Thermophilic fungus cleavable polysaccharide monooxygenase and application thereof

Abstract

The invention discloses thermophilic fungus cleavable polysaccharide monooxygenase and application thereof, and belongs to the technical field of enzyme engineering and microbial engineering. The thermophilic fungus lysing polysaccharide monooxygenase provided by the invention has excellent thermal stability; when the lysable polysaccharide monooxygenase is used for synergistically hydrolyzing phosphoric acid swelling cellulose, microcrystalline cellulose and bagasse, the enzymolysis rate is increased, and the problems of poor thermal stability of the lysable polysaccharide monooxygenase, low enzymolysis rate of synergic cellulase and high LPMOs enzyme dosage are solved. The method has high practicability and can be widely applied to the enzymolysis process of various cellulosic substrates, so that the enzymolysis efficiency of the cellulosic substrates is remarkably improved.

Description

Technical Field

[0001] The invention relates to a thermophilic fungus lytic polysaccharide monooxygenase and application thereof, belonging to the field of enzyme engineering and technology. Background Art

[0002] Cellulose, 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 looking for effective methods to improve the hydrolysis process of cellulose to better release the glucose contained in it, so that it can be used for the production of biofuels or biochemicals.

[0003] Lytic polysaccharide monooxygenases (LPMOs) are a class of enzymes recently discovered that can disrupt the polymer structure of cellulose. In particular, LPMOs from the auxiliary activity 9 family (AA9 LPMOs) are oxidative enzymes that can introduce oxygen atoms during cellulose hydrolysis, thereby improving cellulose degradation. AA9 LPMOs function by disrupting the dense crystalline regions of cellulose, increasing their accessibility and making it easier for other cellulolytic enzymes to degrade cellulose and release glucose. AA9 LPMOs can cleave glycosidic bonds in the crystalline regions of cellulose through a redox pathway, thereby enhancing the efficiency of cellulose hydrolysis by cellulases.

[0004] Despite the enormous potential of AA9 LPMOs in cellulose hydrolysis, their practical application still faces numerous challenges. Currently, AA9 LPMOs are primarily derived from non-thermophilic fungi, making them difficult to maintain activity for extended periods under high temperature conditions. Their thermal stability is a key bottleneck limiting their application. Most AA9 LPMOs exhibit poor stability at high temperatures, which not only compromises their ability to efficiently and effectively hydrolyze cellulose with cellulases over extended periods but also significantly reduces the hydrolysis rate. To fully unlock the potential of AA9 LPMOs, improving their thermal stability is a key goal. This improvement could significantly enhance cellulose degradation efficiency, making their application in industrial biomass conversion more efficient. Notably, thermophilic fungi can survive high temperatures, and the LPMOs they carry often exhibit excellent thermal stability. Therefore, identifying and developing thermostable LPMOs from thermophilic fungi would significantly improve the efficiency of cellulase-assisted hydrolysis of lignocellulose. This strategy would not only help address key challenges in biomass resource utilization but also increase renewable energy production and promote the development of more sustainable biomass conversion processes, thereby bringing significant social and economic benefits in the energy and environmental sectors.

[0005] Therefore, the key to developing LPMOs derived from thermophilic fungi to obtain enzymes that can maintain long-term activity at high temperatures is to achieve optimal results when used in synergy with cellulases. This not only enables efficient hydrolysis of lignocellulose, but also improves the efficiency of cellulase hydrolysis at low LPMO dosages, thereby reducing cellulase usage and costs. Solving this problem will have extremely high economic and practical value, promoting the development of lignocellulose biorefining technology. Summary of the Invention

[0006] In order to solve the problems of poor thermal stability of lytic polysaccharide monooxygenase, low enzymatic hydrolysis rate of synergistic cellulase and high LPMOs enzyme dosage, the present invention provides a thermophilic fungus lytic polysaccharide monooxygenase with excellent thermal stability; the enzymatic hydrolysis rate is improved when synergistically hydrolyzing phosphate-swollen cellulose, microcrystalline cellulose and sugarcane bagasse.

[0007] The first object of the present invention is to provide a use of a lytic polysaccharide monooxygenase in hydrolyzing cellulose. The amino acid sequence of the lytic polysaccharide monooxygenase is shown in SEQ ID NO.3.

[0008] In one embodiment, the lytic polysaccharide monooxygenase can be obtained by heterologous expression.

[0009] In one embodiment, the vector includes pPIC series vectors, pPICZ series vectors, pPICZα, and pET series vectors;

[0010] 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.

[0011] In one embodiment, the cells include Pichia pastoris, Escherichia coli, Saccharomyces cerevisiae, and filamentous fungi;

[0012] Alternatively, the filamentous fungi include Aspergillus niger, Aspergillus oryzae, and the like.

[0013] In one embodiment, the cellulose comprises pretreated bagasse, phosphoric acid swollen cellulose, and microcrystalline cellulose.

[0014] In one embodiment, the pretreated bagasse is divided into alkali-treated bagasse and acid-treated bagasse, and the preparation method includes:

[0015] The dried sugar cane, glycerin and NaOH (or sulfuric acid) are mixed, heated and stirred, filtered and washed to prepare alkali-treated bagasse (acid-treated bagasse).

[0016] The second object of the present invention is to provide a method for hydrolyzing cellulose, using a mixture of a lytic polysaccharide monooxygenase and a cellulase to synergistically hydrolyze cellulose; the amino acid sequence of the lytic polysaccharide monooxygenase is shown in SEQ ID NO.3.

[0017] In one embodiment, the cellulase comprises a thermophilic endoglucanase or a commercial cellulase;

[0018] Optionally, the amino acid sequence of the thermophilic endoglucanase is shown in SEQ ID NO.2;

[0019] Alternatively, commercial cellulases include Celluclast or Ctec2;

[0020] Cellulose includes pretreated bagasse, phosphoric acid swollen cellulose, and microcrystalline cellulose.

[0021] In one embodiment, the added amount of the lytic polysaccharide monooxygenase is 0.2-2 mg / g dry substrate; the added amount of the cellulase (the total amount of the lytic polysaccharide monooxygenase and the commercial cellulase) is 10 mg / g.

[0022] In one embodiment, the amount of cellulose added is 0.5-2% w / v.

[0023] In one embodiment, the enzymatic hydrolysis conditions are 45-50° C. for 12-96 h;

[0024] Preferably, the enzymatic hydrolysis condition is 50° C. for 12 to 96 hours.

[0025] The present invention also provides a lytic polysaccharide monooxygenase mutant, which 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. 3.

[0026] Preferably, the lytic polysaccharide monooxygenase mutant has an amino acid mutation at amino acids 32, 107, 120, 137, 154 and 160 based on the amino acid sequence as shown in SEQ ID NO.3.

[0027] In one embodiment, the glycine at position 32 is mutated to serine, designated as G32S; or,

[0028] The proline at position 107 is mutated to leucine, named P107L; or,

[0029] The glutamine at position 120 is mutated to tyrosine, named Q120Y; or

[0030] The glutamine at position 137 is mutated to isoleucine, named Q137I; or

[0031] The alanine at position 154 is mutated to proline, named A154P; or

[0032] The aspartic acid at position 160 is mutated to glycine and named D160G.

[0033] In one embodiment, the relative enzyme activity of mutants G32S, P107L, Q120Y, Q137I, A154P, and D160G is increased by more than 50% compared with before mutation, and the enzyme activity reaches more than 53.2 U / g; among them, the enzyme activity of the G32S mutant is increased by 65% ​​compared with before mutation, and the enzyme activity reaches 56.7 U / g.

[0034] Beneficial effects

[0035] The present invention heterologously expressed the thermophilic fungus-derived polysaccharide monooxygenase MtLPMO9V in Pichia pastoris GS115 and characterized it using phosphate-swollen cellulose as a substrate. The enzyme was found to possess C4 oxidative cleavage activity. After incubation for 24 hours at 50°C and 70°C, respectively, it retained over 50% and 40% of its original activity, demonstrating excellent thermal stability. Furthermore, MtLPMO9V exhibited a strong synergistic effect when working with cellulase to hydrolyze cellulosic substrates, significantly improving the efficiency of cellulase in hydrolyzing cellulosic feedstocks.

[0036] The main characteristics of MtLPMO9V and its role in synergistic hydrolysis with cellulase are as follows:

[0037] (1) The MtLPMO9V of the present invention has C4 oxidative cleavage activity;

[0038] (2) The MtLPMO9V of the present invention has the best activity at 85°C, with an enzyme activity of 477.7 U / g (pH 7.5);

[0039] (3) When MtLPMO9V of the present invention cooperates with thermophilic endoglucanase DtCelA to hydrolyze phosphate-swollen cellulose and microcrystalline cellulose at 70°C, the synergistic degree can reach up to 127% and 120%, respectively;

[0040] (4) The glucose yields of the present invention when MtLPMO9V and cellulase Celluclast 1.5L synergistically hydrolyze microcrystalline cellulose and sugarcane bagasse pretreated with glycerol organic solvent under acid / base catalysis reach 71%, 66%, and 78%, respectively, which are increased by 22%, 18%, and 28%, respectively, compared with the results without adding MtLPMO9V. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 MtLPMO9V expressed for Pichia pastoris GS115 was analyzed by SDS-PAGE;

[0042] Figure 2 Figure 2 shows the regioselectivity analysis of MtLPMO9V. (a, b, c, d, and e) show the MALDI-TOF / MS spectra of the DP2 to DP6 oxidation products released from PASC by MtLPMO9V. The native product and the oxidized product are indicated by blue and red letters, respectively.

[0043] Figure 3 The optimum temperature and thermal stability analysis of MtLPMO9V; a is the optimum temperature; b is the residual enzyme activity after long-term treatment at different temperatures;

[0044] Figure 4The reducing sugar concentration and synergistic degree DS of MtLPMO9V and DtCelA in the synergistic hydrolysis of phosphate-swollen cellulose (PASC) and microcrystalline cellulose (MCC) are shown in Table 1. a) is the result of synergistic hydrolysis of microcrystalline cellulose; b is the result of synergistic hydrolysis of phosphate-swollen cellulose;

[0045] Figure 5 The changes in the enzymatic hydrolysis rate of microcrystalline cellulose (MCC) and glycerol organic solvent pretreated bagasse under acid / base catalysis by synergistic hydrolysis of microcrystalline cellulose (MCC) with different addition amounts of MtLPMO9V and cellulase Celluclast 1.5L; among them, a is the result of synergistic hydrolysis of microcrystalline cellulose; b is the result of synergistic hydrolysis of acid pretreated bagasse; c is the result of synergistic hydrolysis of alkali pretreated bagasse.

[0046] Figure 6 Activity results of each mutant of MtLPMO9V are shown. DETAILED DESCRIPTION

[0047] The following will clearly and completely describe the technical solutions described in the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0048] Experimental Materials

[0049] Cloning strain: Escherichia coli strain (E. coli JM109) is maintained in this laboratory; expression strain: Pichia pastoris GS115 is maintained in this laboratory; expression plasmid pPIC9K is maintained in this laboratory; thermophilic endoglucanase DtCelA is expressed by retained strains of E. coli BL21 (DE3) constructed in the laboratory in the early stage. The amino acid sequence of thermophilic endoglucanase DtCelA is shown in SEQ ID NO.2. The thermophilic endoglucanase DtCelA can be directly expressed, isolated and purified in E. coli BL21 (DE3).

[0050] The enzyme activity and molecular weight of the enzyme used in the present invention are respectively MtLPMO9V wild type (WT): 34.2 U / g (30°C pH 7.5, i.e., standard conditions for detecting enzyme activity), and a molecular weight of approximately 24.4 kDa; DtCelA: 63 U / g, and a molecular weight of 37.1 kDa; the enzyme addition amount in the examples of the present invention is added in the unit of mg / g dry substrate.

[0051] Unless otherwise specified, w / v in the text is g / mL.

[0052] Detection method

[0053] 1. Determination of reducing sugar concentration (DNS method):

[0054] During the hydrolysis process, 0.4 mL of the hydrolyzate was centrifuged at 8000 rpm for 5 minutes to obtain the supernatant. The supernatant was diluted to an appropriate dilution and the reducing sugar content was determined using DNS, from which the reducing sugar concentration was calculated.

[0055] The calculation formula of synergy degree (DS) is:

[0056]

[0057] 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.

[0058] 2. Glucose yield:

[0059] At specified time intervals, 0.4 mL of sample was extracted and heated at 100°C for 5 minutes to inactivate the enzyme. The sample was then centrifuged at 8000 rpm for 5 minutes, and the supernatant was filtered through a 0.22 μm filter and diluted appropriately. Glucose concentration was determined by high-performance liquid chromatography (HPLC, Chomaster CM5110, Hitachi, Japan) equipped with a refractometer and a Bio-Rad Aminex HPX-87H column (9 μm, 7.8 × 300 mm). The mobile phase consisted of 5 mM dilute H₂SO₄ at a flow rate of 0.6 mL / min, with isocratic elution and a column temperature of 60°C.

[0060] The calculation formula for glucose yield is:

[0061]

[0062] 3. Preparation of phosphoric acid swollen cellulose (PASC), the specific steps are as follows:

[0063] (1) Add 0.2 g of MCC (microcrystalline cellulose) to a 50 mL centrifuge tube and moisten with 0.6 mL of ddH2O to form a suspension;

[0064] (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 must be mixed thoroughly.

[0065] (3) Place on ice for 1 hour, stirring every 10 minutes;

[0066] (4) Centrifuge at 4°C, 5000 rpm for 20 min, resuspend the pellet in cold water, add 10 mL of cold water at a time, and add a total of 40 mL. Stir thoroughly after each addition to produce a white turbid precipitate. Centrifuge at 4°C, 6500 rpm for 25 min.

[0067] (5) Wash the precipitate with 50 mL of cold water, repeat four times;

[0068] (6) Add 0.5 mL of 2 mol / L Na2CO3 solution to neutralize the phosphoric acid, resuspend in 45 mL of cold water, and centrifuge at 4°C, 6500 rpm for 25 min;

[0069] (7) Add 50 mL of cold water to resuspend and centrifuge twice to adjust the pH to approximately 5-7;

[0070] (8) Store in a refrigerator at 4°C or -20°C.

[0071] 4. A method for preparing bagasse by pre-treatment with glycerol organic solvent under acid / base catalysis, the specific steps are as follows:

[0072] (1) Grind the bagasse into fine particles using a grinder, sieve it with a 60-mesh sieve, and dry it in an oven at 60°C until constant weight is reached;

[0073] (2) Place 10 g of oven-dried bagasse in a three-necked flask, add 140 g of glycerol and 4% NaOH (w / w, based on bagasse, 0.4 g) or 6% sulfuric acid (w / w, based on bagasse, 0.6 g);

[0074] (3) Place the three-necked flask in a heating mantle and heat to 195°C (for alkali treatment) or 200°C (for acid treatment), stirring with a stirring paddle to ensure uniform mixing and heating. React for 45 minutes (for alkali treatment) or 15 minutes (for acid treatment).

[0075] (4) When the specified temperature is reached, the timer is started. When the reaction is finished, 100 mL of hot water is added to terminate the reaction. The mixture is filtered through a G1 sand core funnel, and the filter cake is washed with hot water until it is neutral and the glycerol is washed away.

[0076] (5) Place the treated bagasse in a 60°C oven and dry it to constant weight, collect it, dry it and store it for later use.

[0077] 5. Pichia pastoris electroporation

[0078] Preparation and transformation of Pichia pastoris GS115 competent cells (electroporation method) were performed as follows:

[0079] (1) Streak Pichia pastoris GS115 on a YPD plate and incubate at 30°C for 2 days to isolate a single colony. Use YNB minimal medium and His-supplemented medium for spot isolation and purification. Pick a single colony that grows on the supplemented medium but not on the minimal medium and streak it on a YPD plate and store at 4°C.

[0080] (2) A single colony of Pichia pastoris GS115 was picked from a YPD plate and inoculated into a 50 mL Erlenmeyer flask containing 5 mL of YPD medium. The culture was incubated at 30°C and 250–300 rpm / min overnight.

[0081] (3) Take 20 μL of culture and inoculate it into a 50 mL triangular shake flask containing 20 mL of fresh culture medium. Incubate overnight at 28-30°C and 250-300 rpm / min until the OD 600 Reach 1.3-1.5;

[0082] (4) The cell culture was divided into multiple 1.5 mL EP tubes, centrifuged at 1,500 g for 30 s at 4°C, and the cell pellet was resuspended in 1 mL of ice-cold sterile water;

[0083] (5) Centrifuge as in step (4) and resuspend the bacterial pellet in 1 mL of ice-cold sterile water;

[0084] (6) Centrifuge as in step (4) and resuspend the bacterial pellet in 1 mL of ice-cold 1 M sorbitol solution;

[0085] (7) Centrifuge as in step (4), resuspend the bacterial pellet in 60 μL of ice-cold 1 M sorbitol solution, and immediately use for electroporation transformation;

[0086] (8) Dissolve 5–20 μg of linearized DNA in 5–10 μL of TE solution, mix with 80 μL of the cells obtained in step (7), and transfer to a 0.2 cm ice-cold electroporation cuvette;

[0087] (9) Place the electroporation cuvette in an ice bath for 5 minutes;

[0088] (10) Voltage 3 kV, shock duration 6 ms;

[0089] (11) After the electroporation is completed, immediately add 1 mL of 1 mol / L ice-cold sorbitol solution to the electroporation cuvette to mix the cells. Transfer the contents of the electroporation cuvette to a new 1.5 mL centrifuge tube.

[0090] (12) Spread the bacterial suspension onto MD plates, with 200–600 μL applied to one plate.

[0091] (13) Incubate the plate at 30°C until a single colony appears.

[0092] 6. Pichia pastoris protein induction expression and purification

[0093] (1) Inducible expression

[0094] Single colonies grown in MD medium were picked up by toothpicks and placed in YPD medium containing 1 g / L, 3 g / L and 5 g / L of Geneticin for screening. Recombinant bacteria grown in high concentrations of antibiotics were inoculated into BMGY medium and cultured overnight until OD 600 The cells were then transferred into BMMY medium until the OD value of the cells was 2 to 6. 600 The fermentation was carried out in a shaker at 30°C and 260 rpm, and a methanol solution with a final concentration of 1% was added every 24 hours to induce fermentation and enzyme production for 7 consecutive days. After the fermentation was completed, the fermentation broth was centrifuged at 4°C and 10,000 rpm for 20 minutes, and the supernatant was collected.

[0095] (2) Protein purification

[0096] The collected supernatant was precipitated by adding ammonium sulfate with a final concentration of 70%, and then the excess salt was removed using a dialysis bag. + The protein was purified by -NTA nickel column affinity chromatography according to the instructions; the purified protein was stored in a refrigerator at 4°C.

[0097] 7. Enzyme activity detection

[0098] The enzyme activity, optimal temperature and thermal stability of MtLPMO9V wild type (WT) and mutants were determined using 2,6-DMP and H2O2 as co-substrates.

[0099] (1) Prepare 116 mM pH 7.5 phosphate buffer, 10 mM 2,6-DMP solution, and 5 mM H2O2 solution. All solutions should be used within 12 hours of preparation.

[0100] (2) Add 860 μL of phosphate buffer, 100 μL of 2,6-DMP solution, and 20 μL of H2O2 solution into a centrifuge tube, mix thoroughly, and incubate at the corresponding temperature for 15 min.

[0101] (3) LPMO protein sample was added to a final concentration of 1 μM and mixed thoroughly. The activity of LPMO was calculated by measuring the increase in absorbance at 469 nm (ε469 = 53200 L / mol / cm) over 5 min at different temperatures. The residual activity was also determined after incubation at different temperatures and for different times.

[0102] One unit of enzyme activity was defined as the conversion of 2 μmol 2,6-DMP per minute under standard reaction conditions. The relative activity of the mutants was calculated with the activity of the wild-type enzyme as 100%.

[0103] Example 1: Preparation of thermophilic fungal polysaccharide monooxygenase

[0104] 1. Construction of recombinant plasmid vector

[0105] 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.1, and its nucleotide sequence is shown in SEQ ID NO.5.

[0106] The nucleotide sequence shown in SEQ ID NO.5 was inserted between the BamHI restriction site and the NotⅠ restriction site behind the AOXⅠ promoter in the pPIC9K vector, and a His tag was introduced at the C-terminus of the gene for subsequent purification to construct the recombinant plasmid pPIC9K-MtLPMO9V, which was commissioned to Tianlin Biotechnology Wuxi Co., Ltd. for synthesis.

[0107] 2. Expression of the lytic polysaccharide monooxygenase MtLPMO9V

[0108] The recombinant plasmid pPIC9K-MtLPMO9V was digested with SacI restriction enzyme at its optimal temperature for 1 hour and purified using a product recovery kit. The protein was transformed into Pichia pastoris GS115 competent cells, expressed, and purified to obtain the lytic polysaccharide monooxygenase MtLPMO9V. The purified protein was stored at 4°C and analyzed by SDS-PAGE.

[0109] MtLPMO9V (amino acid sequence shown in SEQ ID NO.1) consists of a signal peptide sequence and an enzyme sequence; among them, the first 21 amino acids are the signal peptide, and the amino acid sequence from the 22nd position to the end is the mature enzyme sequence; the amino acid sequence of MtLPMO9V after removing the signal peptide is shown in SEQ ID NO.3.

[0110] The results are as follows Figure 1 As shown, MtLPMO9V is larger than the predicted data at a position around 30 kDa. This difference may be due to the presence of one potential N-glycosylation site and three potential O-glycosylation sites.

[0111] Example 2: Regioselective identification of MtLPMO9V

[0112] The MtLPMO9V prepared in Example 1 was used to determine its oxidative cleavage mode using PASC (phosphoric acid swollen cellulose) as a substrate, as follows:

[0113] 1 μM MtLPMO9V, 2 mM ascorbic acid (AscA) and 4 mg / ml PASC were reacted in 50 mM acetate buffer at 50° C. for 24 h; the oxidation products of the reaction were analyzed by MALDI-TOF / MS.

[0114] The results are as follows Figure 2 As shown, various cellooligosaccharides from DP2 to DP6 were detected as a series of sodium adducts by MALDI-TOF / MS analysis. + (M+Na + Da)], and the oxidation product [M-2+Na + Da]、[M+14+Na + Da] and [M+16+Na + Da] corresponding signal.

[0115] [M-2+Na + The [Da] ion may correspond to either C1 oxidized oligosaccharide (lactone form) or C4 (4-ketoaldose form), but the latter is more acceptable because the δ-lactone is unstable and readily hydrolyzes further to form the aldehyde in MALDI-TOF / MS. [M+16+Na + The Da] ions may represent C1 oxidation products (aldehyde acid form, DPnAA) or C4 oxidation products (glycol form, DPnGM).

[0116] At the same time, the sodium aldehyde adduct [DPnAA+2Na-H] + of [M+38+Na + The absence of [M+14+Na + The [Da] signal may indicate oxidation at both ends of the oligomer, but due to the lack of reliable C1 oxidation characteristics (such as disodium addition signals), it is likely a degradation product. The results show C4 oxidative cleavage activity. Taken together, these results indicate that the regioselectivity of MtLPMO9V for PASC is characterized by C4 oxidative cleavage activity.

[0117] Example 3: Optimum temperature and thermal stability test of MtLPMO9V

[0118] Using the MtLPMO9V prepared in Example 1, the 2,6-DMP rapid enzyme activity method was adopted, and its optimal temperature and thermal stability were determined using 2,6-DMP and H2O2 as co-substrates; the relative enzyme activity of MtLPMO9V at different temperatures and the residual enzyme activity after long-term treatment at different temperatures were respectively detected.

[0119] The results are as follows Figure 3 As shown in the figure, the relative enzyme activity is the percentage of the enzyme activity at the optimal temperature of MtLPMO9V. The optimal temperature of MtLPMO9V is 85°C, and the enzyme activity reaches 477.7U / g (pH 7.5). After incubation at 50°C for 24 hours, it still maintains more than 50% of the original activity, and the enzyme activity reaches 253.2U / g, showing its good thermal stability.

[0120] Example 4: Application of MtLPMO9V in cooperation with cellulase in hydrolysis of cellulose substrates

[0121] 1. Detection of the synergistic effect of MtLPMO9V on cellulase

[0122] The MtLPMO9V prepared in Example 1 was used to detect its synergistic effect with cellulase. The thermophilic endoglucanase DtCelA was expressed in laboratory-preserved E. coli BL21 (DE3) and then ultrasonically crushed to obtain a crude enzyme. + The protein was purified by -NTA nickel column affinity chromatography to obtain the amino acid sequence of DtCelA as shown in SEQ ID NO.2.

[0123] (1) Synergistically hydrolyzes phosphate-swollen cellulose and microcrystalline cellulose by thermophilic endoglucanase DtCelA

[0124] To test the high-temperature synergistic effect of MtLPMO9V and cellulase, MtLPMO9V was used in a synergistic hydrolysis of phosphate-swollen cellulose (PASC) and microcrystalline cellulose (MCC) at 70°C with thermophilic endoglucanase DtCelA for 96 hours. Samples were taken at regular intervals to determine the reducing sugar concentration in the hydrolyzate. The specific method is as follows:

[0125] The enzyme reaction was performed in 5 mL of 50 mM acetate buffer (pH 5) containing 0.5% w / v PASC (2% when using MCC as the substrate), 10 mg / g dry substrate DtCelA, 1 mM ascorbic acid AscA, and 5 mg / g dry substrate MtLPMO9V (1 mg / g dry substrate when using MCC as the substrate). The reaction was incubated on a shaker at 70°C, 180 rpm for 96 hours. 0.4 mL samples were removed at intervals, boiled at 100°C for 10 minutes, and the supernatant was collected by centrifugation at 10,000 rpm for 10 minutes. Reducing sugars in the supernatant were measured using the DNS method.

[0126] The results are as follows Figure 4 As shown, the maximum synergy produced when DtCelA hydrolyzes PASC is 127%, and the maximum synergy produced when DtCelA hydrolyzes MCC is 120%.

[0127] (2) Synergistic cellulase Celluclast 1.5L hydrolyzes MCC and pretreated bagasse substrates

[0128] MtLPMO9V was used in conjunction with Celluclast 1.5L to hydrolyze 2% w / v MCC and pretreated bagasse (acid-hydrolyzed bagasse ac-AGO and alkaline-hydrolyzed bagasse al-AGO) to test the enzymatic hydrolysis rate. The specific steps are as follows:

[0129] The enzyme reaction was carried out in 50 mM acetate buffer, pH 5. The reaction solution included 1 mM gallic acid (no gallic acid was added when the substrate was MCC), 2% w / v substrate, a cellulase mixture with a total concentration of 10 mg / g dry substrate (the total concentration here is: when no MtLPMO9V is added, the total concentration of the cellulase mixture is the concentration of Celluclast; when MtLPMO9V is included, the total concentration is 10 mg / g dry substrate of Celluclast and MtLPMO9V), ​​and MtLPMO9V at different concentrations (0, 0.2, 0.4, 0.75, 1, and 2 mg / g dry substrate) in a shaker at 50°C and 180 rpm for 96 h.

[0130] The results are as follows Figure 5As shown in the figure, different concentrations of MtLPMO9V exhibited different synergistic effects on the three matrices. Among them, MtLPMO9V at 0.4 mg / g dry matrix synergized with Celluclast 1.5L to produce the greatest synergistic effect on the matrices of MCC and ac-AGO, respectively, increasing glucose yields by 22% and 17% compared to no MtLPMO9V addition. For the matrix of al-AGO, MtLPMO9V at only 0.2 mg / g dry matrix produced the greatest synergistic effect with Celluclast, increasing glucose yield by 28% compared to no MtLPMO9V addition (Table 1).

[0131] Table 1 Glucose yields produced by hydrolysis of three substrates in 96 h

[0132]

[0133] 2. Comparison of MtLPMO9V performance with existing LPMOs in the literature

[0134] The MtLPMO9V in Example 1 was compared with the LPMO reported in the existing literature in terms of dosage and performance. The results are shown in Table 2.

[0135] The results show that compared with the enzymes reported in existing literature, MtLPMO9V can achieve a better improvement in the enzymatic hydrolysis effect for the same substrate while significantly reducing the dosage.

[0136] Table 2 Comparison results of dosage and performance

[0137]

[0138]

[0139] Example 5: Construction of MtLPMO9V mutant

[0140] 1. Construction of mutants

[0141] The plasmid pPIC9K-MtLPMO9V constructed in Example 1 was taken, and primers were designed to perform point mutations thereon. The primers and mutant names are shown in Table 3.

[0142] MtLPMO9V (amino acid sequence shown in SEQ ID NO. 1) consists of a signal peptide sequence and an enzyme sequence; the first 21 amino acids constitute the signal peptide (i.e., MRYYFLQLAAAAAFAVNSAAG), while the mature enzyme sequence begins and ends at amino acid position 22. Therefore, position 22 serves as the first amino acid of the wild-type (WT) lytic polysaccharide monooxygenase. Its amino acid sequence is shown in SEQ ID NO. 3, and its nucleotide sequence is shown in SEQ ID NO. 4.

[0143] According to "amino acid before mutation + mutation site + amino acid after mutation", the mutants were named as shown in Table 3. For example, G32S means that based on SEQ ID NO. 3, glycine (G) at position 32 was mutated to serine (S); A170C / A175C means that based on SEQ ID NO. 3, alanine (A) at position 170 was mutated to cysteine ​​(C), and alanine (A) at position 175 was mutated to cysteine ​​(C).

[0144] Table 3 Mutant and primer sequences

[0145]

[0146]

[0147] 2. Detection of mutant activity

[0148] The relative enzyme activity of the mutants was detected according to the method in Example 3. Figure 6 shown.

[0149] The results showed that under standard enzyme activity detection conditions (30℃ pH 7.5), the relative enzyme activities of mutants G32S, P107L, Q120Y, Q137I, A154P, and D160G were all increased by more than 50% compared with the wild type (WT). Among them, the enzyme activity of the G32S mutant was increased by more than 65%, reaching 56.7 U / g (30℃ pH 7.5, WT is 34.2 U / g).

[0150] 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 in the scope of protection of the present invention.

[0151] Sequence Listing used in the present invention

[0152] MtLPMO9V original amino acid sequence SEQ ID NO.1:

[0153] MRYYFLQLAAAAAFAVNSAAGHYIFQQFATGGTKYPPWKYIRRNTNPDWLQNGPVTDLSSTDLRCNVGGQVSNGTETITLDAGDEFSFILDTPVYHAGPTSLYMSKAPGAVADYDGGGAWFKIYDWGPSGTSWTLSGTYTQRIPKCIPDGEYLLRIQQIGLHNPGAAPQFYISCAQVKVVDGGSTNPTPTAQIPGAFHSNDPGLTVNIYNDPLTNYVVPGPRVFSC

[0154] Initial amino acid sequence of DtCelA SEQ ID NO.2:

[0155] MNNLPIKRGINFGDALEAPYEGAWSGYIIKDEYFKIVKDAGFDHVRIPIKWSVYTQKEAPYSIEKRIFDRVDHLIEEGLKNNLHVIINIHHYEEIMEDPLGEKERFLAIWRQISEHYKDYPNNLYFELLNEPTQNLSSELWNQFLKEAIEVIRRTNPERKIIVGPDNWNSLYNLEKLIIPENDENIIITFHYYNPFPFTHQGAGWVKIDLPVGVKWLGTEEEKREIERELDMAVSWAEEHGNIPLYMGEFGAYSKADMESRVRWTDFVARSAEKRGIAWSYWEFYSGFGVFDPEKNEWRTPLLRALIPERNI

[0156] Initial amino acid sequence of MtLPMO9V (signal peptide removed) SEQ ID NO.3:

[0157] HYIFQQFATGGTKYPPWKYIRRNTNPDWLQNGPVTDLSSTDLRCNVGGQVSNGTETITLDAGDEFSFILDTPVYHAGPTSLYMSKAPGAVADYDGGGAWFKIYDWGPSGTSWTLSGTYTQRIPKCIPDGEYLLRIQQIGLHNPGAAPQFYISCAQVKVVDGGSTNPTPTAQIPGAFHSNDPGLTVNIYNDPLTNYVVPGPRVFSC

[0158] Nucleotide sequence of MtLPMO9V (signal peptide removed) SEQ ID NO.4:

[0159] CATTACATTTTTCAGCAGTTCGCAACAGGAGGTACTAAATATCCACCCTGGAAGTATATTCGTCGAAATACGAATCCTGACTGGTTGCAAAACGGCCCTGTTACCGACCTTAGTTCCACGGACCTACGATGCAATGTTGGAGGTCAAGTCTCAAATGGTACAGAGACGATCACTTTAGATGCAGGTGACGAGTTTAGTTTCATCCTAGATACACCAGTTTACCACGCTGGTCCAACAAGTTTGTACATGTCAAAAGCCCCTGGAGCTGTTGCTGACTATGATGGTGGAGGAGCATGGTTCAAGATTTACGATTGGGGACCTTCAGGAACTTCTTGGACCTTGAGTGGTACCTACACCCAGAGAATCCCCAAGTGCATTCCAGACGGTGAGTATCTGTTGAGAATACAACAAATTGGTTTGCATAACCCCGGTGCTGCTCCTCAGTTTTACATTTCCTGTGCCCAGGTTAAGGTCGTGGACGGTGGCTCAACTAACCCTACACCAACTGCTCAAATTCCTGGTGCATTTCATTCCAATGATCCCGGTCTGACCGTTAACATTTACAACGATCCCCTAACTAACTACGTGGTACCAGGTCCTAGAGTTTTCAGTTGC

[0160] Nucleotide sequence of MtLPMO9V with signal peptide SEQ ID NO.5:

[0161] ATGAGGTACTACTTCTTGCAATTGGCTGCTGCAGCTGCTTTTGCTGTTAACTCTGCTGCAGGTCATTACATTTTTCAGCAGTTCGCAACAGGAGGTACTAAATATCCACCCTGGAAGTATATTCGTCGAAATACGAATCCTGACTGGTTGCAAAACGGCCCTGTTACCGACCTTAGTTCCACGGACCTACGATGCAATGTTGGAGGTCAAGTCTCAAATGGTACAGAGACGATCACTTTAGATGCAGGTGACGAGTTTAGTTTCATCCTAGATACACCAGTTTACCACGCTGGTCCAACAAGTTTGTACATGTCAAAAGCCCCTGGAGCTGTTGCTGACTATGATGGTGGAGGAGCATGGTTCAAGATTTACGATTGGGGACCTTCAGGAACTTCTTGGACCTTGAGTGGTACCTACACCCAGAGAATCCCCAAGTGCATTCCAGACGGTGAGTATCTGTTGAGAATACAACAAATTGGTTTGCATAACCCCGGTGCTGCTCCTCAGTTTTACATTTCCTGTGCCCAGGTTAAGGTCGTGGACGGTGGCTCAACTAACCCTACACCAACTGCTCAAATTCCTGGTGCATTTCATTCCAATGATCCCGGTCTGACCGTTAACATTTACAACGATCCCCTAACTAACTACGTGGTACCAGGTCCTAGAGTTTTCAGTTGC

Claims

1. Use of a lytic polysaccharide monooxygenase in hydrolyzing cellulose, characterized in that: The amino acid sequence of the lytic polysaccharide monooxygenase is shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that: The lytic polysaccharide monooxygenase can be obtained by heterologous expression.

3. The use according to claim 2, characterized in that: Plasmid vectors used for heterologous expression include pPIC series vectors, pPICZ series vectors, pPICZα and pET series vectors.

4. The use according to claim 2, characterized in that: Host cells used for heterologous expression include Pichia pastoris, Escherichia coli, Saccharomyces cerevisiae, Aspergillus niger, and Aspergillus oryzae.

5. The use according to claim 1, characterized in that: The cellulose includes pretreated bagasse, phosphoric acid swollen cellulose and microcrystalline cellulose.

6. A method for hydrolyzing cellulose, characterized in that: A lytic polysaccharide monooxygenase is mixed with a cellulase to synergistically hydrolyze cellulose; the amino acid sequence of the lytic polysaccharide monooxygenase is shown in SEQ ID NO.

3.

7. The method according to claim 6, characterized in that The cellulase comprises thermophilic endoglucanase or commercial cellulase; Optionally, the amino acid sequence of the thermophilic endoglucanase is shown in SEQ ID NO.2; Alternatively, commercial cellulases include Celluclast or Ctec2; Optionally, the cellulose comprises pretreated bagasse, phosphoric acid swollen cellulose, microcrystalline cellulose.

8. The method according to claim 6, characterized in that The added amount of the lytic polysaccharide monooxygenase is 0.2-2 mg / g dry substrate; the added amount of the cellulase is 4-10 mg / g.

9. The method according to claim 6, characterized in that The amount of cellulose added is 0.5-2% w / v.

10. The method according to claim 6, characterized in that The enzymatic hydrolysis conditions are 45-50°C for 12-96 hours.

Images