Cracking polysaccharide monooxygenase mutant FpLPMO9A-N160T and application thereof

Abstract

The invention discloses a cleavable polysaccharide monooxygenase mutant FpLPMO9A-N160T and application of the cleavable polysaccharide monooxygenase mutant FpLPMO9A-N160T, and belongs to the field of enzyme engineering. The optimal temperature of the lytic polysaccharide monooxygenase mutant FpLPMO9A-N160T disclosed by the invention is 50 DEG C, the optimal pH is 5, and the enzymatic activity of the mutant FpLPMO9A-N160T is 10.8 times that of a wild type. The thermal stability of the lytic polysaccharide monooxygenase mutant is remarkably improved, IC50 is 101.1 min at the temperature of 50 DEG C and is 1.57 times that of a wild type, the activity of the mutant can be kept for a longer time at the high temperature, and better thermal stability is shown. The improvement has important significance on the expression of the LPMO in industrial application, especially in environments requiring high-temperature operation, such as biomass conversion, biofuel production and other processes. The higher thermal stability not only prolongs the service life of the enzyme, but also improves the efficiency and reliability of the enzyme in practical application.

Description

Technical Field

[0001] The invention belongs to the field of enzymology, and in particular relates to a mutant of a lytic polysaccharide monooxygenase. Background Art

[0002] As a by-product of agricultural production, crop straw has become a biological resource with great potential due to its large output and low cost.

[0003] Traditional straw treatment mainly includes three methods: burning, landfilling and returning to the field. Burning straw can quickly reduce the amount of straw, but it will cause environmental pollution, produce carbon monoxide, sulfur dioxide, nitrogen oxides and volatile organic compounds, etc., which will harm air quality and people's health. Landfilling straw will occupy land resources, produce greenhouse gases, and have a negative impact on the environment.

[0004] Returning straw to the field is an important ecological agricultural practice in agricultural production. It not only significantly improves soil quality and fertility, but also helps prevent soil erosion, reduce the use of chemical fertilizers and promote sustainable agricultural development. Therefore, promoting the practice of returning straw to the field is of great significance for achieving green development of agriculture. However, since the main component of straw is lignocellulose, including cellulose, hemicellulose and lignin, these substances constitute important components of plant cell walls and have high structural strength and stability, making them difficult to degrade quickly. This difficult-to-degrade characteristic makes the straw decompose slowly in the natural environment, causing a certain degree of impact on the soil and the environment.

[0005] The cell wall of straw plants presents a complex cross-linked structure, especially the polysaccharide chains that form highly ordered crystalline regions with lignin through hydrogen bonds. It is difficult for traditional glycoside hydrolases (GH) to effectively degrade straw, resulting in the low catalytic efficiency of current biodegradation catalysts. Further research and development of efficient catalytic enzymes is needed to improve the degradation rate and efficiency.

[0006] The new straw oxidase, lytic polysaccharide monooxygenase (LPMO) (LPMOs, EC 1.14.99.53-56), is a class of enzymes that can degrade cellulose. Studies have shown that LPMO, with the assistance of electron donors, can oxidatively break the glycosidic bonds in the crystalline region of lignocellulose, releasing more reducing ends, thereby providing more binding sites for glycoside hydrolases. When LPMO and glycoside hydrolases act together, the conversion efficiency of lignocellulose can be significantly improved.

[0007] By exploring new LPMO resources and coordinating with straw decomposing agents, it can not only improve the efficiency and quality of decomposing agents, but also make positive contributions to the resource utilization of agricultural waste and environmental protection. It is expected to play a greater role in future agricultural production, promote the efficient utilization of resources and improve soil health. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a polysaccharide monooxygenase mutant capable of efficiently degrading cellulose and its application.

[0009] The technical solution of the present invention is: a lytic polysaccharide monooxygenase mutant FpLPMO9A-N160T, whose amino acid sequence is shown in SEQ ID No.1.

[0010] The gene encoding the mutant FpLPMO9A-N160T described above.

[0011] Furthermore, the nucleotide sequence of the gene is shown in SEQ ID No.2.

[0012] An expression vector containing the gene described above.

[0013] An engineered bacterium containing the above-mentioned expression vector.

[0014] The application of the mutant FpLPMO9A-N160T or the engineered bacteria described above in degrading cellulose.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The optimal temperature of the lytic polysaccharide monooxygenase mutant FpLPMO9A-N160T of the present invention is 50°C, the optimal pH is 5, and the enzyme activity of the mutant is 10.8 times that of the wild type. The thermal stability of the lytic polysaccharide monooxygenase mutant of the present invention is significantly improved, and the IC 50 The mutant can maintain activity for a longer time at higher temperatures, showing better thermal stability. This improvement is of great significance for the performance of LPMO in industrial applications, especially in environments that require high-temperature operation, such as biomass conversion and biofuel production. Higher thermal stability not only extends the service life of the enzyme, but also improves its efficiency and reliability in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Comparison of enzyme activities between FpLPMO9A-WT and FpLPMO9A-N160T; Figure 2 is the relative activity of FpLPMO9A-WT and FpLPMO9A-N160T at different pH values; Figure 3 pH tolerance analysis of FpLPMO9A-WT and FpLPMO9A-N160T; Figure 4 Optimal temperature analysis for FpLPMO9A-WT and FpLPMO9A-N160T; Figure 5 The relative enzyme activities of FpLPMO9A-WT and FpLPMO9A-N160T at 50°C over time.

[0017] Figure 6 The synergistic effect of LPMO and cellulase on the yield of reducing sugars. In the figure, a and b are marked with significant differences (P < 0.05), and the same letters indicate no significant differences. DETAILED DESCRIPTION

[0018] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0019] Example 1 Mining and Optimization of LPMO Genes Relevant datasets encoding LPMO sequences were collected through online databases (CAZy, NCBI, Uniprot, etc.), and data preprocessing was performed. The sequences were uploaded to the online software dbCAN HMMdb v10.0 in FASTA format for functional annotation. The candidate genes obtained from the annotation results were then submitted to the interpro database to predict potential domains and functions. The structural homology model of LPMO was generated using the AlphaFold 3 server.

[0020] Fusarium pyrifolium Fusarium poae LPMO gene (named FpLPMO9A-WT, the nucleotide sequence of which is shown in SEQ ID No. 3, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 4), Fusarium pyrifolium Fusarium poae is an important plant pathogenic fungus that primarily affects wheat, barley, and other cereal crops. It belongs to the genus Fusarium ( Fusarium ), which can cause a variety of diseases, such as root rot, stem base rot and ear blight. F. poae In the process of degradation, CAZy (carbohydrate active enzyme) and LPMO work together to play a key role in the degradation of plant cell wall polysaccharides. CAZy enzymes break down complex polysaccharides into simple oligosaccharides or monosaccharides through hydrolysis, while LPMO further degrades stubborn polysaccharide structures through oxidative cleavage. The synergistic effect of these two enzymes not only improves the efficiency of polysaccharide degradation, but also enhances F. poae The nutrient acquisition ability and pathogenic mechanism of the bacteria enable them to more effectively invade and destroy the defense system of the host plant and adapt to different environmental conditions. F. poae It is of great importance to survive in the soil and infect crops.

[0021] By analyzing the three-dimensional structure of the protein, site-directed mutations were performed on a series of amino acid residues that could affect the catalytic activity of LPMO (Table 1).

[0022] Table 1 Primers

[0023] Example 2 Construction, heterologous expression and activity analysis of recombinant engineering bacteria encoding LPMO genes The wild-type and mutant genes were connected to the pGAPZαA vector by homologous recombination, and the recombinant plasmids were transformed into E. coli DH5α competent cells were plated on LB plates containing 25 µg / ml Zeocin low salt, inverted and cultured overnight at 37°C, positive colonies were picked, and verified by colony PCR and sequencing.

[0024] Plasmid extraction and characterization: About 10 μg of plasmid was extracted using a kit and used Avr II linearized positive plasmid, incubated at 37°C for 15 min, and detected the linearized product by electrophoresis and purified the product.

[0025] Preparation of Pichia pastoris competent cells: Streak GS115 on YPD plates and culture at 30°C for 2-3 days. Inoculate 100 mL YPD liquid medium at 1% inoculum and culture overnight at 30°C, 220 rpm until OD 600 Reach 1.1-1.3; let the bacterial solution stand on ice for 30 min, centrifuge at 4°C, 4000 rpm for 5 min, discard the supernatant, add 100 mL pre-cooled ddH2O to resuspend the bacteria; centrifuge and discard the supernatant, add 50 mL pre-cooled ddH2O to resuspend. Centrifuge and discard the supernatant, resuspend the bacteria with 25 mL 1 M pre-cooled sorbitol; centrifuge and discard the supernatant, add 15 mL 1 M sorbitol to resuspend the bacteria; centrifuge and discard the supernatant, add 0.75 mL 1 M sorbitol to resuspend the bacteria; aliquot 80 µL per tube and use immediately.

[0026] Electroporation transformation: Place a 2 mm electroporation cup on ice and add 10 µL of linearized plasmid; voltage 1500 V, electroporation time >4.5 ms; immediately add 500 µL of 1 M sorbitol to wash the bacteria and resuscitate at 30°C for 1 h; culture at 30°C in the dark for 2-3 days.

[0027] Screening of positive transformants: Pick a single colony and inoculate it into 2 mL YPD, take 1 mL of bacterial solution, incubate at 6000 rpm, 4°C, for 5 min, and harvest the bacteria; extract the genome with benzyl chloride, and use the genome as a template for PCR amplification using gene primers and pGAP / 3'AOX as primers.

[0028] Recombinant protein expression in Pichia pastoris: Pick a single colony and inoculate it in 50 mL YPD, culture it at 30°C, 220 rpm for 8 days, centrifuge it at 10,000 rpm, 4°C, for 15 min, and take the supernatant as the crude enzyme solution. Purify the crude enzyme solution by affinity chromatography (the protein is designed with a His tag, purified by a nickel column, and the target protein is confirmed by molecular weight), determine the protein concentration by a BCA kit, and place it in a 4°C refrigerator for later use.

[0029] Example 3 Activity determination LPMO activity was determined using the DNS method. 15 μL (~1.5 μM) of the enzyme was mixed with 60 μL of 5 mg / mL cellulose substrate solution dissolved in 0.05 M sodium phosphate buffer (pH 5.5), incubated at 37°C and 200 rpm for 24 h, 75 μL of DNS was added, and color was developed at 95°C for 10 min. After cooling to room temperature, 100 μL was taken and the OD was measured. 540 , reactions were performed in triplicate.

[0030] Standard curve drawing: Prepare 10 mg / mL glucose stock solution, dilute to different concentrations (1~5 mg / mL) with deionized water, mix 75 μL glucose of different concentrations with 75 μL DNS, mix well, color develop at 95°C for 10 min, cool to room temperature, take 100 μL, and measure OD 540 The reaction was performed in triplicate. The standard curve was drawn with glucose concentration as the horizontal axis and absorbance as the vertical axis. The regression equation was y=0.3418x-0.02968 (R 2 =0.9959).

[0031] By performing a detailed analysis of the enzyme activity of the wild-type FpLPMO9A-WT and the mutants, we found significant differences in the ability to produce reducing sugars. Specifically, the amount of reducing sugar produced by FpLPMO9A-WT under experimental conditions was 0.78±0.04 μM, while N160T showed significantly higher activity, producing 8.56±0.29 μM of reducing sugar. The amount of reducing sugar produced by N160T was 10.8 times that of the wild type. This result suggests that the N160T mutation significantly enhances the catalytic efficiency of the enzyme, making its reducing sugar production much higher than the wild-type level and other mutants. This may suggest that the amino acid substitution at the N160 position has an important effect on the structure and function of the enzyme, thereby improving its catalytic activity.

[0032] N160T was named mutant FpLPMO9A-N160T, its amino acid sequence was shown in SEQ ID No.1, and the nucleotide sequence of the encoding gene was shown in SEQ ID No.2.

[0033] The mutant with the highest enzyme activity, FpLPMO9A-N160T, was selected for subsequent studies.

[0034] Example 4 Analysis of Enzymatic Properties (1) Optimum pH: 15 μL (~1.5 μM) of LPMO was thoroughly mixed with 60 μL of 5 mg / mL cellulose substrate prepared in pH 2.2–9.0 buffer (pH 2.2-8.0 citrate / phosphate buffer and pH 8.0-9.0 0.2 M Tris-HCl buffer), incubated at 37°C for 24 h, and the optimal pH was determined by the DNS method. The relative activity was analyzed with the highest activity group as 100%.

[0035] The results are as follows Figure 2 As shown, the optimal pH values ​​of the two enzymes, FpLPMO9A-WT and FpLPMO9A-N160T, are similar, both around 5.

[0036] (2) pH tolerance: The enzyme solution was mixed with pH 2.2-8.0 buffer at a ratio of 1:1. After being placed on ice for 60 min, the residual enzyme activity was determined under the optimal conditions. The pH stability was calculated with the untreated enzyme solution as 100%.

[0037] The results are as follows Figure 3As shown in the figure, in the range of pH 2 to 4, the enzyme activity of the mutant N160T was significantly higher than that of the wild type. At pH 5, the activities of the two enzymes reached the maximum, and the difference between the two was the smallest. In the range of pH 6 to 8, the enzyme activity of FpLPMO9A-N160T was still higher than that of the wild type, but the difference between the two gradually decreased. Overall, FpLPMO9A-N160T showed higher enzyme activity in a wider pH range.

[0038] (3) Optimum temperature: At the optimal pH, 15 μL (~1.5 μM) of LPMO was thoroughly mixed with 60 μL of 5 mg / mL cellulose substrate and incubated at 30–80°C for 24 h. The optimal temperature was determined using the DNS method. The relative activity was analyzed with the data of the group with the highest activity as 100%.

[0039] The results are as follows Figure 4 As shown, FpLPMO9A-N160T has slightly higher activity at lower temperatures, and the activities of the two are similar and reach a peak at around 50°C. However, in the higher temperature range of 60°C to 80°C, FpLPMO9A-N160T exhibits higher thermal stability and its activity is significantly higher than that of FpLPMO9A-WT.

[0040] (4) Temperature tolerance: After the appropriately diluted enzyme solution was kept at 50°C for 10, 20, 40 and 60 min, the residual enzyme activity was measured and the residual activity was calculated with the untreated group (0 min) as 100%.

[0041] The results are as follows Figure 5 As shown in the figure, the thermal stability of FpLPMO9A-N160T at 50°C is slightly higher than that of the wild type. Specifically, the residual activity of the wild type after treatment at 50°C for 1 h is about 50%, while the residual activity of the mutant is about 60% under the same conditions. 50 The values ​​show that the wild-type IC 50 The value is 64.4 min, which means that at 50°C, it takes about 64.4 min for the enzyme activity to decrease to 50%. In contrast, the IC 50 The value was 101.1 min, and the thermal stability of FpLPMO9A-N160T at 50 °C was better than that of the wild type, which may mean that the mutation has a positive effect on improving the thermal stability of the enzyme.

[0042] Example 5 Synergy between LPMO and cellulase LPMO was used to catalyze the substrate with cellulase (derived from Aspergillus oryzae). 60 μL of substrate, 7.5 μL (0.5 μM) LPMO and 7.5 μL (1.0 μM) GH were added to each experimental tube. Heat-inactivated GH and LPMO were added to the LPMO and GH treatment groups, and 15 μL of pre-boiled enzyme solution was added to each blank tube. Four parallel experiments were performed and incubated at 37°C for 72 hours. The reaction system contained 1.5 mM ascorbic acid. After the reaction was completed, 75 μL of DNS reagent was added and incubated at 100°C for 10 minutes. After cooling to room temperature, the OD was measured using a spectrophotometer. 540 The absorbance value was measured in triplicate. The standard curve was drawn with glucose concentration as the horizontal axis and absorbance as the vertical axis. The regression equation was y=0.3418x-0.02968 (R 2 =0.9959).

[0043] The results are as follows Figure 6 As shown, compared with cellulase alone, the reducing sugar yields of the synergistic groups of cellulase + FpLPMOA-WT and cellulase + FpLPMOA-N160T were 15.23±0.07 and 17.56±0.52 μmol / L, respectively, and the reducing sugar content increased by 17.83% and 35.83%, respectively. When cellulase acted synergistically with FpLPMOA-N160T, the reducing sugar yield was the highest, which was significantly higher than that of other treatment groups.

Claims

1. A lytic polysaccharide monooxygenase mutant FpLPMO9A-N160T, whose amino acid sequence is shown in SEQ ID No.

1.

2. A gene encoding the mutant FpLPMO9A-N160T according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID No.

2.

4. An expression vector containing the gene according to claim 2 or 3.

5. An engineered bacterium containing the expression vector according to claim 4.

6. Use of the mutant FpLPMO9A-N160T according to claim 1 or the engineered bacteria according to claim 5 in degrading cellulose.

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