Lytic polysaccharide monooxygenase mutants, methods of making and use thereof

Abstract

The application discloses a lytic polysaccharide monooxygenase mutant, a preparation method and application thereof. In view of the problems of a low expression level and poor stability of a lytic polysaccharide monooxygenase in a microbial expression system, a multi-site mutant with an amino acid sequence shown in SEQ ID NO. 2 is obtained by mutating the lytic polysaccharide monooxygenase RsLPMO9, and the expression level of the multi-site mutant is obviously higher than that of a wild-type protein RsLPMO9. The analysis of thermal stability shows that the thermal stability of the multi-site mutant is obviously improved compared with that of the wild-type protein RsLPMO9. The lytic polysaccharide monooxygenase mutant provided by the application has application prospects in the degradation of biomass or plant polysaccharides.

Description

TECHNICAL FIELD

[0001] The present application relates to a lytic polysaccharide monooxygenase mutant, in particular to a mutant of lytic polysaccharide monooxygenase and its application in degrading plant polysaccharides or biomass, and belongs to the field of lytic polysaccharide monooxygenase mutants and their applications. BACKGROUND

[0002] Lytic polysaccharide monooxygenases (LPMOs) are a class of enzymes that catalyze the cleavage of polysaccharide substrates through a monooxygenation reaction. LPMOs play an important role in biomass degradation, especially in the degradation of cellulose and hemicellulose, and unlike traditional hydrolytic enzymes, they break glycosidic bonds through an oxidation reaction, thereby accelerating the decomposition of polysaccharides.

[0003] LPMOs are a class of copper-dependent enzymes, usually containing one or two copper ions in their active center, and their mechanism of action is different from that of traditional glycoside hydrolases. LPMOs do not directly hydrolyze glycosidic bonds, but change the chemical structure of glycosidic bonds through an oxidation reaction, causing the polysaccharide molecule to break. The specific mechanism is that after LPMOs bind to the substrate, they use oxygen to catalyze the reaction through copper ions, introducing an oxygen atom to the non-reducing end of the sugar molecule, thereby initiating the cleavage of the glycosidic bond, producing oligosaccharides or small sugar molecules. LPMOs bind to polysaccharide substrates (such as cellulose, hemicellulose) through their specific structure. The structure of the substrate and the binding site of LPMOs determine the selectivity of the substrate. LPMOs use copper ions and molecular oxygen to oxidize the non-reducing end of the sugar molecule. By introducing an oxygen atom, LPMOs make the carbon-oxygen bond in the glycosidic bond more fragile. After the oxidation reaction, the glycosidic bond breaks at a specific location, generating oligosaccharides, disaccharides, or smaller sugar molecules, ultimately producing small molecules suitable for further degradation.

[0004] LPMOs have important application value in accelerating the degradation of plant polysaccharides, promoting biomass conversion, and improving energy production efficiency. However, there are still many problems and key challenges in the scalable application of LPMOs in large-scale biorefining industries. The expression level of LPMOs in microbial expression systems is low, and currently high-activity, good-thermal-stability, and high-expression-level LPMOs are still scarce. SUMMARY

[0005] One of the purposes of the present application is to provide a mutant RsLPMO9 M1 of the fungal-derived lytic polysaccharide monooxygenase RsLPMO9.

[0006] The second purpose of the present application is to provide a coding gene of the mutant of the fungal-derived lytic polysaccharide monooxygenase RsLPMO9.

[0007] The third object of the present application is to provide an expression cassette containing the coding gene of the mutant, a recombinant expression vector, or a recombinant host cell containing the recombinant expression vector.

[0008] The fourth object of the present application is to apply the mutant RsLPMO9M1 of the lytic polysaccharide monooxygenase RsLPMO9, the coding gene thereof, the expression cassette containing the coding gene of the mutant, the recombinant expression vector, or the recombinant host cell containing the recombinant expression vector, etc. to degradation of plant polysaccharides or biomass.

[0009] In order to achieve the above-mentioned objects, the main technical solutions adopted by the present application include:

[0010] An aspect of the present application is to provide a multi-site mutant RsLPMO9M1 of the lytic polysaccharide monooxygenase RsLPMO9, which is a multi-site mutant obtained by performing D36G, S46Q, F47Y, N84G, P121N, N125D, S149T and D227S multi-site mutations on the lytic polysaccharide monooxygenase RsLPMO9 with the amino acid sequence shown in SEQ ID NO. 1, and has the amino acid sequence shown in SEQ ID NO. 2.

[0011] The multi-site mutant "D36G, S46Q, F47Y, N84G, P121N, N125D, S149T and D227S" of the present application means that the 36th amino acid of the lytic polysaccharide monooxygenase RsLPMO9 with the amino acid sequence shown in SEQ ID NO. 1 is mutated from aspartic acid (D) to glycine (G); the 46th amino acid is mutated from serine (S) to glutamine (Q); the 47th amino acid is mutated from phenylalanine (F) to tyrosine (Y); the 84th amino acid is mutated from asparagine (N) to glycine (G); the 121st amino acid is mutated from proline (P) to asparagine (N); the 125th amino acid is mutated from asparagine (N) to aspartic acid (D); the 149th amino acid is mutated from serine (S) to threonine (T); and the 227th amino acid is mutated from aspartic acid (D) to serine (S); and the rest of the multi-site mutations of the present application are described in the same manner.

[0012] Another aspect of the present application is to provide a coding gene of the multi-site mutant RsLPMO9M1 of the lytic polysaccharide monooxygenase RsLPMO9.

[0013] Another aspect of the present application is an expression cassette containing the coding gene of the multi-site mutant RsLPMO9 M1 of the lytic polysaccharide monooxygenase RsLPMO9, a recombinant expression vector, or a recombinant host cell containing the recombinant expression vector; wherein the recombinant expression vector can be a recombinant prokaryotic expression vector or a recombinant eukaryotic vector.

[0014] The present application further provides a method for preparing the multi-site mutant RsLPMO9 M1 of the lytic polysaccharide monooxygenase RsLPMO9, comprising:

[0015] (1) constructing a recombinant expression vector by operably linking the coding gene of the multi-site mutant of the lytic polysaccharide monooxygenase RsLPMO9 with an expression regulatory element;

[0016] (2) transforming a host cell with the recombinant expression vector, culturing the host cell, inducing the expression of the recombinant protein, and purifying, to obtain the multi-site mutant RsLPMO9 M1 of the lytic polysaccharide monooxygenase RsLPMO9.

[0017] In a preferred embodiment of the present application, the purification in step (2) is the purification of the protein using a nickel column affinity chromatography.

[0018] Another aspect of the present application is the application of the multi-site mutant RsLPMO9 M1 of the lytic polysaccharide monooxygenase RsLPMO9, the coding gene thereof, the expression cassette containing the coding gene of the multi-site mutant, the recombinant expression vector, or the recombinant host cell containing the recombinant expression vector, etc. in the degradation of plant polysaccharides or biomass; comprising: using the multi-site mutant as a catalytic enzyme to catalyze the cleavage of polysaccharide substrates through a monooxygenation reaction; or using the protein encoded by the coding gene as a catalytic enzyme to catalyze the cleavage of polysaccharide substrates through a monooxygenation reaction; or using the recombinant protein prepared by the expression cassette or the recombinant expression vector as a catalytic enzyme to catalyze the cleavage of polysaccharide substrates through a monooxygenation reaction; or using the recombinant protein prepared by the recombinant host cell as a catalytic enzyme to catalyze the cleavage of polysaccharide substrates through a monooxygenation reaction.

[0019] In a preferred embodiment of the present application, the polysaccharide is preferably a plant polysaccharide, and the plant polysaccharide includes cellulose or hemicellulose.

[0020] The present application aims at the problems of low expression level and poor stability of lytic polysaccharide monooxygenase in a microbial expression system, and three multi-site mutants, i.e., mutant RsLPMO9 M1, mutant RsLPMO9 M2 and mutant RsLPMO9 M3, are obtained by mutating lytic polysaccharide monooxygenase RsLPMO9. After protein purification electrophoresis, it is found that there is no band for the mutant RsLPMO9 M3, and the expression amount of the mutant RsLPMO9 M1 is obviously higher than that of the wild-type protein RsLPMO9. Through analysis of the thermal stability of the mutant RsLPMO9 M1, the thermal stability of the mutant RsLPMO9 M1 is obviously improved, i.e., after 30 minutes of treatment at 70 DEG C, the residual enzyme activity of the wild-type protein RsLPMO9 is 70%, and the residual enzyme activity of the mutant RsLPMO9 M1 is about 86%; after 1 hour of treatment at 70 DEG C, the residual enzyme activity of the wild-type protein RsLPMO9 is only 53%, and the residual enzyme activity of the mutant RsLPMO9 M1 is still about 86%. The lytic polysaccharide monooxygenase mutant provided by the present application has application prospect in degradation of biomass or plant polysaccharides.

[0021] Definitions of terms involved in the present invention

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods, devices and materials are now described.

[0023] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides in either single- or double-stranded form, and polymers thereof. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically indicated otherwise, the term also refers to oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, and the like). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of nucleotides or a mixture of deoxyinosine residues (Mol Cell. Probes 8:91-98 (1994)).

[0024] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to mean a polymer of amino acid residues. That is, a description to a polypeptide applies equally to a description of a peptide and to a description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as to amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terms encompass amino acid chains of any length including full-length proteins (i.e., antigens) in which the amino acid residues are connected by covalent peptide bonds.

[0025] The terms "mutation" and "mutant" have their common meaning herein and refer to a genetic, naturally occurring or introduced change in a nucleic acid or polypeptide sequence, in the sense commonly known to persons skilled in the art.

[0026] The term "recombinant host cell strain" or "host cell" means a cell which contains a polynucleotide of the present application, regardless of the method by which it was inserted into the host cell, such as direct uptake, transduction, f-mating, or other methods known in the art. The exogenous polynucleotide can remain as a non-integrated vector, such as a plasmid, or can integrate into the host genome. The host cell can be a prokaryotic or eukaryotic cell.

[0027] The term "transformation" refers to the genetic transformation of a host cell with a polynucleotide or polypeptide by introducing the coding gene into the host cell in such a way that the polynucleotide or polypeptide is genetically transformed into the host cell.

[0028] The term "expression": transcription and / or translation of an endogenous gene or a transgene in a host cell. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Figure for the three-dimensional structure model of the encoded protein of the RsLPMO9 gene.

[0030] Figure 2 Figure for the electrophoresis results of the wild-type protein RsLPMO9 and the mutant RsLPMO9 M1; wherein 1 is a protein electrophoresis molecular weight standard Marker; 2 is the fermentation supernatant of the recombinant strain containing the RsLPMO9 expression cassette; 3 is the fermentation supernatant of the recombinant strain containing the RsLPMO9 M1 expression cassette; 4 is the fermentation supernatant of the Trichoderma reesei SUS7 strain.

[0031] Figure 3 Figure for the electrophoresis results of the mutant RsLPMO9 M3; wherein 1 is a protein electrophoresis molecular weight standard Marker; 2-10 are the fermentation supernatants of the recombinant strain containing the RsLPMO9 M3 expression cassette; 11 is the fermentation supernatant of the Trichoderma reesei SUS7 strain.

[0032] Figure 4Figure of the results of the relative enzyme activity determination of the wild-type protein RsLPMO9 and the mutant RsLPMO9 M1. DETAILED DESCRIPTION

[0033] The present application will be further described below in connection with specific embodiments, and its advantages and characteristics will become more apparent from the description. However, it should be understood that the described embodiments are only exemplary, and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present application without departing from the spirit and scope of the present application, and such modifications or substitutions all fall within the protection scope of the present application.

[0034] Reagents and media

[0035] Sorbitol and PEG6000 were purchased from Sigma; 2,6-dimethylphenol (2,6-DMP) was purchased from Aldrich; recombinant enzymes were purchased from Beijing Zoman Biotech Co., Ltd.; DNA gel recovery kit was purchased from Beijing Tiangen Biotech Co., Ltd.; restriction endonuclease NotI was purchased from TaKaRa Co., Ltd.; and all other reagents were domestic analytical reagents.

[0036] Minimal medium (MM) (1 L): (NH4)2SO4 5.0 g, KH2PO4 1.5 g, MgSO4·7H2O 0.6 g, CaCl2·2H2O 0.6 g, FeSO4·7H2O 0.005 g, ZnSO4·7H2O 0.0014 g, MnSO4·H2O 0.0016 g, carbon source was 2% glucose (volume fraction), and the natural pH value.

[0037] Cellulose induction medium: the carbon source in the minimal medium was replaced with 2% microcrystalline cellulose, and the natural pH value.

[0038] LB liquid medium: 0.5% yeast extract, 1% NaCl, 1% peptone; LB solid medium additionally added 2% agar powder.

[0039] Bioinformatics analysis of lytic polysaccharide monooxygenase RsLPMO9 in test example 1

[0040] The RsLPMO9 gene co-encodes 253 amino acids, and the encoded protein of the RsLPMO9 gene has a conserved domain of the AA9 superfamily (27-243). This enzyme family was initially classified as glycoside hydrolase (GH61), and has now been reclassified as the auxiliary activity family 9 (AA9) of CAZy. The signal peptide of the encoded protein of the RsLPMO9 gene is predicted by SingnalP-5.0 (https: / / services.healthtech.dtu.dk / service.php?SignalP-5.0), and the first 25 amino acids at the N-terminus thereof are the possible signal peptide sequence thereof, and the theoretical molecular weight of the mature protein thereof is 24.3 kDa. The three-dimensional structure modeling prediction of the encoded protein of the RsLPMO9 gene is performed by SWISS-MODEL (https: / / swissmodel.expasy.org / ) using A0A0F4YK14 as a template, and the prediction result is shown in Figure 1 .

[0041] The amino acid sequence of the encoded protein RsLPMO9 of the RsLPMO9 gene is shown in SEQ ID NO. 1:

[0042]

[0043] The nucleotide sequence of the RsLPMO9 gene is shown in SEQ ID NO. 5:

[0044]

[0045]

[0046] Test Example 2 Design, screening, expression, and determination of enzymatic properties of lysing polysaccharide monooxygenase mutants

[0047] 1. Design of lysing polysaccharide monooxygenase mutants

[0048] Three protein RsLPMO9 multiple point mutants RsLPMO9M1, RsLPMO9M2 and RsLPMO9M3 are designed and screened by means of the protein expression amount prediction model MPB-EXP and the protein multiple point mutant generation model MPB-MUT fine-tuned by MP-TRANS.

[0049] The multiple point mutation positions of the mutant RsLPMO9M1 are D36G, S46Q, F47Y, N84G, P121N, N125D, S149T and D227S, respectively.

[0050] The positions of the multiple point mutations of mutant RsLPMO9 M2 are T12A, F38T, I43L, S51P, N125D, N193Q, D235N and P249A, respectively.

[0051] The positions of the multiple point mutations of mutant RsLPMO9 M3 are F11L, D36G, S40G, P55A, T91S and T133Q, respectively.

[0052] The amino acid sequence of mutant RsLPMO9 M1 is shown as SEQ ID NO. 2:

[0053]

[0054]

[0055] The nucleotide sequence of the coding gene of mutant RsLPMO9 M1 is shown as SEQ ID NO. 6:

[0056]

[0057] The amino acid sequence of mutant RsLPMO9 M2 is shown as SEQ ID NO. 3:

[0058]

[0059]

[0060] The nucleotide sequence of the coding gene of mutant RsLPMO9 M2 is shown as SEQ ID NO. 7:

[0061]

[0062] The amino acid sequence of mutant RsLPMO9 M3 is shown as SEQ ID NO. 4:

[0063]

[0064] The nucleotide sequence of the coding gene of mutant RsLPMO9 M3 is shown as SEQ ID NO. 8:

[0065]

[0066] 2 Construction and expression and purification of mutants

[0067] Trichoderma reesei transformation method: spores of Trichoderma reesei SUS7 were inoculated on PDA plates and incubated at 28°C. After 5 days, fresh spores were collected. The spore suspension was inoculated into 50 mL PDB medium and incubated at 28°C with 200 rpm shaking for 12 h. The germinated mycelium was collected by 200 mesh sieve, washed with sterile water and 0.8 mol / L MgSO4solution, and then resuspended in 20 mL cell wall lysing solution. After 3 h of digestion at 28°C with 90 rpm, the protoplasts were collected. The linearized DNA fragments were introduced into the protoplasts of Trichoderma reesei by PEG6000-mediated protoplast transformation.

[0068] The genes encoding the wild-type protein RsLPMO9 and its mutants were synthesized, and expression plasmids Ppcbh-RsLPMO9, Ppcbh-RsLPMO9 M1, Ppcbh-RsLPMO9 M2 and Ppcbh-RsLPMO9 M3 of the wild-type protein RsLPMO9 and its mutants in Trichoderma reesei were constructed. The expression cassettes of the wild-type protein RsLPMO9 and its mutants containing the promoter and terminator sequences of the CBHI gene of Trichoderma reesei were introduced into the Trichoderma reesei strain. The single colonies grown on the selection plates were transferred to 24-well plates, and the mycelium after 48 h of culture was treated with fungal lysis solution to extract the genomic DNA of the transformants. Colony PCR was performed to verify whether the expression cassettes of the wild-type protein RsLPMO9 and its mutants had been introduced into the Trichoderma reesei strain.

[0069] The electrophoresis results showed that the transformants had a single band at 1 kb, and there was no band in the negative control strain, indicating that the expression cassettes of the wild-type protein RsLPMO9 and its mutants had been integrated into the genome of Trichoderma reesei.

[0070] The Trichoderma reesei transformants were scraped from the 24-well plates with a sterile cotton swab and inoculated into the basal medium. After 48 h of culture at 28°C with 200 rpm, they were inoculated into the cellulose induction medium at 10% and cultured for another 120 h. The supernatant crude enzyme solution was collected by centrifugation at 12000 rpm for 10 min.

[0071] The wild-type protein RsLPMO9 and its mutants have 6×His tags at their ends, so the proteins were purified by Ni + -NTA nickel column affinity chromatography. The purified proteins were identified by SDS-PAGE electrophoresis and stored in a 4°C refrigerator.

[0072] The protein electrophoresis results are shown in Figure 2 、 Figure 3 The wild-type protein RsLPMO9 and the mutant RsLPMO9 M1 showed obvious bands at about 35 kDa in the fermentation supernatant. Figure 2), while the target band did not appear in the fermentation supernatant of the mutant RsLPMO9 M3 recombinant strain Figure 3 ). The target protein was purified by nickel column affinity chromatography. The purified wild-type protein RsLPMO9 and mutant RsLPMO9 M1 had LPMO enzyme activity, and the protein mass spectrum identification confirmed that the amino acid sequence of the target band was consistent with the reference sequence. The above results showed that the wild-type protein RsLPMO9 and mutant RsLPMO9 M1 successfully realized the heterologous expression in Trichoderma reesei, and the expression amount of the mutant RsLPMO9 M1 was significantly higher than that of the wild-type protein RsLPMO9. The theoretical molecular weight of the wild-type protein RsLPMO9 was about 25 kDa, but the electrophoretic bands of the purified recombinant wild-type protein RsLPMO9 and mutant RsLPMO9 M1 were larger than the theoretical molecular weight and had differences, which may be caused by different degrees of glycosylation modification of the target protein in the Trichoderma reesei host.

[0073] 3 Thermal stability analysis of the mutant

[0074] The purified wild-type protein RsLPMO9 and mutant RsLPMO9 M1 were concentrated by ultrafiltration tube. Before use, the concentrated wild-type protein RsLPMO9 or mutant RsLPMO9 M1 was mixed with CuSO4 at a molar ratio of 1:3, and incubated at 4°C for 1 h with shaking, so that the wild-type protein RsLPMO9 or mutant RsLPMO9 M1 could fully bind Cu 2+ , forming Cu 2+ saturated protein. The excess CuSO4 in the solution was removed by dialysis method: using pH 7 phosphate buffer as dialysis solution, dialysis at 4°C for 3 times. After dialysis, the enzyme solution was collected for subsequent enzyme activity and application research.

[0075] The 2,6-DMP rapid enzyme activity determination method was used to determine the enzyme activity of the wild-type protein RsLPMO9 and mutant RsLPMO9 M1 using 2,6-DMP and H2O2 as co-substrates:

[0076] (1) Prepare 116 mmol·L -1 of pH 7 phosphate buffer; 10 mmol·L -1 of 2,6-DMP solution; 5 mmol·L -1 of H2O2 solution. All solutions should be prepared and used within 12 h.

[0077] (2) Add 860 μL of phosphate buffer, 100 μL of 2,6-DMP solution and 20 μL of H2O2 solution into a centrifuge tube and mix well, and incubate at the corresponding temperature.

[0078] (3) After adding 20 μL of the sample of wild-type protein RsLPMO9 and mutant RsLPMO9 M1, mix well, and measure the increase in absorbance at a wavelength of 469 nm within 5 min (ε469=53200 L·mol -1 ·cm -1 ) to calculate the activity of the LPMO. One enzyme activity unit is defined as 1 μmol of oxidation product generated per minute under the reaction conditions.

[0079] The diluted enzyme solution was immediately placed on ice after being treated at 70°C for 5 min, 10 min, 30 min, and 60 min, respectively, and the residual enzyme activity of the treated enzyme solution was tested at room temperature. The enzyme activity of the untreated enzyme was taken as 100%, and the residual enzyme activity of the sample after being treated at different temperatures for different times was calculated to determine the stability of the enzyme at different temperatures.

[0080] The test results are shown in Table 2. Figure 4 As shown in Table 2, the thermal stability of the mutant protein RsLPMO9 M1 is significantly improved compared with the wild-type RsLPMO9 protein. After being treated at 70°C for 30 min, the residual enzyme activity of the wild-type protein RsLPMO9 is 70%, and the residual enzyme activity of the mutant RsLPMO9 M1 is about 86%. After being treated at 70°C for 1 hour, the residual enzyme activity of the wild-type protein RsLPMO9 is only 53%, while the residual enzyme activity of the mutant RsLPMO9 M1 is still about 86%, showing excellent thermal stability.

Claims

1. A multisite mutant of the polysaccharide monooxygenase RsLPMO9, RsLPMO9 M1, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

2. The encoding gene of the multisite mutant RsLPMO9 M1 as described in claim 1.

3. An expression cassette or recombinant expression vector containing the coding gene as described in claim 2.

4. A recombinant host cell containing the expression cassette or recombinant expression vector as described in claim 3.

5. A method for preparing a multisite mutant RsLPMO9M1 of the cleaving polysaccharide monooxygenase RsLPMO9 according to claim 1, characterized in that, include: Step (1): The coding gene described in claim 2 is operably linked with the expression regulatory element to construct a recombinant expression vector; Step (2): Transform the recombinant expression vector into host cells, culture the host cells, induce the expression of recombinant protein, and purify it to obtain the final product.

6. The method according to claim 5, characterized in that, The purification described in step (2) is the purification of the protein using nickel column affinity chromatography.

7. The application of the multisite mutant RsLPMO9 M1 according to claim 1 in the degradation of plant polysaccharides, characterized in that, Using the aforementioned multi-site mutant RsLPMO9 M1 as a catalytic enzyme, the plant polysaccharide substrate is cleaved via a monooxygenation reaction; the plant polysaccharide is cellulose or hemicellulose.

8. The application of the encoding gene according to claim 2 in the degradation of plant polysaccharides, characterized in that, The protein encoded by the aforementioned gene is used as a catalytic enzyme to catalyze the cleavage of plant polysaccharide substrates via a monooxygenation reaction; the plant polysaccharide is cellulose or hemicellulose.

9. The application of the expression cassette or recombinant expression vector according to claim 3 in the degradation of plant polysaccharides, characterized in that, The recombinant protein prepared using the aforementioned expression cassette or recombinant expression vector serves as a catalytic enzyme, catalyzing the cleavage of plant polysaccharide substrates via a monooxygenation reaction; the plant polysaccharide is cellulose or hemicellulose.

10. The application of the recombinant host cell according to claim 4 in the degradation of plant polysaccharides, characterized in that, The recombinant protein prepared from the recombinant host cell is used as a catalytic enzyme to catalyze the cleavage of plant polysaccharide substrates via a monooxygenation reaction; the plant polysaccharide is cellulose or hemicellulose.

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