Lytic polysaccharide monooxygenase mutant FpLPMO9A-N160T and its application

Abstract

The present invention discloses a lytic polysaccharide monooxygenase mutant FpLPMO9A-N160T and its application, belonging to the field of enzyme engineering. The optimal temperature of the lytic polysaccharide monooxygenase mutant FpLPMO9A-N160T of the present invention is 50 °C, and the optimal pH is 5. The enzyme activity of the mutant FpLPMO9A-N160T 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. At 50 °C, the IC 50 is 101.1 min, which is 1.57 times that of the wild type. The mutant can maintain its activity for a longer time at a higher temperature, 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 operations, such as biomass conversion and biofuel production processes. Higher thermal stability not only extends the service life of the enzyme but also improves its efficiency and reliability in practical applications.

Description

Technical Field

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

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

[0003] Traditional straw treatments mainly include incineration, landfill, and returning to the field. Incinerating straw can quickly reduce the amount of straw, but it will cause environmental pollution, generating harmful substances such as carbon monoxide, sulfur dioxide, nitrogen oxides, and volatile organic compounds, which pose a threat to air quality and people's health. Landfilling straw will occupy land resources and produce greenhouse gases, having 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, which are important components of plant cell walls and have high structural strength and stability, it is difficult to degrade them quickly. This intractable degradation property makes the decomposition rate of straw in the natural environment slow, having a certain impact on the soil and the environment.

[0005] The plant cell wall of straw presents a complex cross-linked structure. Especially, hydrogen bonds between polysaccharide chains and lignin form highly ordered crystalline regions. Traditional glycoside hydrolases (GHs) are difficult to effectively degrade straw, resulting in the problem of low catalytic efficiency of current biodegradation catalysts. There is a need to further research and develop highly efficient catalytic enzymes to improve the degradation rate and efficiency.

[0006] The novel straw oxidase - lytic polysaccharide monooxygenase (LPMO) (LPMOs, EC 1.14.99.53 - 56) is a class of enzymes capable of degrading cellulose. Research shows that with the assistance of an electron donor, LPMO can oxidatively cleave 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 hydrolase act together, the conversion efficiency of lignocellulose can be significantly improved.

[0007] By exploring new LPMO resources and collaborating with straw decomposing agents, this not only improves the efficiency and quality of the decomposing agents, but also makes a positive contribution to the resource utilization of agricultural waste and environmental protection. It is expected to play a greater role in future agricultural production, promoting the efficient use of resources and the improvement of soil health. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a lytic polysaccharide monooxygenase mutant with high efficiency in 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] A gene encoding the above-mentioned mutant FpLPMO9A-N160T.

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

[0012] An expression vector containing the above-mentioned gene.

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

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

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The optimal temperature of the lytic polysaccharide monooxygenase mutant FpLPMO9A-N160T of the present invention is 50 °C, and the optimal pH is 5. 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. At 50 °C, the IC 50 is 101.1 min, which is 1.57 times that of the wild type. The mutant can maintain its activity for a longer time at a higher temperature, 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 operations, such as biomass conversion and biofuel production processes. 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

[0017] Figure 1 It is a comparison chart of the enzyme activities of FpLPMO9A-WT and FpLPMO9A-N160T;

[0018] Figure 2Relative activities of FpLPMO9A-WT and FpLPMO9A-N160T at different pH values;

[0019] Figure 3 pH tolerance analysis of FpLPMO9A-WT and FpLPMO9A-N160T;

[0020] Figure 4 Optimum temperature analysis of FpLPMO9A-WT and FpLPMO9A-N160T;

[0021] Figure 5 Relative enzyme activities of FpLPMO9A-WT and FpLPMO9A-N160T over time at 50 °C.

[0022] Figure 6 Effect of the synergy between LPMO and cellulase on the reducing sugar yield. In the figure, a and b indicate significant differences (P < 0.05), and the same letters indicate no significant differences. Specific implementation mode

[0023] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from commercial channels unless otherwise specified.

[0024] Example 1 Mining and optimization of LPMO gene

[0025] Collect relevant datasets encoding LPMO sequences through online databases (CAZy, NCBI, Uniprot, etc.), and perform data preprocessing. The sequences are uploaded in FASTA format to the online software dbCAN HMMdb v10.0 for functional annotation. Then, the candidate genes obtained from the annotation results are submitted to the interpro database to predict potential domains and functions. A structural homology model of LPMO was generated using the AlphaFold 3 server.

[0026] A LPMO gene (named FpLPMO9A-WT, whose nucleotide sequence is shown in SEQ ID No. 3 and the amino acid sequence of the encoded protein is shown in SEQ ID No. 4) was screened from Fusarium poae Fusarium poae Fusarium poae Fusarium poae Fusarium is an important plant pathogenic fungus that mainly affects wheat, barley and other cereal crops. It belongs to the genus Fusarium F. poaeIn it, CAZy (carbohydrate-active enzymes) and LPMO cooperate 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 recalcitrant polysaccharide structures through oxidative cleavage. The synergistic action of these two enzyme classes not only improves the efficiency of polysaccharide degradation but also enhances F. poae its nutrient acquisition ability and pathogenic mechanism, enabling it to more effectively invade and disrupt the host plant's defense system and adapt to different environmental conditions. This efficient enzyme system is of great significance for F. poae surviving in the soil and infecting crops.

[0027] By analyzing the three-dimensional structure of this protein, site-directed mutagenesis was performed on a series of amino acid residues that can affect the catalytic activity of LPMO (Table 1).

[0028] Table 1 Primers

[0029]

[0030] Example 2 Construction, heterologous expression, and activity analysis of recombinant engineering bacteria encoding the LPMO gene

[0031] By homologous recombination, the wild-type and mutant genes were ligated to the pGAPZαA vector. Through heat shock transformation, the recombinant plasmids were respectively transformed into E. coli DH5α competent cells, and the competent cells were spread on an LB plate containing 25 µg / ml Zeocin with low salt and cultured overnight at 37°C in an inverted position. Positive colonies were picked and verified by colony PCR and sequencing.

[0032] Plasmid linear extraction and linearization: Use a kit to extract about 10 μg of plasmid and linearize the positive plasmid with Avr II at 37°C for 15 min, and perform electrophoresis detection on the linearized product to purify the product.

[0033] Preparation of Pichia pastoris competent cells: Streak the GS115 strain on a YPD plate and culture it at 30°C for 2 - 3 d. Inoculate the bacterial solution at an inoculation amount of 1% into 100 mL of YPD liquid medium and culture it overnight at 30°C and 220 rpm until OD 600Reach 1.1 - 1.3; Let the bacterial solution stand on ice for 30 min, centrifuge at 4°C and 4000 rpm for 5 min, discard the supernatant, add 100 mL of pre - cooled ddH2O to resuspend the bacteria; Centrifuge and discard the supernatant, add 50 mL of pre - cooled ddH2O to resuspend. Centrifuge and discard the supernatant, resuspend the bacteria with 25 mL of 1 M pre - cooled sorbitol; Centrifuge and discard the supernatant, add 15 mL of 1 M sorbitol to resuspend the bacteria; Centrifuge and discard the supernatant, add 0.75 mL of 1 M sorbitol to resuspend the bacteria; Aliquot 80 µL per tube and use immediately.

[0034] Electroporation transformation: Place a 2 mm electroporation cuvette on ice, add 10 µL of linearized plasmid; Voltage 1500 V, electroporation time > 4.5 ms; Immediately after electroporation, add 500 µL of 1 M sorbitol to rinse the bacterial solution, and recover at 30°C for 1 h; Incubate in the dark at 30°C for 2 - 3 d.

[0035] Screen positive transformants: Pick a single colony and inoculate it into 2 mL of YPD, take 1 mL of the bacterial solution, centrifuge at 6000 rpm and 4°C for 5 min to collect the bacteria; Extract the genome by benzyl chloride, and perform PCR amplification using the genomic DNA as a template with gene primers and pGAP / 3’AOX as primers respectively.

[0036] Pichia pastoris recombinant protein expression: Pick a single colony and inoculate it into 50 mL of YPD, culture at 30°C and 220 rpm for 8 d, centrifuge at 10000 rpm and 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 through a nickel column and the target protein is confirmed by molecular weight), measure the protein concentration using a BCA kit, and store it in a 4°C refrigerator for later use.

[0037] Example 3 Activity determination

[0038] Determine the LPMO activity using the DNS method. Incubate 15 μL (~1.5 μM) of the enzyme with 60 μL of a cellulose substrate solution dissolved at 5 mg / mL in 0.05 M sodium phosphate buffer (pH 5.5) at 37°C and 200 rpm for 24 h, add 75 μL of DNS, and develop color at 95°C for 10 min. After cooling to room temperature, take 100 μL and measure the OD 540 The reaction is performed in triplicate.

[0039] Standard curve drawing: Prepare a 10 mg / mL glucose stock solution, dilute it with deionized water to different concentrations (1 - 5 mg / mL). The reaction system is a mixture of 75 μL of glucose at different concentrations and 75 μL of DNS, mix well, develop color at 95°C for 10 min. After cooling to room temperature, take 100 μL and measure the OD 540, The reaction was performed in triplicate. A standard curve was plotted with glucose concentration on the x-axis and absorbance on the y-axis. The regression equation was y = 0.3418x - 0.02968 (R 2 = 0.9959).

[0040] By detailed analysis of the enzyme activities of wild-type FpLPMO9A-WT and mutants, we found significant differences in the ability to produce reducing sugars. Specifically, the amount of reducing sugars produced by FpLPMO9A-WT under the experimental conditions was 0.78 ± 0.04 μM, while N160T showed significantly higher activity, with the amount of reducing sugars produced reaching 8.56 ± 0.29 μM. The amount of reducing sugars produced by N160T was 10.8 times that of the wild type. This result indicates that the N160T mutation significantly enhanced the catalytic efficiency of the enzyme, resulting in a much higher reducing sugar yield than the wild-type level and other mutants. This may imply that the amino acid substitution at the N160 site has an important impact on the structure and function of the enzyme, thereby improving its catalytic activity.

[0041] The N160T was named mutant FpLPMO9A-N160T. Its amino acid sequence is shown in SEQ ID No.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID No.2.

[0042] The mutant FpLPMO9A-N160T with the highest enzyme activity was selected for subsequent studies.

[0043] Example 4 Enzymatic Property Analysis

[0044] (1) Optimal pH: 15 μL (~1.5 μM) of LPMO was thoroughly mixed with 60 μL of 5 mg / mL cellulose substrate prepared in a buffer with a pH of 2.2–9.0 (citrate / phosphate buffer at pH 2.2 - 8.0 and 0.2 M Tris-HCl buffer at pH 8.0 - 9.0), incubated at 37°C for 24 h, and the optimal pH was determined using the DNS method. The relative activity was analyzed with the data of the group with the highest activity taken as 100%.

[0045] The results are as Figure 2 shown. The optimal pH values of the two enzymes, FpLPMO9A-WT and FpLPMO9A-N160T, are similar, both around 5.

[0046] (2) pH tolerance: The enzyme solution was mixed with a buffer at pH 2.2 - 8.0 in a 1:1 ratio, placed on ice for 60 min, and the residual enzyme activity was measured under the optimal conditions. The pH stability was calculated with the untreated enzyme solution taken as 100%.

[0047] The results are as Figure 3As shown, in the pH range of 2 to 4, the enzyme activity of the mutant N160T is significantly higher than that of the wild type. At pH 5, the activities of both enzymes reach the maximum, and the difference between them is the smallest. In the pH range of 6 to 8, the enzyme activity of FpLPMO9A-N160T is still higher than that of the wild type, but the difference between them gradually decreases. Overall, FpLPMO9A-N160T exhibits higher enzyme activity in a wider pH range.

[0048] (3)Optimal 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 DNS method was used to determine the optimal temperature. The data of the group with the highest activity was taken as 100% for analyzing the relative activity.

[0049] The results are as Figure 4 shown. FpLPMO9A-N160T has slightly higher activity at lower temperatures. The activities of both are similar and reach the peak at around 50 °C. In the higher temperature range of 60 °C to 80 °C, FpLPMO9A-N160T shows higher thermal stability, and its activity is significantly higher than that of FpLPMO9A-WT.

[0050] (4)Temperature tolerance: After appropriately diluted enzyme solution was incubated at 50 °C for 10, 20, 40, and 60 min respectively, the residual enzyme activity was measured. The untreated group (0 min) was taken as 100% to calculate the residual activity.

[0051] The results are as Figure 5 shown. 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 is about 50% after being treated at 50 °C for 1 h, while the residual activity of the mutant is about 60% under the same conditions. The IC 50 value shows that the IC 50 value of the wild type is 64.4 min, which means that at 50 °C, it takes about 64.4 min to reduce the enzyme activity to 50%. In contrast, the IC 50 value of the mutant is 101.1 min. The thermal stability of FpLPMO9A-N160T at 50 °C is better than that of the wild type, which may mean that this mutation has a positive effect on improving the thermal stability of the enzyme.

[0052] Example 5 Synergy between LPMO and cellulase

[0053] LPMO was co-catalyzed with cellulase (from Aspergillus oryzae) for the substrate. 60 μL of the substrate, 7.5 μL (0.5 μM) of LPMO and 7.5 μL (1.0 μM) of GH were added to each experimental tube. In the groups treated with LPMO and GH alone, heat-inactivated GH and LPMO were added respectively. 15 μL of pre-boiled and inactivated enzyme solution was added to the blank tube. Four parallel experiments were conducted and incubated at 37°C for 72 h. The reaction system contained 1.5 mM ascorbic acid. After the reaction, 75 μL of DNS reagent was added and incubated at 100°C for 10 min. After cooling to room temperature, the absorbance at OD 540 was measured with a spectrophotometer. The reaction was performed in triplicate. A standard curve was plotted with glucose concentration on the abscissa and absorbance on the ordinate. The regression equation was y = 0.3418x - 0.02968 (R 2 = 0.9959).

[0054] The results are as Figure 6 shown. Compared with cellulase alone, the reducing sugar yields of the co-treatment groups cellulase + FpLPMOA-WT and cellulase + FpLPMOA-N160T were 15.23 ± 0.07 and 17.56 ± 0.52 μmol / L respectively, and the reducing sugars increased by 17.83% and 35.83% respectively. When cellulase was co-treated with FpLPMOA-N160T, the yield of reducing sugar was the highest, 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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