Aa10 family lytic polysaccharide monooxygenase and use thereof

Abstract

This invention discloses an AA10 family cleaving polysaccharide monooxygenase and its applications. It is formed by fusing the AA10 family catalytic domain 5I1 with a cellulose-binding module CB3-dFN3 lacking the FN3 domain via a flexible linker peptide, and its amino acid sequence is shown in SEQ ID NO.1. This recombinant enzyme significantly outperforms its parent CB3-dFN3 in key performance indicators such as specific enzyme activity, thermal stability, and catalytic efficiency, solving the core problems of low catalytic activity and poor thermal stability of natural LPMOs, and possessing greater potential for industrial applications.

Description

Technical Field

[0001] This invention belongs to the fields of bioengineering and enzyme engineering technology, and in particular relates to an AA10 family cleaving polysaccharide monooxygenase and its applications. Background Technology

[0002] Biomass resources are among the most abundant renewable resources on Earth, mainly including lignocellulose and chitin. The efficient degradation and conversion of these polysaccharides is a key step in realizing the high-value utilization of biomass resources. Traditional glycoside hydrolases often have limited efficiency in degrading crystalline polysaccharides (such as cellulose and chitin) due to the high crystallinity and dense structure of the substrates, thus limiting the potential for industrial-scale application of biomass.

[0003] The discovery of polysaccharide monooxygenases (LPMOs) has introduced an innovative oxidative cleavage mechanism to the field of polysaccharide degradation. LPMOs can significantly improve the accessibility and subsequent hydrolysis efficiency of stubborn crystalline polysaccharides such as cellulose and chitin by oxidatively breaking glycosidic bonds, particularly introducing cleavage sites in the crystalline regions of polysaccharides. This makes them an important component of highly efficient biomass inversion enzyme systems.

[0004] AA10 family LPMOs are widely found in bacteria and typically consist of one or more functional domains (such as catalytic domains and carbohydrate-binding modules), exhibiting substrate diversity for cellulose, chitin, and even some hemicellulose. However, naturally derived AA10 family LPMOs generally suffer from insufficient catalytic activity, poor thermal stability, and easy deactivation under industrial reaction conditions, limiting their direct application in actual industrial biomass conversion processes.

[0005] In recent years, protein engineering, including domain recombination, rational design, and directed evolution, has become an effective means of modifying and optimizing the performance of LPMOs. Although there are reports of improving the properties of LPMOs by introducing or replacing specific domains (such as the carbohydrate-binding module CBM) or by performing point mutations, most studies focus on the effects of a single modification strategy or a single domain, lacking in-depth research on systematic domain recombination and comprehensive performance optimization of multi-domain LPMOs.

[0006] Therefore, there is an urgent need in this field to develop a new AA10 family LPMO that combines high catalytic activity, excellent thermal stability and broad substrate adaptability to meet the industrial demand for efficient degradation of complex biomass and promote the green and economical transformation of biomass resources. Summary of the Invention

[0007] In view of this, the present invention aims to propose an AA10 family cleaving polysaccharide monooxygenase and its application, aiming to overcome the technical defects of low enzymatic hydrolysis efficiency of crystalline polysaccharides (such as cellulose and chitin) and insufficient catalytic activity and thermal stability of natural cleaving polysaccharide monooxygenases (LPMOs) in the existing biomass degradation process. This invention provides a novel, efficient, and thermally stable reassembled cleaving polysaccharide monooxygenase 5I1-CB3-dFN3 and its application method. Constructed through rational recombination design of structural domains, this enzyme possesses excellent catalytic activity, broad substrate adaptability, and good thermal stability. It can also synergistically enhance cellulase activity, significantly improving the degradation efficiency and reducing sugar yield of cellulose and lignocellulosic biomass, providing an efficient enzymatic hydrolysis tool for the high-value industrial conversion of biomass resources.

[0008] To achieve the above objectives, the technical solution of the present invention is implemented as follows: One of the technical solutions provided by the present invention is an AA10 family cleaving polysaccharide monooxygenase, which is formed by fusing the AA10 family catalytic domain 5I1 with the cellulose binding module CB3-dFN3 lacking the FN3 domain through a flexible linker peptide. Preferably, its amino acid sequence is shown in SEQ ID NO.1.

[0009] SEQ ID NO.1 is: HGYIYGSRAHIGHTKENNSLNWQQAENKYGEAVSTPQGVEGPKGFPLNGPVDGQLASGGNSGYSLLNIQTPLHWVKKDMKTGKNTLVWEYTAAHKTTKWHYYIT KQGWDPSQPLNRDSLELIQTIDWDGTTAIPGKQSGTGLPHTINIPNDRSGYHVIYAVWDVDDTVNAFYQVLDVNGGGGSGGGGSHGYIESPASRAYKCKLGENK NCGRIIYEPQSLEGKGNFPTGGPADGQITGAGIFTELYEQTPTRWSKVNMNGGPNTFKWVLTAPHATSDWKYYITKKGWDSNKPLARADLELFCSFNDGGKRPP NTVTHTCNVPNDRSGYYLILAVWEIADTGNAFYNVIDVNLNNGGGNQDTQPPSSAPEVPAWAPNTSYQQGALVTYDGRTYECRQAHTSLPGWEPANVPALWLLK Further, the linker peptide is (GGGGS)n, where n is an integer from 1 to 5, preferably n is 2.

[0010] Preferably, the polysaccharide monooxygenase has at least one of the following properties: (a) The optimal reaction temperature is 65~75℃ and the optimal pH is 7.0~8.0; its optimal reaction temperature is 70℃ and the optimal pH is 7.5. Under optimal conditions, its specific enzyme activity reaches 91.63 U / g, which is about 2.14 times higher than that of the parent CB3-dFN3.

[0011] (b) The specific enzyme activity is not less than 80 U / g, preferably not less than 85 U / g, and more preferably 91.63 U / g; (c) After incubation at 50°C for 8 hours, the residual enzyme activity is not less than 40%, preferably not less than 45%, and more preferably 49%, showing significantly better thermal stability than the original enzyme.

[0012] (d) It can degrade at least one of microcrystalline cellulose, sodium carboxymethyl cellulose, filter paper, corn cob, beech xylan and chitin.

[0013] The second technical solution provided by the present invention is an encoding nucleotide that encodes the aforementioned AA10 family cleaving polysaccharide monooxygenase, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0014] SEQ ID NO.2 is: The third technical solution provided by the present invention is an expression vector containing the above-mentioned encoded nucleotides; preferably, the expression vector is a pET-28a(+) plasmid.

[0015] The fourth technical solution provided by the present invention is a host cell containing the above-mentioned expression vector, or having the above-mentioned encoded nucleotides integrated into its genome; preferably, the host cell is Escherichia coli.

[0016] The fifth technical solution provided by this invention is the application of the aforementioned AA10 family cleaving polysaccharide monooxygenase in the degradation of cellulosic biomass, wherein the cellulosic biomass is selected from one or more of microcrystalline cellulose, sodium carboxymethyl cellulose, filter paper, corn cob, or beech xylan. The recommended enzymatic hydrolysis reaction conditions are: substrate concentration 1% (w / v), enzyme dosage 1 mg / mL, ascorbic acid concentration 10 mM, reaction pH 5.0, temperature 50℃, and reaction time 36 h.

[0017] The sixth technical solution provided by this invention is the application of the above-mentioned AA10 family cleaving polysaccharide monooxygenase and cellulase in the synergistic degradation of lignocellulose biomass. The reaction conditions for the synergistic degradation are as follows: the amount of AA10 family cleaving polysaccharide monooxygenase added is 0.5~2 mg / mL, the amount of cellulase added is 0.5~2 FPU / mL, the reaction temperature is 40~60℃, and the reaction pH is 4.0~6.0.

[0018] The preferred synergistic reaction system includes: 1 mg / mL of 5I1-CB3-dFN3, 1.1 FPU / mL of commercial cellulase (such as Cellic®CTec3), 1% (w / v) substrate concentration, 10 mM ascorbic acid, pH 5.0, temperature 50℃, and reaction time 36 h. Experiments show that when 5I1-CB3-dFN3 and cellulase act together on filter paper, the synergy reaches 1.80, and the reducing sugar yield is significantly increased by 179.68% compared to when cellulase acts alone.

[0019] The seventh technical solution provided by the present invention is a method for improving the efficiency of cellulase in degrading lignocellulose, which includes the step of using the above-mentioned AA10 family cleaving polysaccharide monooxygenase in combination with cellulase, wherein the mass ratio of the AA10 family cleaving polysaccharide monooxygenase to cellulase is (0.5-2):1.

[0020] The eighth technical solution provided by the present invention is a biological enzyme preparation containing the above-mentioned AA10 family cleaving polysaccharide monooxygenase as an active ingredient, and a pharmaceutically or industrially acceptable carrier or diluent. Preferably, the formulation further comprises one or more of cellulase, hemicellulase, or pectinase.

[0021] The above-mentioned bioenzyme preparations are used in the fields of biomass energy, biofuels, bio-based chemicals, or feed processing. The reassembled cleaving polysaccharide monooxygenase is used to improve the thermal stability and catalytic efficiency of the enzyme preparation. This enzyme remains stable over a wide temperature and pH range, and its high thermal stability and high catalytic efficiency make it more suitable for industrial biomass degradation processes requiring higher temperatures or longer reaction times.

[0022] Compared with existing technologies, the AA10 family cleaving polysaccharide monooxygenase and its application described in this invention have the following advantages: This application demonstrates a significant improvement in enzyme performance. By rationally reassembling the highly efficient predictive screening AA10 catalytic domain 5I1 with the functional module CB3-dFN3, a novel LPMO-5I1-CB3-dFN3 was successfully constructed. This recombinant enzyme significantly outperforms its parent CB3-dFN3 in key performance indicators such as specific enzyme activity, thermal stability, and catalytic efficiency, solving the core problems of low catalytic activity and poor thermal stability in natural LPMOs, and possessing greater potential for industrial applications.

[0023] 2. This application exhibits a broad substrate spectrum and significant synergistic effects. 5I1-CB3-dFN3 can act on a variety of crystalline polysaccharide substrates, broadening the application range of LPMO. More importantly, this enzyme can produce highly efficient synergistic degradation with commercial cellulases. When acting together with cellulase on filter paper, the synergy is as high as 1.80, increasing the yield of reducing sugars by 179.67%. This can significantly reduce the amount of cellulase used or shorten the reaction time, thereby reducing the cost of the biomass saccharification process.

[0024] 3. This invention employs an innovative strategy combining artificial intelligence-assisted predictive screening with rational domain recombination, overcoming the efficiency bottleneck of traditional single mutation or random screening. It provides a new and valuable approach for the molecular design and functional enhancement of AA10 family and other LPMO families. 4. This enzyme was constructed by recombinantly fusing an AI-assisted screened, highly efficient AA10 catalytic domain (5I1) with a mutant of PlLPMOCB3 lacking the FN3 module (CB3-dFN3) from Bacillus subtilis via a flexible linker peptide (GGGGS)2, and then heterologously expressed in Escherichia coli. This reassembled enzyme exhibits broad substrate specificity and can efficiently degrade various biomass substrates, including microcrystalline cellulose, corn cobs, CMC, filter paper, and beech xylan. Compared to the enzyme before assembly and fusion, its catalytic performance is significantly improved: reducing sugar yield is increased by 34.55% on microcrystalline cellulose, 41.37% on CMC, 92.31% on corn cobs, and 76.14% on filter paper. Furthermore, this novel enzyme demonstrates excellent synergistic effects with cellulase in the degradation of various substrates, with a synergy ratio reaching up to 1.80, significantly improving biomass degradation efficiency. This invention provides a new choice of enzyme elements for constructing high-performance, efficient biomass degradation enzyme systems, which has important application value in fields such as biofuel production, feed additives, and biomass resource conversion. Attached Figure Description

[0025] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This invention provides colony PCR verification of the reassembled plasmid in Example 1 of this invention (M stands for DNA Marker, and lane 1 represents the reassembled protein 5I1-CB3-dFN3). Figure 2 This is an SDS-PAGE result of the reassembled protein in Example 2 of the present invention (M is Protein Marker, lane 1 represents the reassembled protein 5I1-CB3-dFN3). Figure 3 This is a graph showing the yield of reducing sugars when the reassembled protein of Example 3 of the present invention acts on different substrates; Figure 4 This is a graph showing the optimal reaction temperature and pH for protein reassembly in Example 4 of the present invention (a is the optimal reaction temperature; b is the optimal reaction pH). Figure 5 The temperature stability and pH stability of the reassembled protein in Example 4 of this invention (a is temperature stability; b is pH stability). Figure 6 This is a graph showing the relationship between the protein reassembly reaction rate and substrate concentration in Example 5 of the present invention. Figure 7The yield of reducing sugars from different substrates in the synergistic degradation of reassembled protein and cellulase in Example 6 of the present invention is shown in Figure 6 (a is microcrystalline cellulose; b is CMC; c is filter paper; d is corn cob). Detailed Implementation

[0026] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0027] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0028] The culture media and solutions used in the embodiments of this invention are as follows: LB medium: 10.00 g peptone, 5.00 g yeast extract, 10.00 g sodium chloride, add 1000 mL distilled water to make up to volume, autoclave at 121℃ for 20 min. For solid medium, add 2% agar powder.

[0029] PBS buffer: Accurately weigh 7.16 g disodium hydrogen phosphate, 0.48 g potassium dihydrogen phosphate, 0.40 g potassium chloride and 16.00 g sodium chloride into a beaker, add 800 mL deionized water, stir to dissolve, bring the volume to 2 L, and adjust the pH to 7.4 for later use.

[0030] 0.5 mol / L IPTG solution: Dissolve 2.40 g IPTG in deionized water, bring the volume to a 20 mL volumetric flask, filter sterilize, and dispense into sterile 2 mL Eppendorf tubes. Store in a -20°C freezer, protected from light. The working concentration is 0.5 mmol / L.

[0031] 50 mmol / L sodium citrate buffer solution (pH 5.0): 1.47 g sodium citrate was dissolved in 100 mL deionized water and the pH was adjusted to 5.0 with citric acid.

[0032] 50 mmol / L phosphate buffer solution at pH 6.0: (A) 50 mM disodium hydrogen phosphate solution: Dissolve 1.79 g of disodium hydrogen phosphate in deionized water and bring the volume to 100 mL in a volumetric flask. (B) 50 mM sodium dihydrogen phosphate: Dissolve 0.78 g of sodium dihydrogen phosphate in deionized water and bring the volume to 100 mL in a volumetric flask. Adjust the pH to 6.0 using a certain amount of (A) and (B).

[0033] Kanamycin (100 mg / mL): Dissolve 50.00 g of kanamycin in 50 mL of deionized water, bring the volume to 100 mL, filter to sterilize, aliquot into sterile 2 mL Eppendorf tubes, and store at -20°C for later use. The working concentration is 100 μg / mL.

[0034] 100 mM ascorbic acid solution: Dissolve 1.76 g of ascorbic acid in 100 mL of deionized water, store protected from light, and prepare fresh before use.

[0035] PAHBAH stock solution: Dissolve 5.00 g of PAHBAH in 0.5 M hydrochloric acid solution and dilute to 100 mL in a brown volumetric flask. Prepare a 5% (w / v) PAHBAH solution and store at 4°C protected from light.

[0036] PAHBAH working solution: Take 1 mL of PAHBAH stock solution and dilute to 100 mL with 0.5 M NaOH solution to prepare a 1% (w / v) solution. Prepare fresh before use.

[0037] 10 mmol / L 2,6-DMP solution: Dissolve 0.15 g of 2,6-DMP in deionized water, dilute to 100 mL in a volumetric flask, protect from light, and prepare fresh before use.

[0038] In this invention, the amino acid sequence of the reassembled AA10 family cleaving polysaccharide monooxygenase 5I1-CB3-dFN3 is shown in SEQ ID NO.1: HGYIYGSRAHIGHTKENNSLNWQQAENKYGEAVSTPQGVEGPKGFPLNGPVDGQLASGGNSGYSLLNIQTPLHWVKKDMKTGKNTLVWEYTAAHKTTKWHYYIT KQGWDPSQPLNRDSLELIQTIDWDGTTAIPGKQSGTGLPHTINIPNDRSGYHVIYAVWDVDDTVNAFYQVLDVNGGGGSGGGGSGYIESPASRAYKCKLGENKN CGRIIYEPQSLEGKGNFPTGGPADGQITGAGIFTELYEQTPTRWSKVNMNGGPNTFKWVLTAPHATSDWKYYITKKGWDSNKPLARADLELFCSFNDGGKRPPN TVTHTCNVPNDRSGYYLILAVWEIADTGNAFYNVIDVNLNNGGGNQDTQPPSSAPEVPAWAPNTSYQQGALVTYDGRTYECRQAHTSLPGWEPANVPALWLLK; In this invention, the nucleotide sequence encoding the reassembled cleaving polysaccharide monooxygenase 5I1-CB3-dFN3 is shown in SEQ ID NO.2. The technical solution of the present invention will be further described below through specific embodiments.

[0039] Example 1: Construction of reassembled protein 5I1-CB3-dFN3 (1) Screening of highly efficient AA10 catalytic domains 141 AA10 family catalytic domains were screened from the Interpro database. A combination of deep learning prediction (based on cellohexasaccharide substrate affinity, solubility, and Kcat value) and randomized uniform screening (using TM-Score clustering analysis and UPGMA to construct a phylogenetic tree and select candidates from different branches) was used to obtain the highly active AA10 catalytic domain 5I1. Based on PlLPMOCB3 derived from Paenibacillus lactis 154, the FN3 domain (amino acids 219-306 and 315-402) was removed to obtain the CB3-dFN3 fragment. The linker peptide (GGGGS)2 was selected as the bridge connecting the catalytic domain to CB3-dFN3.

[0040] (2) Obtaining the target gene The biotechnology company Anshengda was commissioned to synthesize the nucleotide encoding the AA10 catalytic domain 5I1. Specific primers containing the EcoRI restriction site were designed. The nucleotide sequences of the upstream and downstream primers are shown below: Upstream primer: 5'-agctc gaattc TTATTTCAGGAGCCATAAAGCAGG-3'; Downstream primer: 5'-TggcggcggtggcagcgGCTATATTGAATCGCCTG-3'; Using the synthesized 5I1 gene fragment as a template, PCR amplification was performed with a high-fidelity enzyme. The reaction conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 20 s, and 72℃ extension for 30 s, for a total of 30 cycles; 72℃ final extension for 10 min; and storage at 4℃.

[0041] (3) Plasmid linearization pET-28a(+)-CB3-dFN3 was selected as the recombinant plasmid (pre-inserted with the CB3-dFN3 fragment). The vector was treated with the restriction endonuclease EcoRI. The digestion system (20 μL) contained plasmid DNA (≤1 μg), EcoRI enzyme 1 μL, 10× Buffer 2 μL, and ddH2O was added to a final volume of 20 μL. The reaction was carried out at 37°C for 2 h. The linearized plasmid was purified using a Novizan purification kit to remove impurities.

[0042] (4) Homologous recombination and plasmid construction The linearized vector (0.02 × vector base pairs ng), 5I1 gene amplification product (0.04 × insert base pairs ng), 5× CE Buffer 4 μL, and Exnase II enzyme 2 μL were mixed in the specified proportions, and ddH2O was added to a final volume of 20 μL. The mixture was incubated at 37°C for 30 min. The 5I1 catalytic domain was seamlessly linked to CB3-dFN3 via Exnase II enzyme to construct the recombinant plasmid pET-28a(+)-5I1-CB3-dFN3.

[0043] (5) Validation of recombinant plasmids Take 10 μL of recombinant pET-28a(+) plasmid and gently mix it with 100 μL of E. coli BL21(DE3) competent cells. Incubate on ice for 30 min, heat shock at 42℃ for 90 s, and immediately cool on ice for 4 min. Under sterile conditions, add 1 mL of antibiotic-free LB medium and incubate at 37℃ and 220 rpm for 1 h. Centrifuge and discard 800 μL of supernatant, resuspend the culture, and spread approximately 50 μL of the bacterial culture evenly on LB agar plates containing kanamycin resistance. Incubate upside down at 37℃ for 12 h, and pick positive transformants.

[0044] The positive transformants were validated by colony PCR, and the results are as follows: Figure 1 As shown, the band size is approximately 1500 bp, consistent with expectations. Colonies with the correct band size were sent for sequencing, and the glycerol tubes containing the strains with correct sequencing results were stored for later use.

[0045] Example 2: Heterologous expression of reassembled protein 5I1-CB3-dFN3 The correctly identified recombinant strain was transferred to LB broth containing kanamycin resistance and cultured at 37°C and 220 rpm for 12 h. The seed culture was then inoculated at a rate of 1% into 100 mL Erlenmeyer flasks and cultured at 37°C and 220 rpm until OD (dose elapsed). 600 To achieve a pH of 0.6-0.8, add 100 μL of 0.5 mol / L IPTG and incubate at 16℃ and 200 rpm for 20 h. After induction, centrifuge the bacterial culture at 4℃ and 6800 rpm to collect the cells. Wash the cells with PBS buffer to remove any adhering culture medium. Resuspend the collected cells in pre-chilled PBS buffer at 4℃ at a 1:10 ratio and sonicate on ice for 20 min (sonication conditions: power, 300 W; disruption, 3 s; stop, 4 s). After sonication, centrifuge at 4℃ and 6800 rpm for 20 min and collect the supernatant.

[0046] The supernatant and purified protein were analyzed by SDS-PAGE, such as... Figure 2As shown, the protein size is approximately 43 kDa, which is consistent with the theoretical value, proving that the protein expression was successful and can be used for subsequent determination of enzymatic properties.

[0047] Example 3: Substrate specificity analysis of reassembled protein 5I1-CB3-dFN3 To evaluate the hydrolytic activity of the target enzyme on various substrates, purified CB3-dFN3 and 5I1-CB3-dFN3 were reacted with microcrystalline cellulose, corn cob, CMC, filter paper, and beech xylan substrates, respectively. The reducing sugar content was measured after the reaction. Several 1.5 mL centrifuge tubes were used, and 0.01 g of microcrystalline cellulose, carboxymethyl cellulose (CMC), filter paper (cut into 5×5 mm squares), beech xylan, and corn cob (all pulverized, dried, and sieved through a 20-mesh sieve) were added to each tube. 1 mg / mL of purified enzyme solution was added to each centrifuge tube, and ascorbic acid was added to a final concentration of 10 mM. Subsequently, the total volume of the reaction system was adjusted to 1 mL using 50 mM sodium citrate buffer (pH 5.0). The reaction system was sealed with sealing film and incubated at 50 °C and 220 rpm with shaking for 36 h. After the reaction was completed, the reaction system was heated in boiling water for 5 min to terminate the reaction, followed by centrifugation at 12000 rpm for 1 min, and the supernatant was collected. The yield of reducing sugars in the supernatant was determined by the PAHBAH method to evaluate the hydrolytic efficiency of the enzyme on different substrates. A reaction system without enzyme was set up as a control group to eliminate the interference of spontaneous substrate hydrolysis. All samples were tested in triplicate to ensure the accuracy and reliability of the data.

[0048] The results are as follows Figure 3 As shown, 5I1-CB3-dFN3 produced a 34.55% higher reducing sugar content than CB3-dFN3 when applied to microcrystalline cellulose substrates; a 41.37% higher reducing sugar content when applied to CMC substrates; a 92.31% higher reducing sugar content when applied to corn cob substrates; and a 76.14% higher reducing sugar content when applied to filter paper substrates. In summary, the reassembled protein 5I1-CB3-dFN3 significantly increased the reducing sugar content compared to CB3-dFN3 when applied to microcrystalline cellulose, CMC, corn cob, and filter paper. Substrate-specific analysis demonstrated that CB3-dFN3 and its reassembled protein have a broad substrate spectrum.

[0049] Example 4: Enzymatic property analysis of reassembled protein 5I1-CB3-dFN3 The activity of LPMOs was determined using the 2,6-DMP method. The total reaction volume was 1 mL, containing the following components: 780 μL of 50 mmol / L phosphate buffer (pH 7.5), 100 μL of 10 mM 2,6-DMP solution, 20 μL of 50 mmol / L H2O2, and 100 μL of purified LPMO enzyme solution (protein concentration 0.5 mg / mL). The reaction was carried out at the set temperature for 5 min, and the absorbance was then measured at 469 nm using a UV spectrophotometer.

[0050] Enzyme activity (U) is defined as the amount of enzyme required to produce 1 μmol of xylenoid per minute under the given reaction conditions. Specific enzyme activity is calculated using the following formula: In the formula: X (U / g) is the specific enzyme activity, A is the change in absorbance, V1 (mL) is the reaction volume, c (mg / mL) is the protein concentration, V2 (μL) is the volume of protein added, N is the dilution factor for measuring the absorbance, and ℇ 469 (53200 Lmol) -1 cm -1 ) is the molar absorptivity of xylenoid, b (cm) is the optical path length of the cuvette, and t is the reaction time (min).

[0051] Using the methods described above, the enzymatic properties of the reassembled protein 5I1-CB3-dFN3 were systematically characterized, including optimal reaction conditions, thermal stability, and pH stability.

[0052] (1) Determination of optimal reaction temperature and pH The activity of the reassembled protein 5I1-CB3-dFN3 was determined using the 2,6-dimethylphenol (2,6-DMP) method at temperatures ranging from 30 to 110 °C and pH values ​​ranging from 3.0 to 8.5 (acetic acid buffer for pH 3.0–5.5, and phosphate buffer for pH 6.0–8.5). The highest enzyme activity was defined as 100%, and the relative enzyme activity was calculated. Results are as follows: Figure 4 As shown, the optimal reaction temperature for 5I1-CB3-dFN3 is 70℃, and the optimal pH is 7.5. Under optimal conditions, its specific enzyme activity is 91.63 U / g, which is 2.14 times higher than that of the parent protein CB3-dFN3.

[0053] Thermal stability analysis 5I1-CB3-dFN3 was incubated at 40℃ and 50℃ for 8 hours, respectively. Samples were taken periodically and residual enzyme activity was measured under optimal reaction conditions (with the enzyme activity of unincubated samples as 100%). The results are as follows: Figure 5As shown, after 8 hours of incubation, the relative enzyme activities at 40℃ and 50℃ were 60.69% and 49.04%, respectively, indicating that it has good thermal stability, especially at 40℃ its stability is significantly better than CB3-dFN3.

[0054] (3) pH stability analysis 5I1-CB3-dFN3 was incubated at 37°C in different pH buffers for 1 hour, and then the residual enzyme activity was measured under optimal conditions. The results are as follows: Figure 5 As shown, its residual relative enzyme activity at the optimal pH of 7.5 is 89.47%. Although this value is slightly lower than that of CB3-dFN3, which is speculated to be related to changes in protein surface charge distribution, hydrogen bonds, or hydrophobic interactions after reassembly, it still shows a wide pH adaptation range.

[0055] In summary, the reassembled protein 5I1-CB3-dFN3 exhibits the highest catalytic activity at 70℃ and pH 7.5, and also demonstrates good thermal stability and certain pH stability, providing experimental evidence for its application in industrial biomass conversion.

[0056] Example 5: Catalytic kinetics of the reassembled protein 5I1-CB3-dFN3 Under optimal reaction conditions, the Michaelis constant (Km) and maximum reaction rate (Vmax) of enzymes CB3-dFN3 and 5I1-CB3-dFN3 were determined using different concentrations of 2,6-DMP (1–40 mmol / L) as substrates. Each measurement was repeated three times to ensure data reliability. Kinetic parameters were analyzed using GraphPad Prism software.

[0057] The results are as follows Figure 6 As shown, the Vmax of CB3-dFN3 is 44.72 μmol / g·min, and the Vmax of 5I1-CB3-dFN3 is 113.7 μmol / g·min. The Kcat / Km of 5I1-CB3-dFN3 is 42.3 s. -1 mM -1 The Kcat / Km of CB3-dFN3 is 87.69 s. -1 mM -1 Therefore, it can be seen that the Vmax of 5I1-CB3-dFN3 is higher than that of CB3-dFN3, and is 2.54 times higher than that of CB3-dFN3.

[0058] Example 6: Synergistic properties of reassembled protein 5I1-CB3-dFN3 and cellulase To evaluate the activity of CB3-dFN3 and the synergistic hydrolysis of the reassembled protein with cellulase (Cellic®CTec3, a commercial cellulase from Novozymes), the following reaction systems were used. A 1 mL cellulase reaction system contained only 0.01 g of substrate, 1.1 FPU / mL cellulase, and 10 mM ascorbic acid, followed by adjustment to 1 mL with 50 mM sodium citrate buffer (pH 5.0). A 1 mL synergistic reaction system contained AA10 (CB3-dFN3 or the reassembled protein) and cellulase, comprising 1 mg / mL of purified reassembled protein, 1.1 FPU / mL Cellic®CTec3, 0.01 g of substrate, and 10 mM ascorbic acid, and was finally brought to 1 mL with 50 mmol / L sodium citrate buffer (pH 5.0). The reaction was carried out at 50 °C and 200 rpm for 36 h. After the reaction was complete, the mixture was boiled for 5 min to terminate the reaction, and then centrifuged at 13000 rpm for 1 min to remove the solids. The reducing sugar content in the supernatant was determined by the PAHBAH method.

[0059] The synergy calculation formula of this invention is the reducing sugar production when reassembled protein and cellulase act together / the sum of reducing sugar production when they act alone.

[0060] The results are as follows Figure 7 As shown, when the reassembled protein 5I1-CB3-dFN3 and cellulase act together on microcrystalline cellulose substrates, the reducing sugar yield is increased by 112.43% compared to cellulase alone, with a synergy of 1.12. When they act separately with cellulase on corn cob substrates, the reducing sugar yield is increased by 154.68% compared to cellulase alone, with a synergy of 1.32; when acting on CMC, the reducing sugar yield is increased by 126.64%, with a synergy of 1.20; and when acting on filter paper substrates, the reducing sugar yield is increased by 179.67%, with a synergy of 1.80. The results indicate that the reassembled protein and cellulase have the best synergistic effect when acting together on filter paper substrates. When LPMO of the AA10 family is directly applied to biomass substrates alone, the content of reducing sugars produced by hydrolysis is low. When cellulase and LPMO are acted together on biomass substrates, the synergy is greater than 1, indicating that the two can mutually promote each other when acting together, thereby improving the degradation efficiency of biomass.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An AA10 family cleaving polysaccharide monooxygenase, characterized in that: The cellulose-binding module CB3-dFN3, which lacks the FN3 domain, is formed by the fusion of the 5I1 catalytic domain of the AA10 family with a flexible linker peptide. Preferably, its amino acid sequence is shown in SEQ ID NO.

1.

2. The AA10 family cleaving polysaccharide monooxygenase according to claim 1, characterized in that: The linker peptide is (GGGGS)n, where n is an integer from 1 to 5, and preferably, n is 2; Preferably, the polysaccharide monooxygenase has at least one of the following properties: (a) The optimal reaction temperature is 65~75℃, and the optimal pH is 7.0~8.0; (b) The specific enzyme activity is not less than 80 U / g, preferably not less than 85 U / g, and more preferably 91.63 U / g; (c) After incubation at 50°C for 8 hours, the residual enzyme activity is not less than 40%, preferably not less than 45%, and more preferably 49%; (d) It can degrade at least one of microcrystalline cellulose, sodium carboxymethyl cellulose, filter paper, corn cob, beech xylan and chitin.

3. A nucleotide encoding a AA10 family cleaving polysaccharide monooxygenase as described in claim 1 or 2, the nucleotide sequence of which is shown in SEQ ID NO.

2.

4. An expression vector, characterized in that: It comprises the encoding nucleotide as described in claim 3; preferably, the expression vector is a pET-28a(+) plasmid.

5. A host cell, characterized in that: The expression vector of claim 4 is included, or the genome thereof is integrated with the encoding nucleotide of claim 4; preferably, the host cell is Escherichia coli.

6. The application of the AA10 family cleaving polysaccharide monooxygenase according to claim 1 or 2 in the degradation of cellulosic biomass, characterized in that: The cellulose biomass is selected from one or more of microcrystalline cellulose, sodium carboxymethyl cellulose, filter paper, corn cob, or beech xylan.

7. The application of the AA10 family cleaving polysaccharide monooxygenase and cellulase as described in claim 1 or 2 in the synergistic degradation of lignocellulose biomass, characterized in that, The reaction conditions for the synergistic degradation are as follows: the amount of AA10 family cleaving polysaccharide monooxygenase added is 0.5~2 mg / mL, the amount of cellulase added is 0.5~2 FPU / mL, the reaction temperature is 40~60℃, and the reaction pH is 4.0~6.

0.

8. A method for improving the efficiency of cellulase in degrading lignocellulose, comprising the step of using the AA10 family cleaving polysaccharide monooxygenase as described in claim 1 or 2 in combination with cellulase, characterized in that, The mass ratio of the AA10 family cleaving polysaccharide monooxygenase to cellulase is (0.5-2):

1.

9. A biological enzyme preparation comprising, as the active ingredient, the AA10 family cleaving polysaccharide monooxygenase of claim 1 or 2, and a pharmaceutically or industrially acceptable carrier or diluent. Preferably, the formulation further comprises one or more of cellulase, hemicellulase, or pectinase.

10. The application of the bio-enzyme preparation according to claim 8 or 9 in the fields of biomass energy, biofuels, bio-based chemicals or feed processing.

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