Cracking polysaccharide monooxygenase mutant and application thereof

Abstract

The invention discloses a cleavable polysaccharide monooxygenase mutant and application thereof, which can increase the enzymatic activity of the cleavable polysaccharide monooxygenase mutant under the condition of not damaging the original thermal stability, contribute to reducing the enzyme amount in application, and are more economical. Under the reaction conditions that the temperature is 50 DEG C and the pH value is 6.0, the enzyme activity of the mutant TaLPMO-E160V is about 2.8 times that of a wild type, and the mutant TaLPMO-E160V shows remarkably enhanced catalytic efficiency. The mutant is suitable for the degradation process of lignocellulose, can effectively improve the degradation efficiency and reduce the use cost of an enzyme preparation, and has excellent industrial application prospects.

Description

(I) Technical Field

[0001] This invention belongs to the field of enzymology, specifically relating to a mutant of cleaving polysaccharide monooxygenase (LPMO) with enhanced catalytic activity and its applications. (II) Background Technology

[0002] Biomass energy, as a renewable carbon source, is the fourth largest energy source after coal, oil, and natural gas. The development and utilization of biomass energy is the most promising solution to address global climate change, energy shortages, and environmental pollution. my country is a major agricultural country with hundreds of millions of tons of usable crop straw resources annually. Straw, wood, and other lignocellulose are the largest existing biomass resources on Earth, possessing enormous potential for conversion into green biofuels and value-added chemical raw materials. Their high-value and resource-based utilization plays a crucial role in maintaining the global carbon cycle and the development of the biorefining industry. Cellulose is the main component of lignocellulose, and its complex crystal structure restricts the efficiency of enzymatic hydrolysis. In 2010, it was discovered that LPMO, with the participation of oxygen and reducing agents, can catalyze the oxidative depolymerization of cellulose, and the relevant research results were published in the journal *Science*. This discovery opened a new avenue for the enzymatic degradation of lignocellulose. Recent studies have further shown that LPMO destroys the crystalline structure of cellulose through oxidative cleavage (…). Figure 1 This loose structure provides more binding sites for glycoside hydrolases, playing a crucial role in the efficient enzymatic hydrolysis of cellulose. It is a cellulose-degrading enzyme with great development potential.

[0003] LPMOs are widely derived, with up to eight helper activators discovered from bacteria, archaea, and eukaryotes. However, their structure is highly conserved, featuring a core β-sandwich structure containing 10 typical β-sheets linked by loops. The active site consists of two conserved histidine residues, one tyrosine residue, and one copper ion. The copper ion is attached to the side chains of the two histidine residues and the nitrogen atom at the amino terminus of one of the histidine residues, forming a "T"-shaped structure known as the histidine scaffold. Unlike other cellulases, the active site of LPMOs is planar, which may be related to their large substrates (such as crystalline cellulose).

[0004] Although existing studies have reported the crystal structure and structure-activity relationship of LPMO, its weak affinity for substrates leads to low catalytic activity, making it difficult to meet the demands of industrial applications. This has become a major technical bottleneck for the large-scale application of LPMO in biomass conversion. Developing high-performance enzyme preparations is crucial for achieving efficient and economical conversion of lignocellulose. To address these issues, and to obtain LPMO with high catalytic activity that meets the requirements of industrial applications, thereby fulfilling the need for efficient and robust industrial biocatalysis, molecular modification of existing LPMO using protein engineering technology has become an important strategy for improving its catalytic performance and promoting its industrial application. (III) Summary of the Invention

[0005] The purpose of this invention is to provide a mutant of a highly efficient polysaccharide monooxygenase that degrades lignocellulose and its application, thereby solving the problem of low activity of existing polysaccharide monooxygenases.

[0006] The technical solution adopted in this invention is:

[0007] In a first aspect, the present invention provides a mutant of a cleavable polysaccharide monooxygenase that efficiently degrades lignocellulose, the amino acid sequence of which is shown in SEQ ID NO.3.

[0008] The present invention also relates to the gene encoding the cleavable polysaccharide monooxygenase mutant, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0009] Secondly, the present invention also provides a recombinant plasmid expressing the gene encoding the cleavable polysaccharide monooxygenase mutant, and a recombinant genetically engineered bacterium constructed from the recombinant plasmid, wherein the recombinant plasmid is a pET28a vector, and the recombinant genetically engineered bacterium is a pET28a vector. E.coli BL21(DE3) is the host bacterium.

[0010] Thirdly, the present invention provides an application of the cleavage polysaccharide monooxygenase mutant in the degradation of lignocellulose.

[0011] Furthermore, the application uses copper-saturated pure enzyme prepared from the pure enzyme extracted after induced culture of recombinant genetically engineered bacteria expressing the cleaving polysaccharide monooxygenase mutant as a catalyst, phosphate-swellable cellulose (PASC) as a substrate, ascorbic acid and cellulase are added, and a pH 4-7 buffer solution is used as the reaction medium to form a reaction system. The reaction is carried out at 40-55℃ and 100-300 rpm (preferably 50℃ and 200 rpm) to achieve the degradation of lignocellulose.

[0012] Furthermore, in the reaction system, the final concentration of the substrate is 0.1-6 g / mL (preferably 0.16 g / mL), the amount of catalyst added is 0.1-6 mg / mL (preferably 0.14 mg / mL) based on the protein content; the final concentration of the ascorbic acid is 0.5-3 mM (preferably 2 mM); and the final concentration of the cellulase volume is 0.08-0.4%.

[0013] Furthermore, the reaction medium is preferably a pH 6, 200mM phosphate buffer.

[0014] Further, the preparation of phosphate-swellable cellulose (PASC) was carried out as follows: microcrystalline cellulose was added to ultrapure water and mixed thoroughly. Pre-cooled 85% H3PO4 aqueous solution was added, and the mixture was stirred vigorously and allowed to stand for 5 min. Pre-cooled 85% H3PO4 solution was then added, and the mixture was stirred vigorously until it became transparent. After standing at 4°C overnight, ice water was added, and the mixture was stirred vigorously until a white turbid precipitate formed. The precipitate was centrifuged at 8000 r / min at 4°C for 20 min, and the supernatant was discarded. The process of adding ice water and centrifuging was repeated three times. Phosphoric acid was neutralized with 2M NaCO3 aqueous solution, and the cellulose was resuspended in ice water. The mixture was centrifuged at 8000 r / min at 4°C for 20 min, and the supernatant was discarded. The process of adding ice water and centrifuging was repeated twice, and the pH was adjusted to 6.0. The precipitate was collected to obtain phosphate-swellable cellulose. The volume of ultrapure water used was 3 mL / g based on the mass of microcrystalline cellulose; the total volume of 85% H3PO4 aqueous solution used was 3.5 mL / g based on the mass of microcrystalline cellulose, and the volume ratio of the two additions was 5:2.

[0015] Furthermore, the purified enzyme is prepared according to the following method:

[0016] (1) The recombinant genetically engineered bacteria expressing the lysin monooxygenase mutant were inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance and cultured at 37°C for 12 h. Then, they were inoculated into fresh LB liquid medium containing 50 μg / mL kanamycin resistance at a volume concentration of 1% and cultured at 37°C until the bacterial cell concentration reached OD500. 600 When the concentration is 0.6-0.8, add IPTG to a final concentration of 0.5 mM, induce culture at 26℃ for 12 h, centrifuge the culture medium at 4℃ and 8000 r / min for 10 min, discard the supernatant, collect the precipitate, and obtain wet bacterial cells containing recombinant lysine monooxygenase.

[0017] (2) Protein purification: The wet bacterial cells were resuspended in binding buffer (pH 8.0, 10 mM imidazole, 200 mM NaCl, 40 mmol / L Tris-HCl buffer) and then sonicated for 30 min. The sonication power was 400 W, the sonication time was 3 s and the interval was 4 s. The mixture was centrifuged at 8000 r / min for 10 min at 4 ℃ to obtain the crude enzyme solution.

[0018] (3) The crude enzyme solution was separated and purified using a Ni-NTA 6FF pre-packed gravity column. First, the column was equilibrated with binding buffer (pH 8.0, 10 mM imidazole, 200 mM NaCl, 40 mmol / L Tris-HCl buffer), and then the crude enzyme solution was loaded with 10 column volumes. Next, the impurities were eluted with washing buffer (pH 8.0, 50 mM imidazole, 200 mM NaCl, 40 mmol / L Tris-HCl buffer), and finally the target protein was eluted with elution buffer (pH 8.0, 250 mM imidazole, 200 mM NaCl, 40 mmol / L Tris-HCl buffer) to obtain the pure enzyme.

[0019] Furthermore, the copper-saturated pure enzyme was prepared as follows: the pure enzyme was equilibrated in 100 mM, pH 5.0 sodium acetate buffer at room temperature for 5 h; the equilibrated enzyme solution was placed in 100 mM, pH 5.0 sodium acetate buffer containing 1 mM CuSO4 and incubated at room temperature for 30 min; dialyzing was then performed in 100 mM, pH 5.0 sodium acetate buffer (molecular weight cutoff of 8 kDa) for 24 h to remove excess Cu. 2+ The dialysis solution was changed every 8 hours; the dialyzed enzyme solution (retained solution) was aliquoted into 1.5 mL centrifuge tubes to obtain copper-saturated pure enzyme.

[0020] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0021] This invention provides a highly efficient cleaving polysaccharide monooxygenase mutant for degrading lignocellulose. It increases enzyme activity without compromising the original thermal stability, thus reducing the amount of enzyme needed in applications and making it more economical. Under reaction conditions of 50°C and pH 6.0, the mutant… The The enzyme activity of LPMO-E160V is approximately 2.8 times that of the wild type, exhibiting significantly enhanced catalytic efficiency. This mutant is suitable for the degradation of lignocellulose, effectively improving degradation efficiency and reducing enzyme preparation costs, demonstrating excellent prospects for industrial applications. (iv) Description of the attached drawings

[0022] Figure 1 A schematic diagram of the degradation of the three components of lignocellulose.

[0023] Figure 2 Recombinant plasmid pET28a- The Plasmid map of LPMO.

[0024] Figure 3 , The LPMO-WT and TheReducing sugar concentration (a) and relative enzyme activity (b) of LPMO-E160V.

[0025] Figure 4 , The LPMO-WT and The Thermostability diagram of LPMO-E160V mutant at 50℃.

[0026] Figure 5 pH The LPMO-WT and The The effect of LPMO-E160V on enzyme activity.

[0027] Figure 6 Metal ion pairs The LPMO-WT and The The effect of LPMO-E160V on enzyme activity. (V) Detailed Implementation Methods

[0028] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0029] Unless otherwise specified, the methods in the embodiments of this invention are conventional methods; the experimental materials used are all conventional materials, and unless otherwise specified, they are all purchased from commercial channels.

[0030] The culture media and reagents involved in the embodiments of this invention are as follows:

[0031] (1) LB liquid medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, solvent is deionized water. If preparing solid medium, 15 g / L-20 g / L of agar powder needs to be added.

[0032] (2) Preparation and storage of kanamycin (Kan): Dissolve 50 mg Kan in 1 mL of sterile water. Filter sterilize using a syringe and a 0.22 µm pore size aqueous filter. Aliquot into 1.5 mL EP tubes to form a 50 mg / mL stock solution. Store at -20°C for later use.

[0033] (3) Preparation and storage of isopropyl-β-D-thiogalactoside (IPTG): Dissolve 0.238 g of IPTG powder in 10 mL of ultrapure water to prepare a 0.1 mM solution. Filter the solution sterile using a syringe and a 0.22 µm pore size aqueous filter. Aliquot into 1.5 mL EP tubes. Store at -20°C for later use.

[0034] (4) Preparation and storage of phosphate-buffered saline (PBS): Dissolve PBS tablets in 100 mL of ultrapure water and then sterilize at 115 °C for 30 min. Store at room temperature.

[0035] Example 1: Cleavage Polysaccharide Monooxygenase The Obtaining the LPMO gene, constructing and expressing the starting strain

[0036] 1. The Acquisition of the LPMO gene

[0037] According to the NCBI database, it originates from thermophilic ascomycetes. The The LPMO gene sequence (ID: MK359139.1) was obtained through optimization and synthesis of the target gene by Qingke Biotechnology Co., Ltd. The The LPMO gene has the nucleotide sequence shown in SEQ ID NO.2 and the amino acid sequence of the encoded protein shown in SEQ ID NO.1, with a histidine tag added to the C-terminus.

[0038] 2. Construction of the starting strain

[0039] (1) Preparation of competent states: E.coli BL21(DE3) was activated on LB agar plates and incubated overnight at 37°C. Single colonies were selected from the plates and inoculated into 5 mL of LB liquid medium, incubated overnight at 37°C. Seed culture was then inoculated into 50 mL of fresh LB liquid medium at a concentration of 1% by volume and incubated at 37°C until OD500 reached. 600 The concentration should be 0.3-0.4. The bacterial culture was placed on ice and cooled for 10 min, shaking every 2 min during this period. The cooled bacterial culture was transferred to sterilized 50 mL centrifuge tubes and centrifuged at 4000 r / min, 4°C for 10 min. The supernatant was discarded, and 10 mL of pre-chilled 0.1 M CaCl2 aqueous solution was added. The mixture was gently mixed and then placed on ice again for 15 min. The culture was centrifuged again at 4000 r / min, 4°C for 10 min, and the supernatant was discarded. 10 mL of pre-chilled 0.1 M CaCl2 aqueous solution containing 10% glycerol was added and gently mixed. The culture was aliquoted into 100 µL tubes and stored at -80°C for later use.

[0040] (2) Construction of the starting strain: The The LPMO gene was inserted between the NcoI / XhoI sites in plasmid pET28a to construct the intracellular expression recombinant plasmid pET28a- The LPMO ( Figure 2 Transformed competent cells were introduced via heat shock. E. coli BL21(DE3) was plated on LB agar plates containing 50 µg / mL kanamycin resistance and incubated overnight at 37°C with the plates inverted. Positive colonies were picked and verified by colony PCR and sequencing to obtain the originating strain. E. coliBL21(DE3) / pET28a- The LPMO, denoted as WT. A control strain containing an empty plasmid was also constructed. E. coli BL21(DE3) / pET28a.

[0041] 3. Expression and purification of recombinant proteins

[0042] (1) Recombinant protein expression: The starting strain E.coli BL21(DE3) / pET28a- The LPMO was inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance and cultured at 37°C for 12 h. Then, it was inoculated at a 1% (v / v) inoculation rate into fresh LB liquid medium containing 50 μg / mL kanamycin resistance and cultured at 37°C until the bacterial concentration reached OD500. 600 When the concentration is 0.6-0.8, add IPTG to a final concentration of 0.5 mM, induce culture at 26℃ for 12 h, centrifuge the culture medium at 4℃ and 8000 r / min for 10 min, discard the supernatant, collect the precipitate, and obtain wet bacterial cells containing recombinant lysin monooxygenase.

[0043] (2) Crude enzyme solution: The wet bacterial cells were resuspended in binding buffer (pH 8.0, 10 mM imidazole, 200 mM NaCl, 40 mmol / L Tris-HCl buffer) at a concentration of 20 g / L and then sonicated for 30 min. The sonication power was 400 W, the sonication time was 3 s and the interval was 4 s. The mixture was centrifuged at 8000 r / min for 10 min at 4℃ to obtain the crude enzyme solution.

[0044] (3) Protein purification: The crude enzyme solution was purified using a Ni-NTA 6FF pre-packed gravity column. The column was first equilibrated with binding buffer (pH 8.0, 10 mM imidazole, 200 mM NaCl, 40 mmol / L Tris-HCl buffer), and then the crude enzyme solution was loaded at a volume of 10 column volumes. Next, impurities were eluted with washing buffer (pH 8.0, 50 mM imidazole, 200 mM NaCl, 40 mmol / L Tris-HCl buffer), and finally, the target protein was eluted with elution buffer (pH 8.0, 250 mM imidazole, 200 mM NaCl, 40 mmol / L Tris-HCl buffer) to obtain the purified enzyme. The protein concentration was then measured and recorded as follows: The LPMO-WT (wild type).

[0045] 4. Determination of monooxygenase activity in cleaving polysaccharides

[0046] (1) Preparation of Phosphate-Swellable Cellulose (PASC): Weigh 0.2 g of microcrystalline cellulose (Avicel series, model PH101) into a 50 mL centrifuge tube and add 0.6 mL of ultrapure water and mix well. Add 5 mL of pre-cooled 85% H3PO4 aqueous solution, stir vigorously, and let stand for 5 min. Continue to add 2 mL of pre-cooled 85% H3PO4 solution and stir vigorously until it becomes transparent. After standing at 4℃ overnight, add 40 mL of ice water and stir vigorously until a white turbid precipitate forms. Centrifuge at 8000 r / min at 4℃ for 20 min and discard the supernatant. Repeat the addition of ice water and centrifugation 3 times, then add 3 mL of 2M NaCO3 aqueous solution to neutralize the phosphoric acid. Add 45 mL of ice water to resuspend the cellulose. Centrifuge at 8000 r / min at 4℃ for 20 min and discard the supernatant. Repeat the addition of ice water and centrifugation 2 times, adjust the pH to 6.0, collect the precipitate, and obtain 4 g of phosphate-swellable cellulose, which is stored at 4℃ for later use.

[0047] (2) Copper saturation of LPMO protein: The 2.5 mL of LPMO purified enzyme solution (protein concentration 0.2 mg / mL) was equilibrated in 3.5 mL of 100 mM, pH 5.0 sodium acetate buffer at room temperature for 5 h. The equilibrated enzyme solution was then placed in 3.5 mL of 100 mM, pH 5.0 sodium acetate buffer containing 1 mM CuSO4 and incubated at room temperature for 30 min. Dialysis was then performed in 100 mM, pH 5.0 sodium acetate buffer (molecular weight cutoff 8 kDa) for 24 h to remove excess Cu. 2+ The dialysis buffer was changed every 8 hours. The dialyzed enzyme solution (withheld fluid) was aliquoted into 1.5 mL centrifuge tubes to obtain 2.5 mL of copper-saturated solution. The LPMO purified enzyme, adjusted to a protein concentration of 1.0 mg / mL with 100 mM, pH 5.0 sodium acetate buffer, and stored at -20℃ for later use.

[0048] (3) Standard conditions for enzyme activity determination of cleaving polysaccharide monooxygenase: Prepare the reaction system according to Table 1, and react at 50℃ and 200 r / min for 24 h. Take 1 mL of sample, centrifuge at 10000 r / min for 5 min, collect the supernatant, and determine the glucose content using a glucose content detection kit (manufacturer Solarbio, batch number BC2500).

[0049] Enzyme activity is defined as the amount of enzyme required to degrade PASC to produce 1 μmol of reducing sugar (calculated as glucose) per minute under standard reaction conditions (usually 50°C and pH 6). One enzyme activity unit (U) is defined as the amount of enzyme required to degrade PASC to produce 1 μmol of reducing sugar (calculated as glucose) per minute.

[0050] Table 1: Synergistic Reaction System of LPMO and Cellulase

[0051]

[0052] 5. Detection of reducing sugar release

[0053] The reducing sugar content was determined using a glucose content assay kit (Solarbio). The steps are as follows: First, preheat the spectrophotometer for at least 30 minutes, adjust the wavelength to 505 nm, and zero the instrument with distilled water. Second, mix equal volumes of reagents two and three from the kit (prepare fresh before use). Finally, perform the sample analysis. Dilute the supernatant of the sample 10 times with distilled water and mix all components thoroughly according to Table 2 below.

[0054] Table 2: Reducing Sugar Determination System

[0055]

[0056] After reacting each group in a 37℃ incubator for 15 min, the absorbance A was read at a wavelength of 505 nm and recorded as Ablank, Astandard, and Adetermined, respectively. Calculate ΔAdetermined = Adetermined - Ablank, Δstandard = Astandard - Ablank. Glucose content (μmol / g) = ΔAdetermined / Δstandard Dilution factor

[0057] Example 2 The Construction and screening of LPMO mutants

[0058] 1. Screening for mutation sites

[0059] Using Discovery Studio software The The LPMO protein docks with the substrate molecule, defining the substrate binding plane. Based on the location of the active site, this planar structure is determined to consist of four loops. Virtual saturation mutations were then performed on sites within these four loops, selecting the 50 mutants with the lowest mutation energies. Simultaneously, the EVOLVEpro AI model was used to predict these 50 sites. Combining these predictions, repetitive sites were selected for site-directed mutagenesis, ultimately choosing site 160 for mutation.

[0060] 2. Construction of mutants

[0061] Based on the site to be mutated, corresponding primers were designed and synthesized. Plasmids from the starting strain of Example 1 were extracted using a plasmid extraction kit (Shanghai Bioengineering Co., Ltd.) with pET28a- TheUsing the LPMO whole plasmid as a template, whole plasmid PCR amplification was performed using 2×Phanta Master Mix high-fidelity polymerase (Vayme Biotechnology Co., Ltd.). The reaction system preparation is shown in Table 4. The annealing temperature was adjusted according to the primer Tm value, and the extension time was adjusted according to the length of the target gene fragment (1 min can amplify 1-2 Kb fragment). After the PCR product was positive by 10% agarose gel electrophoresis, it was purified using a purification kit (manufacturer: Vazyme, model DC301) before subsequent experimental operations.

[0062] Table 3: Primers

[0063]

[0064] Table 4: PCR amplification system

[0065]

[0066] 3. Screening for mutants

[0067] The purified PCR products were converted to different PCR products by heat shock. E.coli BL31(DE3) competent cells were plated on LB agar plates containing 50 µg / mL natriuretic peptide and incubated overnight at 37°C with the plates inverted. Positive colonies were picked and verified by colony PCR and sequencing to obtain the mutant strain. E.coli BL31(DE3)-pET28a- The LPMO-E160V, denoted as E160V.

[0068] The starting strain WT, control strain, and mutant strain E160V were induced to express enzymes using the method described in Example 1. The enzyme activity and reducing sugar release (i.e., glucose content) of WT and E160V were then measured, with the crude enzyme solution of the control strain added as a control. The results are shown in [Figure 1]. Figure 3 .

[0069] like Figure 3 As shown, in experiments degrading PASC substrates, the following were evaluated: The LPMO-WT (wild type) and The The synergistic effect of LPMO-E160V (mutant) and cellulase. When cellulase alone is used, the amount of reducing sugar released after 24 hours of reaction is approximately 10.05 μmol / mL. However, when wild-type cellulase is added... The LPMO-WT or mutant TheAfter LPMO-E160V was applied, the reducing sugar release increased to approximately 12.96 μmol / mL and 18.36 μmol / mL, respectively. Based on the wild-type co-enzyme activity as 100%, the co-enzyme activity of the mutant E160V was approximately 2.8 times that of the wild-type, confirming its superior performance in improving the degradation efficiency of lignocellulose. Superior mutants were screened to obtain... The LPMO-E160V, nucleotide sequence as shown in SEQ ID NO.4, amino acid sequence as shown in SEQ ID NO.3.

[0070] SEQ ID NO. 3

[0071] MHGFVQNIVIDGKKYVIARRNQYPYMSNPPEVIAWSTTATDLGFVDGTGYQTPDIICHRGAKPGALTAPVSPGGTVELQWTPWPDSHHGPVINYLAPCNGDCSTVDKTQLEFFK IAESGLINDDNPPGIWASDNLIAANNSWTVTIPTTIAPGNYVLRHVIIALHSAQNQDGAQNYPQCINLQVTGGGSDNPAGTLGTALYHDTDPGTLINIYQKLSSYIIPGPPLYTG

[0072] Example 3: Enzymatic Properties Analysis

[0073] 1. Horseradish peroxidase assay to determine the enzyme activity of LPMO

[0074] Table 5 Reaction System

[0075]

[0076] Experimental group: Add each component from Table 5 above to a black-background 96-well plate and mix thoroughly. Then add 17 μL of ascorbic acid (50 μM) and detect the sample at 30°C for 1 h using a microplate reader under the conditions of excitation wavelength (Ex) 560 nm and emission wavelength (Em) 590 nm. Simultaneously... The The purified LPMO enzyme was replaced with the crude enzyme solution of the control strain as a control group. The obtained fluorescence intensity data were fitted, and the slope of the linear phase in the early stage of the reaction was calculated. The slopes of the experimental group and the control group were compared.

[0077] Enzyme activity is defined as the amount of enzyme required to produce 1 μmol of H2O2, calculated from the fluorescence increment (ΔRFU / min) generated per minute under standard reaction conditions and converted using the H2O2 standard curve.

[0078] 2. Temperature tolerance:

[0079] The method prepared in Example 1 The LPMO-WT (wild type) and The The purified LPMO-E160V (mutant) enzyme solution was diluted with phosphate buffer (20 mM, pH 6.0) to a protein concentration of 0.2 mg / mL. After incubation at 80 °C for 10, 20, 30, 40, 50 and 60 min, the residual enzyme activity was determined using the method in step 1. The residual activity was calculated with the untreated group (i.e., unincubated) as 100%.

[0080] The results are as follows Figure 4 As shown, The The thermal stability of LPMO9-E160V at 80℃ is similar to that of the wild type. Specifically, the residual activity of both the wild type and the mutant was approximately 50% after treatment at 80℃ for 30 min. In traditional protein modification, there is often a trade-off between catalytic efficiency and thermal stability; mutations typically increase specific activity but also negatively impact thermal stability. However, the E160V mutant still exhibits a significant increase in enzyme activity compared to the wild type without compromising thermal stability.

[0081] 3. Optimal pH:

[0082] Buffer solutions with different pH values ​​were prepared: 50 mM phosphate buffer (pH 5.0, 6.0, 7.0), 50 mM Tis-HCl buffer (pH 8.0, 9.0), and 50 mM citrate buffer (pH 3.0, 4.0). The buffer solution prepared using the method in Example 1 was then used. The LPMO-WT (wild type) and The The pure enzyme solution of LPMO-E160V (mutant) was diluted with phosphate buffer (20mM, pH6.0) to a protein concentration of 0.2mg / mL. The enzyme activity was detected using the method in step 1, and the data with the highest activity was used as 100% for relative activity analysis.

[0083] The results are as follows Figure 5 As shown, The LPMO9A-WT and The The optimal pH values ​​for LPMO9A and E160V are similar, both around 6.

[0084] 4. The effects of different metal ions on enzyme activity:

[0085] Prepare sulfate aqueous solutions of different metal ions (Zn) 2+ Cu 2+ Co 2+ Ni 2+ Ca 2+ K + Mn 2+ Mg2+ The sample prepared using the method of Example 1 will be used. The LPMO-WT (wild type) and The The purified LPMO-E160V (mutant) enzyme solution was diluted with phosphate buffer (20 mM, pH 6.0) to a protein concentration of 0.2 mg / mL. Sulfate solutions of different metal ions were added to each component to bring the final concentration of each metal ion to 0.5 mM. Enzyme activity was determined using the method in step 1, and the data with the highest activity was used as 100% for relative activity analysis.

[0086] The results are as follows Figure 6 As shown, most metal ions can positively influence protease activity by improving enzyme stability, forming metal bonds with functional groups, and promoting hydrogen bonding. To investigate the effects of different metal ions on the activity of cleaving polysaccharide monooxygenase (LPMO), this invention added various metal ions to the reaction system for testing. Experimental results showed that Zn... 2+ Cu 2+ Co 2+ Ni 2+ Ca 2+ K + Mn 2+ Mg 2+ All of them have a promoting effect on enzyme activity, among which Mg 2+ The promoting effect of K is the most significant. + It has a slight activating effect. The above-mentioned metal ions have a generally consistent trend in their influence on the activity of WT and E160V, but differ in the degree of promotion: K + The promoting effect on wild type is stronger, while Zn 2+ The promoting effect on mutant E160V is even better.

Claims

1. A mutant of a highly efficient polysaccharide monooxygenase that degrades lignocellulose, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.

3.

2. A recombinant genetically engineered bacterium expressing the gene encoding the cleavage polysaccharide monooxygenase mutant of claim 1.

3. The application of the cleavable polysaccharide monooxygenase mutant of claim 1 in the degradation of lignocellulose.

4. The application as described in claim 3, characterized in that, The application uses copper-saturated pure enzyme prepared from the pure enzyme extracted after induction culture of recombinant genetically engineered bacteria expressing the cleavage polysaccharide monooxygenase mutant as a catalyst, phosphate-swollen cellulose as a substrate, ascorbic acid and cellulase are added, and a pH 4-7 buffer solution is used as the reaction medium to form a reaction system. The reaction is carried out at 40-55℃ and 100-300 rpm to achieve the degradation of lignocellulose.

5. The application as described in claim 4, characterized in that, In the reaction system, the final concentration of the substrate is 0.1-6 g / mL, the amount of catalyst added is 0.1-6 mg / mL based on protein content, the final concentration of ascorbic acid is 0.5-3 mM, and the final concentration of cellulase volume addition is 0.08-0.4%.

6. The application as described in claim 4, characterized in that, Preparation of phosphate-swellable cellulose: Microcrystalline cellulose was mixed with ultrapure water, and pre-cooled 85% H3PO4 aqueous solution was added. The mixture was stirred vigorously and allowed to stand for 5 min. Pre-cooled 85% H3PO4 solution was added and stirred vigorously until the mixture became transparent. After standing at 4°C overnight, ice water was added and the mixture was stirred vigorously until a white turbid precipitate formed. The precipitate was centrifuged at 8000 r / min at 4°C for 20 min and the supernatant was discarded. The process of adding ice water and centrifuging was repeated 3 times. Phosphoric acid was neutralized with 2M NaCO3 aqueous solution, and the cellulose was resuspended in ice water. The mixture was centrifuged at 8000 r / min at 4°C for 20 min and the supernatant was discarded. The process of adding ice water and centrifuging was repeated 2 times. The pH was adjusted to 6.0, and the precipitate was collected to obtain phosphate-swellable cellulose.

7. The application as described in claim 6, characterized in that, The volume of ultrapure water used is 3 mL / g based on the mass of microcrystalline cellulose; the total volume of 85% H3PO4 aqueous solution used is 3.5 mL / g based on the mass of microcrystalline cellulose, and the volume ratio of the two additions is 5:

2.

8. The application as described in claim 4, characterized in that, The purified enzyme was prepared according to the following method: (1) The recombinant genetically engineered bacteria expressing the lysin monooxygenase mutant were inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance and cultured at 37°C for 12 h. Then, they were inoculated into fresh LB liquid medium containing 50 μg / mL kanamycin resistance at a volume concentration of 1% and cultured at 37°C until the bacterial cell concentration reached OD500. 600 When the concentration is 0.6-0.8, add IPTG to a final concentration of 0.5 mM, induce culture at 26℃ for 12 h, centrifuge the culture medium at 4℃ and 8000 r / min for 10 min, discard the supernatant, collect the precipitate, and obtain wet bacterial cells containing recombinant lysine monooxygenase. (2) Protein purification: The wet bacterial cells were resuspended in binding buffer and then sonicated for 30 min. The sonication power was 400 W, the sonication time was 3 s and the interval was 4 s. The mixture was centrifuged at 8000 r / min for 10 min at 4 ℃ to obtain crude enzyme solution. The binding buffer composition was: pH 8.0, 10 mM imidazole, 200 mM NaCl, 40 mmol / L Tris-HCl buffer. (3) The crude enzyme solution was separated and purified using a Ni-NTA 6FF pre-packed gravity column. The column was first equilibrated with binding buffer, and then the crude enzyme solution was loaded with 10 column volumes. Next, the impurity proteins were eluted with washing buffer, and finally the target protein was eluted with elution buffer to obtain the pure enzyme. The binding buffer consisted of pH 8.0, 10 mM imidazole, 200 mM NaCl, and 40 mmol / L Tris-HCl buffer. The washing buffer consisted of pH 8.0, 50 mM imidazole, 200 mM NaCl, and 40 mmol / L Tris-HCl buffer. The elution buffer consisted of pH 8.0, 250 mM imidazole, 200 mM NaCl, and 40 mmol / L Tris-HCl buffer.

9. The application as described in claim 4, characterized in that, The copper-saturated pure enzyme was prepared as follows: the pure enzyme was equilibrated in 100 mM, pH 5.0 sodium acetate buffer at room temperature for 5 h; the equilibrated enzyme solution was placed in 100 mM, pH 5.0 sodium acetate buffer containing 1 mM CuSO4 and incubated at room temperature for 30 min; further dialyzing was performed in 100 mM, pH 5.0 sodium acetate buffer for 24 h to remove excess Cu. 2+ The dialysis solution was changed every 8 hours; the choked solution was used to obtain copper-saturated pure enzyme.

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