the enzymatic hydrolysis rate is higher. Compared with the wild type, when the addition amount is 0.3 μmol·L -1 the enzymatic hydrolysis rate increases by 17%;

[0032] (3) The lytic polysaccharide monooxygenase mutant D17W of the present invention shows higher hydrolysis activity when synergistically enzymatically hydrolyzing bagasse. Compared with the wild type, the enzymatic hydrolysis rates at 6, 12, and 24 h increase by 29%, 16%, and 12% respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : Enzyme activity of lytic polysaccharide monooxygenase mutants at different mutation sites;

[0034] Figure 2 : Detection of the optimum temperature and pH of mutant D17W;

[0035] Figure 3 : Synergistic enzymatic hydrolysis of phosphoric acid swollen cellulose by different concentrations of mutant D17W, where the left column in each group is WT and the right is D17W;

[0036] Figure 4 : Change in the enzymatic hydrolysis rate of mutant D17W synergistically enzymatically hydrolyzing bagasse. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] 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 of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0038] 1. Experimental materials

[0039] The E. coli strains (E. coli DH5α, E. coli BL21(DE3)) are all preserved in our laboratory; the plasmid pET-20b(Amp+) is preserved in our laboratory; the thermophilic endoglucanase TnCelB and the thermophilic xylanase Xyn10A are both expressed from the strains reserved after being previously constructed in E. coli BL21(DE3) in the laboratory. The amino acid sequences of the thermophilic endoglucanase TnCelB and the thermophilic xylanase Xyn10A are shown in SEQ ID NO.5 - 6, and the thermophilic endoglucanase TnCelB and the thermophilic xylanase Xyn10A can be directly obtained by expression, separation and purification through E. coli BL21(DE3).

[0040] The enzyme activities and molecular weights of the enzymes used in the present invention are as follows: mgLPMO10 wild type (WT): 25.5 U / g, molecular weight 34821.14; TnCelB: 571.3 U / g, molecular weight 31752.04; Xyn10A: 78.7 U / g, molecular weight of Xyn10A 131243.65; in the examples of the present invention, the enzyme addition amounts are all added in the unit of μmol·L -1 for addition.

[0041] 2. The method (DNS method) for measuring the enzymatic hydrolysis rate involved in the present invention is as follows:

[0042] During the enzymatic hydrolysis process, 0.4 mL of the enzymatic hydrolysis solution is taken respectively, and the supernatant is obtained by centrifugal separation. The centrifugation conditions are: rotation speed 8000 - 10000 rpm, time 5 - 15 min. The supernatant is diluted by 10 - 100 times, and the reducing sugar content is measured by high-performance liquid chromatography, and the enzymatic hydrolysis rate of the reducing sugar is calculated therefrom.

[0043] 3. Preparation of phosphoric acid swollen cellulose (PASC), the specific steps are as follows:

[0044] (1) Add 0.2 g of MCC into a 50 mL centrifuge tube, add 0.6 mL of ddH2O to wet it to form a suspension;

[0045] (2) Slowly add 10 mL of cold 86.2% phosphoric acid, stir well, and finally it is 83.2%. Before adding the last 2 mL, the cellulose suspension should be mixed evenly;

[0046] (3) Place it on ice for 1 h, and stir it every 10 min;

[0047] (4) Centrifuge at 4℃ and 5000 rpm for 20 min, suspend the particles with cold water, add 10 mL of cold water each time, add a total of 40 mL, stir well after each addition, and finally produce a white turbid precipitate. Centrifuge at 4℃ and 6500 rpm for 25 min;

[0048] (5) Wash the precipitate with 50 mL of cold water and repeat four times;

[0049] (6) Add 0.5 mL of 2 mol·L-1 Na2CO3 solution to neutralize phosphoric acid, resuspend with 45 mL of cold water, and centrifuge at 4 °C and 6500 rpm for 25 min;

[0050] (7) Resuspend with 50 mL of cold water and centrifuge twice to make the pH about 5 - 7;

[0051] (8) Storage: Store in a refrigerator at 4 °C or -20 °C.

[0052] 4. Preparation method of pretreated bagasse, the specific steps are as follows:

[0053] (1) Grind the bagasse with a grinder into fine particles, sieve it through a 60-mesh sieve, and place it in an oven at 60 °C until constant weight;

[0054] (2) Put 10 g of dried bagasse into a three-necked flask, add 140 g of glycerol and 0.3 g of NaOH;

[0055] (3) Place the three-necked flask in a heating mantle and heat to 240 °C, while stirring with a stirrer paddle to make the mixture heated evenly, and react for 30 min;

[0056] (4) After the reaction, wait for it to cool naturally, and filter and wash it three times with 4500 mL of H 2 2O to remove the glycerol solution attached to the bagasse, and collect the bagasse;

[0057] (5) Place the treated bagasse in an oven at 60 °C until constant weight, collect it and store it dry for later use.

[0058] Example 1 Preparation of wild-type lytic polysaccharide monooxygenase

[0059] 1. Acquisition of wild-type lytic polysaccharide monooxygenase gene

[0060] The complete gene sequence of mgLPMO10 (metagenome, mg) derived from compost metagenome was obtained from the Integrated Microbial Genomes / M database of the Joint Genome Institute (https: / / img.jgi.doe.gov / cgi-bin / m / main.cgi; IMG genome ID 2199352008)

[0061] Delete the native signal peptide of mgLPMO10 and replace it with the signal peptide of pelB. Optimize the codon bias before synthesizing the gene, add restriction enzyme sites Nde I (sequence: CATATG) and Xho I (sequence: CRCGAG) at both ends of the optimized target gene, and synthesize it by Tianlin Biotechnology Wuxi Co., Ltd.

[0062] 2. Construct the mgLPMO10 expression vector

[0063] (1) PCR amplification of the mgLPMO10 gene

[0064] Obtain the mgLPMO10 recombinant plasmid with pelB as the signal peptide by homologous recombination. Delete the TAA base at the end to ensure that the expression will not be interrupted prematurely, and obtain a protein with a 6×His tag for subsequent protein purification experiments. The primers used for plasmid construction are shown in Table 1

[0065] Table 1 Primers required in this study

[0066]

[0067] Using the synthesized gene sequence as a template, amplify the target gene with the two pairs of designed primers respectively. The PCR reaction system is shown in Table 2.

[0068] Table 2 PCR system

[0069]

[0070] The PCR reaction program is shown in Table 3.

[0071] Table 3 PCR reaction program

[0072]

[0073] Detect and verify the PCR products by agarose gel nucleic acid electrophoresis. After verifying the accurate bands by nucleic acid electrophoresis, add 2 μL of Dpn I to digest the plasmid template in the PCR products. After verifying the concentration and purity of the PCR products digested by Dpn I with a Nanodrop instrument, store them at -20 °C in the refrigerator.

[0074] (2) Extraction and linearization of the empty vector

[0075] In this experiment, the pET-20b vector was selected for heterologous expression of the target gene. E. coli DH5α containing the pET-20b plasmid was cultured overnight in LB medium, and the plasmid pET-20b was extracted from the bacterial solution. The extraction method referred to the operation manual of the plasmid extraction kit, and the extracted plasmid was verified for its concentration and purity with a Nanodrop instrument.

[0076] The pET-20b was linearized by double digestion, and the linearization system is shown in Table 4.

[0077] Table 4 Plasmid linearization system

[0078]

[0079] After linearization, the mixture was subjected to gel extraction and concentration by agarose gel electrophoresis. The method of gel extraction referred to the operation manual of the kit. The concentration and purity of the linearized plasmid after gel extraction were verified using a Nanodrop instrument.

[0080] 3. Ligation of the mgLPMO10 gene fragment and the vector

[0081] The target gene obtained in "Step 2 (1) PCR amplification of the mgLPMO10 gene" and the above-obtained linearized vector were subjected to homologous recombination at a molar ratio of 3:1. The recombination reaction system was added to a PCR tube as shown in Table 5. After mixing evenly, it was reacted at 50 °C for 15 min. The mgLPMO10 recombinant vector pET-20b (pelB-mgLPMO10) with a signal peptide was obtained. After the reaction, it was stored at -20 °C.

[0082] Table 5 Homologous recombination ligation system

[0083]

[0084] 4. Transformation of the recombinant vector

[0085] Take the prepared E. coli DH5α competent cells frozen at -80 °C and perform the transformation of the recombinant vector pET-20b (pelB-mgLPMO10):

[0086] (1) Pipette 10 μL of the recombinant vector and add it to the E. coli DH5α competent cells melted on ice. Gently mix and incubate on ice for 30 min;

[0087] (2) Heat shock in a metal bath at 42 °C for 90 s and then place on ice for 2 min;

[0088] (3) Add 1 mL of SOC recovery medium, mix gently, and culture in a shaker at 37 °C and 200 rpm for 45 min;

[0089] (4) Centrifuge the bacterial solution, discard 90% of the supernatant, resuspend by pipetting, and spread it on an LB solid plate with ampicillin (Amp) resistance. Incubate it upside down at 37 °C overnight.

[0090] After colonies grow on the plate, pick a monoclonal transformant and dissolve it in 10 μL of ddH2 Using 5 μL of the bacterial solution as a template, perform bacterial solution PCR, observe the bands by agarose gel electrophoresis, and verify whether it is a positive transformant. Inoculate the transformant with accurate bands into an LB test tube containing Amp and culture it overnight at 37°C. Extract the plasmid from the bacterial solution and send it to Tianlin Biotechnology Company for sequencing. Use the DNAMAN software to compare the base sequence obtained by sequencing with the optimized target gene sequence to verify the correctness of the positive transformant and obtain the E. coli DH5α strain carrying the recombinant plasmid of mgLPMO10.

[0091] 5. Protein expression

[0092] Extract the plasmid of the verified recombinant and re-introduce it into Escherichia coli E. coli BL21(DE3), and inoculate it into an LB liquid medium containing Amp and culture it overnight. Inoculate it into a conical flask containing 200 mL of LB culture medium at an inoculation amount of 1%, and perform shaking fermentation on a shaker at 37°C and 220 rpm for about 3 h. When the OD600 reaches 0.6 - 0.8, add IPTG with a final concentration of 0.5 mmol·L-1, and then ferment it on a shaker at 37°C for 6 h. After the fermentation is completed, centrifuge the fermentation broth at 4°C and 10,000 rpm for 10 min to collect the bacterial cells.

[0093] 6. Protein purification

[0094] Wash the collected bacterial cells with PBS buffer and resuspend them in PBS buffer with a volume equivalent to 1 / 2 of the fermentation broth, and place them in an ultrasonic cell disruptor to completely disrupt. Centrifuge the cell disruption solution at 4°C and 12,000 rpm to collect the supernatant, which is the crude enzyme solution of mgLPMO10 protein. Ultrasonic disruption parameters: power 70%, on for 5 s, off for 5 s, total time 45 min.

[0095] The recombinant mgLPMO10 protein has a 6×His tag at the end, so the method of Ni+-NTA nickel column affinity chromatography is used for protein purification. The specific operation is as follows:

[0096] (1) Let the 20% ethanol preservation solution in the nickel resin flow out, and equilibrate the column with Binding Buffer. Add the cell disruption supernatant to bind with the nickel resin and pour it into a centrifuge tube, and slowly vertically shake it in a 4°C molecular hybridization instrument for 1 h to make the mgLPMO10 protein fully bind to the nickel resin;

[0097] (2) Pour the binding solution into the column. After the resin settles, open the stopper to let the liquid flow out through the column;

[0098] (3) Pipette 10 mL of Binding buffer to elute the unbound or weakly bound miscellaneous proteins;

[0099] (4) Slowly add 10 mL of Elution buffer with imidazole concentrations of 50, 100, 200, and 400 mmol·L -1 to the column to elute the mgLPMO10 protein from the nickel resin. Collect the eluate to obtain the purified target protein, and its amino acid sequence is shown in SEQ ID NO.1. The purified protein is stored in a refrigerator at 4°C.

[0100] Example 2 Preparation of Lytic Polysaccharide Monooxygenase Mutants with Different Mutation Sites

[0101] Site-directed mutagenesis was completed by designing site-directed mutagenesis primers for PCR. Using the recombinant plasmid pET-20b(pelB-mgLPMO10) constructed in Example 1 as a template, the primers are shown in Table 6. The remaining construction and expression methods are the same as those in Example 1. Ten mutants with corresponding position mutations based on the amino acid sequence shown in SEQ ID NO.1 were prepared. According to "amino acid before mutation + mutation site + amino acid after mutation", the mutants were named D17W, V6R, T89H, D17F, T89I, V6M, D17Y, L88R, V6Y, and D17H respectively.

[0102] Table 6 Mutation Primers

[0103]

[0104] Example 3 Detection of Mutant Enzyme Activity

[0105] Using the mgLPMO10 wild type and its mutants prepared in Example 1 and Example 2, the 2,6-DMP rapid method for determining enzyme activity was adopted, and 2,6-DMP and H 2 O 2 were used as co-substrates to determine the enzyme activities of the mgLPMO10 wild type (WT) and mutants:

[0106] (1) Prepare a phosphate buffer with a concentration of 116 mmol·L -1 and a pH of 7.5; a 2,6-DMP solution with a concentration of 10 mmol·L -1 ; and an H -1 solution with a concentration of 5 mmol·L 2 O 2 . All solutions should be used within 12 hours after preparation.

[0107] (2) Add 860 μL of phosphate buffer, 100 μL of 2,6-DMP solution, and 20 μL of H 2 O 2 solution to a centrifuge tube and mix well, and incubate at the corresponding temperature for 15 minutes.

[0108] (3) After adding 20 μL of the mgLPMO10 protein sample and mixing well, measure the increase in absorbance at a wavelength of 469 nm within 5 min (ε469 = 53200 L·mol -1 ·cm -1 ) to calculate the activity of LPMO. One enzyme activity unit is defined as the conversion of 2 μmol of 2,6-DMP per minute under the reaction conditions.

[0109] The results are as Figure 1 shown. The mutants D17W, V6R, D17F, T89I, and L88R are all positive mutants. Compared with the wild enzyme before mutation, their enzyme activities are increased by 55%, 13%, 11%, 30%, and 6% respectively; among them, the mutant D17W has the highest activity among all mutants, with a 55% increase in enzyme activity compared to the mgLPMO10 wild-type (WT) enzyme.

[0110] Example 4 Detection of Enzymatic Properties of Mutants

[0111] The enzymatic properties of the mgLPMO10 wild-type prepared in Example 1 and the mutant D17W prepared in Example 2 were detected. The relative enzyme activities of the mutant D17W at 40 - 90 °C and at pH 4 - 9 were detected respectively. The method for detecting enzyme activity was the same as that in Example 2.

[0112] The results are as Figure 2 shown. Compared with the wild-type (WT), the mutant D17W has higher enzyme activity under various temperatures ( Figure 2 (a)) or various pH values ( Figure 2 (b)). The optimal temperature of the mutant WT is 70 °C, and the optimal pH is pH 6, which is the same as that of the wild-type. Under these conditions, the enzyme activity of D17W is increased by 55% compared to WT.

[0113] Example 5 Application of Mutant Synergistic Cellulase in Hydrolyzing Cellulose Substrates

[0114] The mgLPMO10 wild-type and its mutants prepared in Examples 1 and 2 were used to detect their synergistic effects with cellulase. The thermophilic endoglucanase TnCelB and the thermophilic endoxylanase Xyn10A can be expressed and isolated through the laboratory-preserved Escherichia coli E. coli BL21(DE3). The steps are the same as those in "5. Protein Expression and 6. Protein Purification" in Example 1. The amino acid sequences of TnCelB and Xyn10A are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0115] (1) Enzymatic hydrolysis of phosphoric acid-swollen cellulose with the thermophilic endoglucanase TnCelB

[0116] Detect the high-temperature synergistic effect of mutant D17W and cellulase. Mutant D17W and the thermophilic endoglucanase TnCelB were used for the synergistic hydrolysis of phosphoric acid swollen cellulose (PASC) at 70 °C, and the reducing sugar concentration in the hydrolysis solution was measured after 1 h of reaction. The specific method is as follows:

[0117] The mutant D17W and the thermostable endoglucanase TnCelB were used for the synergistic hydrolysis of the cellulose matrix to detect the synergistic effect between them. The enzyme reaction was carried out in 1 mL of phosphate buffer (50 mmol·L -1 , pH 6), and the reaction solution contained 0.5% PASC, 0.3 μmol·L -1 TnCelB, 1 mmol·L -1 AscA, and different concentrations (0, 0.03, 0.075, 0.15, 0.3, 0.75, 1.5 mmol·L -1 ) of mgLPMO10. The reaction solution was placed in a hybridization oven and reacted vertically at 70 °C for 1 h. After the reaction, it was 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 reducing sugar content in the supernatant was measured by the DNS method.

[0118] The results are as Figure 3 shown. With the addition amount of TnCelB unchanged, the synergistic hydrolysis efficiency of mutant D17W with various protein concentrations and TnCelB is higher than that of the wild type (WT). When the concentration of D17W protein is 0.3 μmol·L -1 , the enzymatic hydrolysis rate is increased by 17% compared with WT.

[0119] (2) Synergistic enzymatic hydrolysis of pretreated sugarcane bagasse by thermophilic endoglucanase TnCelB and thermophilic endoxylanase Xyn10A

[0120] The wild type of mgLPMO10 and its mutants were used to synergistically hydrolyze the pretreated sugarcane bagasse with a concentration of 15% w / v by thermophilic endoglucanase TnCelB and thermophilic endoxylanase Xyn10A, and their enzymatic hydrolysis rates were detected. The specific steps are as follows:

[0121] mgLPMO10, thermophilic endoglucanase TnCelB and thermophilic xylanase Xyn10A were used to carry out a hydrolysis reaction on 15% w / v pretreated sugarcane bagasse at 70 °C for 3 h. The enzyme reaction was carried out in a phosphate buffer of 50 mmol·L -1 , pH 6, and the reaction solution included 3 μmol·L -1 TnCelB, 3 μmol·L -1 mgLPMO10, 3 μmol·L -1 Xyn10A, 1 mmol·L-1 AscA, 15% w / v pretreated sugarcane bagasse was placed in a hybridization oven for vertical rotation reaction; the reaction solution was cooled to 50 °C, and Celluclast (10 mg·g-1 substrate) was added, and the reaction was carried out at 50 °C for 72 h.

[0122] The results are as Figure 4 shown. The mutant D17W and two high-temperature enzymes synergistically hydrolyzed the pretreated sugarcane bagasse, and the hydrolysis rate was higher than that of the wild type, increasing by 29%, 16%, and 12% at 6, 12, and 24 h, respectively.

[0123] The initial amino acid sequence of mgLPMO10 SEQ ID NO.1:

[0124] HGAAMVPGSRTYLCWR D GLSPTGQIIPNNPACAAAVAVSGANSLYNWFSVLRSDAGGRTVGYIPDGQLCSGGNPGFLGYDLARDDWPLTHLTAGATIEFRYSNWAHHPGTFYFYVTRDSWSPTRPLAWSDLESEPFLTVTNPPQRGAVGTNDGHYYFTGRLPNKSGRHIIYSRWVRSDSQENFFGCSDVVFDGGNGEVTGIGSGSGPTTPPTTPPTTPPTTPPTTPPTTPGGSTGCAATYQVVGSWTGGFQAEVTVRNTGTAPLNGWTIQWTFANGETVGSLWNGQHSQSGSTVTVRNVDHNGSLAPGASTSFGFVGSGSTGATPTPTCTSA

[0125] The sequence of mutant D17W SEQ ID NO.2:

[0126] HGAAMVPGSRTYLCWR WGLSPTGQIIPNNPACAAAVAVSGANSLYNWFSVLRSDAGGRTVGYIPDGQLCSGGNPGFLGYDLARDDWPLTHLTAGATIEFRYSNWAHHPGTFYFYVTRDSWSPTRPLAWSDLESEPFLTVTNPPQRGAVGTNDGHYYFTGRLPNKSGRHIIYSRWVRSDSQENFFGCSDVVFDGGNGEVTGIGSGSGPTTPPTTPPTTPPTTPPTTPPTTPGGSTGCAATYQVVGSWTGGFQAEVTVRNTGTAPLNGWTIQWTFANGETVGSLWNGQHSQSGSTVTVRNVDHNGSLAPGASTSFGFVGSGSTGATPTPTCTSA

[0127] Mutant D17W nucleotide sequence SEQ ID NO.3:

[0128] CATGGAGCTGCGATGGTACCCGGTTCCCGTACTTATTTATGCTGGCGT TGGGGCTTATCCCCTACAGGACAGATCATCCCTAATAATCCCGCGTGCGCTGCTGCAGTGGCGGTGAGTGGAGCTAACTCTTTGTATAACTGGTTCAGCGTCCTTCGTTCTGACGCCGGTGGCCGTACCGTAGGATACATCCCGGATGGTCAGTTGTGTTCAGGTGGAAATCCAGGGTTCCTGGGGTACGACTTGGCACGTGATGATTGGCCGCTGACGCATCTTACGGCTGGAGCTACCATCGAGTTTCGTTACTCAAACTGGGCGCATCATCCGGGAACGTTTTATTTCTACGTGACCCGCGACAGCTGGTCGCCGACCCGCCCTCTGGCTTGGTCTGACTTAGAATCCGAACCATTTTTAACCGTGACAAACCCGCCACAGCGTGGGGCCGTGGGTACAAATGATGGACATTACTACTTTACTGGTCGCTTACCGAACAAGAGCGGGCGCCACATCATCTACAGCCGTTGGGTTCGCTCGGACTCACAAGAGAATTTCTTCGGGTGCTCCGACGTTGTATTTGACGGAGGGAACGGTGAGGTGACCGGCATCGGATCAGGTTCGGGGCCTACGACTCCACCAACGACCCCACCTACAACGCCTCCAACTACACCACCAACCACACCCCCTACTACACCGGGCGGCTCCACGGGCTGCGCGGCGACATATCAAGTCGTAGGGTCGTGGACTGGCGGATTCCAAGCAGAAGTTACGGTACGCAACACTGGCACTGCTCCTTTGAACGGTTGGACAATTCAGTGGACGTTTGCAAATGGTGAGACTGTAGGCTCATTATGGAACGGCCAACACAGTCAGAGCGGCTCCACGGTCACTGTGCGCAACGTTGACCATAATGGAAGCCTTGCACCGGGGGCCTCCACGTCGTTTGGATTCGTTGGAAGCGGTTCTACTGGGGCTACGCCTACTCCCACATGCACAAGCGCGTAA

[0129] Amino acid sequence of TnCelB SEQ ID NO.4:

[0130] MRLVVSFLLVVSAFLFSAEVVLTDIGATDITFKGFPVTMELNFWNVKSYEGETWLKFDGEKVQFYADIYNIVLQNPDSWVHGYPEIYYGYKPWAAHNSGTEILPVKVKDLPDFYVTLDYSIWYENDLPINLAMETWITRKPDQTSVSSGDVEIMVWFYNNILMPGGQKVDEFTTTIEINGSPVETKWDVYFAPWGWDYLAFRLTTPMKDGRVKFNVKDFVEKAAEVIKKHSTRVENFDEMYFCVWEIGTEFGDPNTTAAKFGWTFKDFSVEIGE

[0131] Amino acid sequence of Xyn10A: SEQ ID NO.5

[0132]

[0133] The initial nucleotide sequence of mgLPMO10 SEQ ID NO.28:

[0134] CATGGAGCTGCGATGGTACCCGGTTCCCGTACTTATTTATGCTGGCGT GACGGCTTATCCCCTACAGGACAGATCATCCCTAATAATCCCGCGTGCGCTGCTGCAGTGGCGGTGAGTGGAGCTAACTCTTTGTATAACTGGTTCAGCGTCCTTCGTTCTGACGCCGGTGGCCGTACCGTAGGATACATCCCGGATGGTCAGTTGTGTTCAGGTGGAAATCCAGGGTTCCTGGGGTACGACTTGGCACGTGATGATTGGCCGCTGACGCATCTTACGGCTGGAGCTACCATCGAGTTTCGTTACTCAAACTGGGCGCATCATCCGGGAACGTTTTATTTCTACGTGACCCGCGACAGCTGGTCGCCGACCCGCCCTCTGGCTTGGTCTGACTTAGAATCCGAACCATTTTTAACCGTGACAAACCCGCCACAGCGTGGGGCCGTGGGTACAAATGATGGACATTACTACTTTACTGGTCGCTTACCGAACAAGAGCGGGCGCCACATCATCTACAGCCGTTGGGTTCGCTCGGACTCACAAGAGAATTTCTTCGGGTGCTCCGACGTTGTATTTGACGGAGGGAACGGTGAGGTGACCGGCATCGGATCAGGTTCGGGGCCTACGACTCCACCAACGACCCCACCTACAACGCCTCCAACTACACCACCAACCACACCCCCTACTACACCGGGCGGCTCCACGGGCTGCGCGGCGACATATCAAGTCGTAGGGTCGTGGACTGGCGGATTCCAAGCAGAAGTTACGGTACGCAACACTGGCACTGCTCCTTTGAACGGTTGGACAATTCAGTGGACGTTTGCAAATGGTGAGACTGTAGGCTCATTATGGAACGGCCAACACAGTCAGAGCGGCTCCACGGTCACTGTGCGCAACGTTGACCATAATGGAAGCCTTGCACCGGGGGCCTCCACGTCGTTTGGATTCGTTGGAAGCGGTTCTACTGGGGCTACGCCTACTCCCACATGCACAAGCGCGTAA

[0135] 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.

Claims

1. A lytic polysaccharide monooxygenase mutant, characterized in that, in the lytic polysaccharide monooxygenase with the amino acid sequence shown in SEQ ID NO.1, the aspartic acid at the 17th position is mutated to tryptophan.

2. A gene encoding the lytic polysaccharide monooxygenase mutant according to claim 1.

3. A recombinant plasmid carrying the gene according to claim 2.

4. The recombinant plasmid according to claim 3, characterized in that, the vector of the recombinant plasmid is a pET vector.

5. A host cell carrying the gene according to claim 2 or the recombinant plasmid according to any one of claims 3 to 4.

6. The host cell according to claim 5, characterized in that, the host cell is a bacterium or a fungus.

7. Use of the lytic polysaccharide monooxygenase mutant according to claim 1 or the host cell according to any one of claims 5 to 6 in hydrolyzing cellulose.

8. Use of the gene according to claim 2 or the recombinant plasmid according to any one of claims 3 to 4 in hydrolyzing cellulose.

9. A method for hydrolyzing cellulose, characterized in that, the lytic polysaccharide monooxygenase mutant according to claim 1 or the host cell according to any one of claims 5 to 6 and other cellulases are added to cellulose for enzymatic hydrolysis; the other cellulases are any one or combination of endoglucanase, endoxylanase or cellulase.

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