Cracking polysaccharide monooxygenase mutant as well as preparation method and application thereof

Abstract

The invention discloses a lytic polysaccharide monooxygenase mutant as well as a preparation method and application thereof. In order to solve the problems of low expression level, poor stability and the like of the existing lysing polysaccharide monooxygenase in a microbial expression system, the lysing polysaccharide monooxygenase RsLPMO9 is mutated to obtain a multi-site mutant of which the amino acid sequence is shown as SEQ ID NO.2, and the expression quantity of the multi-site mutant is obviously higher than that of a wild type protein RsLPMO9. The analysis of the thermal stability shows that the thermal stability of the multi-site mutant is obviously improved compared with that of the wild protein RsLPMO9, and the lytic polysaccharide monooxygenase mutant provided by the invention has an application prospect in the aspects of degrading biomass or plant polysaccharide and the like.

Description

Technical Field

[0001] The present invention relates to mutants of lytic polysaccharide monooxygenase, and particularly to mutants of lytic polysaccharide monooxygenase and their applications in degrading plant polysaccharides or biomass, belonging to the field of lytic polysaccharide monooxygenase mutants and their applications. Background Art

[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. Different from traditional hydrolases, they break glycosidic bonds through an oxidation reaction, thus accelerating the decomposition of polysaccharides.

[0003] LPMOs are a class of copper-dependent enzymes, usually containing one or two copper ions in their active centers. 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 breakage of polysaccharide molecules. The specific mechanism is that after LPMOs bind to the substrate, they use oxygen to catalyze the reaction through copper ions, introduce an oxygen atom into the non-reducing end of the sugar molecule, thereby triggering the cleavage of glycosidic bonds, and generating oligosaccharides or small molecule sugars. LPMOs bind to polysaccharide substrates (such as cellulose and hemicellulose) through their specific structures. The structure of the substrate and the binding sites of LPMOs determine the substrate selectivity. LPMOs use the combined action of 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 position, generating oligosaccharides, disaccharides or smaller sugar molecules, and finally producing small molecule products suitable for further degradation.

[0004] LPMOs have important application values in accelerating the degradation of plant polysaccharides, promoting biomass conversion, improving energy production efficiency, etc. However, there are still many problems and key challenges in the scalable application of LPMOs in the large-scale biorefinery industry. The expression level of LPMOs in microbial expression systems is relatively low, and currently, LPMOs with high activity, excellent thermal stability and high expression level are still scarce, etc. Summary of the Invention

[0005] One object of the present invention is to provide a mutant RsLPMO9 M1 of a lytic polysaccharide monooxygenase RsLPMO9 derived from fungi.

[0006] Another object of the present invention is to provide a coding gene of the mutant of the lytic polysaccharide monooxygenase RsLPMO9 derived from fungi.

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

[0008] The fourth object of the present invention is to apply the mutant RsLPMO9M1 of the lytic polysaccharide monooxygenase RsLPMO9, its encoding gene, an expression cassette containing the encoding gene of the mutant, a recombinant expression vector or a recombinant host cell containing the recombinant expression vector to degrade plant polysaccharides or biomass.

[0009] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0010] One aspect of the present invention is to provide a multi-site mutant RsLPMO9M1 of the lytic polysaccharide monooxygenase RsLPMO9. The multi-site mutant RsLPMO9 M1 is a multi-site mutant obtained by subjecting the lytic polysaccharide monooxygenase RsLPMO9 with the amino acid sequence shown in SEQ ID NO.1 to multi-site mutations of D36G, S46Q, F47Y, N84G, P121N, N125D, S149T and D227S, and its amino acid sequence is shown in SEQ ID NO.2.

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

[0012] Another aspect of the present invention is to provide a gene encoding a multi-site mutant RsLPMO9 M1 of the lytic polysaccharide monooxygenase RsLPMO9.

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

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

[0015] (1) operably linking the encoding gene of the multi-site mutant of the lytic polysaccharide monooxygenase RsLPMO9 to an expression regulatory element to construct a recombinant expression vector;

[0016] (2) transforming the recombinant expression vector into a host cell, culturing the host cell, inducing the expression of the recombinant protein, and purifying to obtain the product.

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

[0018] Another aspect of the present invention is to apply the multi-site mutant RsLPMO9 M1 of the lytic polysaccharide monooxygenase RsLPMO9, its encoding gene, the expression cassette containing the encoding gene of the multi-site mutant, the recombinant expression vector or the recombinant host cell containing the recombinant expression vector, etc. to degrade plant polysaccharides or biomass; including: using the multi-site mutant as a catalytic enzyme to catalyze the cleavage of a polysaccharide substrate through a monooxygenation reaction; or using the protein encoded by the encoding gene as a catalytic enzyme to catalyze the cleavage of a polysaccharide substrate through a monooxygenation reaction; or using the recombinant protein prepared from the expression cassette or the recombinant expression vector as a catalytic enzyme to catalyze the cleavage of a polysaccharide substrate through a monooxygenation reaction; or using the recombinant protein prepared from the recombinant host cell as a catalytic enzyme to catalyze the cleavage of a polysaccharide substrate through a monooxygenation reaction.

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

[0020] In view of the problems of low expression level and poor stability of lytic polysaccharide monooxygenase in microbial expression systems, three multi-site mutants were obtained by mutating lytic polysaccharide monooxygenase RsLPMO9, namely mutant RsLPMO9 M1, mutant RsLPMO9 M2, and mutant RsLPMO9 M3. After protein purification electrophoresis, it was found that there was no band for mutant RsLPMO9 M3, while the expression level of mutant RsLPMO9 M1 was significantly higher than that of the wild-type protein RsLPMO9. Through the analysis of the thermal stability of mutant RsLPMO9 M1, the thermal stability of mutant RsLPMO9 M1 was significantly improved. That is, after treatment at 70 °C for 30 minutes, the remaining enzyme activity of the wild-type protein RsLPMO9 was 70%, and the remaining enzyme activity of mutant RsLPMO9 M1 was about 86%; after treatment at 70 °C for 1 hour, the remaining enzyme activity of the wild-type protein RsLPMO9 was only 53%, and the remaining enzyme activity of mutant RsLPMO9 M1 was still about 86%. The lytic polysaccharide monooxygenase mutants provided by the present invention have application prospects in aspects such as degrading biomass or plant polysaccharides.

[0021] Term definitions related to the present invention

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention 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 invention, the preferred methods, devices, and materials are now described.

[0023] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides in single-stranded or double-stranded form, and their polymers. Unless specifically restricted, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically restricted, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs (such as phosphorothioates, phosphoroamidates, etc.) used in antisense technology. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (including, but not limited to, degenerate codon substitutions) and complementary sequences, as well as the explicitly specified sequence. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (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 of a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The terms apply to both naturally occurring amino acid polymers and 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 linked by covalent peptide bonds.

[0025] The terms "mutation" and "mutant" have their ordinary meanings herein and refer to a genetic, naturally occurring or introduced change in a nucleic acid or polypeptide sequence, which has the same meaning as is commonly understood by a person skilled in the art.

[0026] The term "recombinant host cell line" or "host cell" means a cell that contains a polynucleotide of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, f-mating or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector such as a plasmid or may be integrated into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell.

[0027] The term "transformation" refers to the manner in which a coding gene is introduced into a host cell such that a polynucleotide or polypeptide is genetically transformed into the host cell.

[0028] The term "expression": the transcription and / or translation of an endogenous gene or a transgene in a host cell. Description of the Drawings

[0029] Figure 1 It is a three-dimensional structure model diagram of the encoded protein of the RsLPMO9 gene.

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

[0031] Figure 3 It is an electrophoresis result diagram of mutant RsLPMO9 M3; wherein, 1 is the protein electrophoresis molecular weight standard Marker; 2-10 are the supernatants of the fermentation broth of the recombinant strains containing the RsLPMO9 M3 expression cassette; 11 is the fermentation supernatant of Trichoderma reesei SUS7 strain.

[0032] Figure 4It is a graph showing the results of measuring the relative enzyme activity of wild-type protein RsLPMO9 and mutant RsLPMO9 M1. Detailed implementation manners

[0033] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the described embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. 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 invention without departing from the spirit and scope of the present invention, but these modifications or substitutions all fall within the protection scope of the present invention.

[0034] Reagents and culture media

[0035] Sorbitol and PEG6000 were purchased from Sigma; 2,6-dimethylphenol (2,6-DMP) was purchased from Aladdin; the recombinant enzyme was purchased from Beijing TransGen Biotech Co., Ltd.; the DNA gel extraction kit was purchased from Beijing Tiangen Biotech Co., Ltd.; the restriction endonuclease NotI was purchased from TaKaRa Co., Ltd.; all other reagents were domestic analytical pure reagents.

[0036] Minimal medium (MM) (1 L): (NH4)2SO4 5.0 g, KH2PO4 15 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, and the carbon source was 2% glucose (volume fraction), with natural pH.

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

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

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

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

[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] Experimental Example 2 Design, Screening, Expression and Enzymatic Property Determination of Lytic Polysaccharide Monooxygenase Mutants

[0047] 1 Design of Lytic Polysaccharide Monooxygenase Mutants

[0048] With the help of the protein expression level prediction model MPB-EXP and the protein multi-site mutant generation model MPB-MUT fine-tuned by MP-TRANS, three multi-site mutants RsLPMO9M1, RsLPMO9M2, and RsLPMO9M3 of the protein RsLPMO9 were designed and screened.

[0049] The multi-site mutation positions of the mutant RsLPMO9 M1 are: D36G, S46Q, F47Y, N84G, P121N, N125D, S149T, and D227S.

[0050] The multiple mutation positions of mutant RsLPMO9 M2 are: T12A, F38T, I43L, S51P, N125D, N193Q, D235N, and P249A.

[0051] The multiple mutation positions of mutant RsLPMO9 M3 are: F11L, D36G, S40G, P55A, T91S, and T133Q.

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

[0053]

[0054]

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

[0056]

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

[0058]

[0059]

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

[0061]

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

[0063]

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

[0065]

[0066] Construction, expression, and purification of 2 mutants

[0067] Trichoderma reesei transformation method: Inoculate the spores of Trichoderma reesei SUS7 on a PDA plate and incubate statically at 28°C. After 5 days, collect the fresh spores. Inoculate the spore suspension into 50 mL of PDB medium and incubate with shaking at 28°C and 200 rpm for 12 h. Filter through a 200-mesh sieve to collect the germinated hyphae, wash them with sterile water and 0.8 mol / L MgSO4 solution, and then resuspend them in 20 mL of cell wall lysis solution for the mycelium. Digest at 28°C and 90 rpm for 3 h and then collect the protoplasts. Mix PEG6000 and the protoplasts, incubate in an ice bath for 30 min, and incubate at room temperature for 20 min. Transfer the linearized DNA fragment into the protoplasts of Trichoderma reesei by the PEG6000-mediated protoplast transformation method.

[0068] Synthesize the coding genes of the wild-type protein RsLPMO9 and its mutants, and construct their expression plasmids Ppcbh-RsLPMO9, Ppcbh-RsLPMO9 M1, Ppcbh-RsLPMO9 M2, and Ppcbh-RsLPMO9 M3 in Trichoderma reesei respectively. Transfer the expression cassettes of the wild-type protein RsLPMO9 and its mutants containing the promoter and terminator sequences of the Trichoderma reesei CBHI coding gene into the Trichoderma reesei strain. Transfer the monoclonal colonies grown on the screening plate to a 24-well plate, and after culturing for 48 h, treat the mycelium with a fungal lysis solution to extract the genomic DNA of the transformants. Verify by colony PCR whether the expression cassettes of the wild-type protein RsLPMO9 and its mutants have been transferred into the Trichoderma reesei strain.

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

[0070] Scrape the Trichoderma reesei transformants from the 24-well plate with a sterilized cotton swab and inoculate them into a basic medium. After culturing at 28°C and 200 rpm for 48 h, inoculate them at 10% into a cellulose induction medium and culture for another 120 h, then centrifuge at 12000 rpm for 10 min to collect the crude enzyme solution of the supernatant.

[0071] Both the wild-type protein RsLPMO9 and its mutants have a 6×His tag at the end, so Ni + -NTA nickel column affinity chromatography is used to purify the protein. Identify the purified protein by SDS-PAGE electrophoresis, and store the purified protein in a 4°C refrigerator.

[0072] The protein electrophoresis results are as Figure 2 、 Figure 3 shown. An obvious band appears at around 35 kDa in the fermentation supernatant of the wild-type protein RsLPMO9 and the mutant RsLPMO9 M1 ( Figure 2),while the target band did not appear in the fermentation supernatant of the recombinant strain of mutant RsLPMO9 M3 Figure 3 )。Purify the target protein by nickel column affinity chromatography. The purified wild-type protein RsLPMO9 and mutant RsLPMO9 M1 have LPMO enzyme activity, and the amino acid sequence of the target band is confirmed to be consistent with the reference sequence by protein mass spectrometry identification. The above results show that the wild-type protein RsLPMO9 and mutant RsLPMO9 M1 have been successfully heterologously expressed in Trichoderma reesei, and the expression level of mutant RsLPMO9 M1 is significantly higher than that of the wild-type protein RsLPMO9. The theoretical molecular weight of the wild-type protein RsLPMO9 is about 25 kDa, but the electrophoretic bands of the purified recombinant wild-type protein RsLPMO9 and mutant RsLPMO9 M1 are larger than the theoretical molecular weight and different, which may be due to different degrees of glycosylation modification of the target protein in the Trichoderma reesei host

[0073] 3 Thermal stability analysis of mutants

[0074] Concentrate the purified wild-type protein RsLPMO9 and mutant RsLPMO9 M1 through an ultrafiltration tube. Before use, mix the concentrated wild-type protein RsLPMO9 or mutant RsLPMO9 M1 with CuSO4 at a molar ratio of 1:3 and incubate with shaking at 4 °C for 1 h to allow the wild-type protein RsLPMO9 or mutant RsLPMO9 M1 to fully bind to Cu 2+ to form Cu 2+ -saturated protein. Remove the excess CuSO4 in the solution by dialysis for the protein with complete binding: use a phosphate buffer with a pH of 7 as the dialysis solution and dialyze 3 times at 4 °C. After dialysis, collect the enzyme solution for subsequent enzyme activity and application research

[0075] Adopt the 2,6-DMP rapid enzyme activity assay method to measure the enzyme activities of the wild-type protein RsLPMO9 and mutant RsLPMO9 M1 using 2,6-DMP and H2O2 as co-substrates

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

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

[0078] (3) After adding 20 μL of the samples of wild-type protein RsLPMO9 and mutant RsLPMO9 M1 and mixing them, 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 LPMO. One enzyme activity unit is defined as the formation of 1 μmol of oxidation product per minute under the reaction conditions.

[0079] Dilute the enzyme solution and treat it at 70 °C for 5 min, 10 min, 30 min, and 60 min respectively, then immediately place it on ice, and measure the remaining enzyme activity of the treated enzyme solution under normal temperature conditions. Taking the enzyme activity of the untreated enzyme as 100%, calculate the remaining enzyme activity of the samples after treatment at different temperatures for different times to determine the stability of the enzyme at different temperatures.

[0080] The test results are as Figure 4 shown. The thermal stability of the mutant protein RsLPMO9 M1 is significantly improved compared with that of the wild-type RsLPMO9 protein. After treatment at 70 °C for 30 minutes, the remaining enzyme activity of the wild-type protein RsLPMO9 is 70%, and the remaining enzyme activity of the mutant RsLPMO9 M1 is about 86%; after treatment at 70 °C for 1 hour, the remaining enzyme activity of the wild-type protein RsLPMO9 is only 53%, while the remaining enzyme activity of the mutant RsLPMO9 M1 is still about 86%, showing excellent thermal stability.

Claims

1. The multi-site mutant RsLPMO9 M1 of the lytic polysaccharide monooxygenase RsLPMO9, characterized in that, Its amino acid sequence is shown as SEQ ID NO.

2.

2. The coding gene of the multi-site mutant RsLPMO9 M1 according to claim 1.

3. An expression cassette or recombinant expression vector containing the coding gene according to claim 2.

4. A recombinant host cell containing the expression cassette or recombinant expression vector according to claim 3.

5. A method for preparing a multi-site mutant RsLPMO9M1 of the lytic polysaccharide monooxygenase RsLPMO9 as claimed in claim 1, characterized in that, Comprising: (1) A recombinant expression vector is constructed by operably connecting the coding gene according to claim 2 with an expression regulatory element; (2) The recombinant expression vector is transformed into a host cell, the host cell is cultured, the recombinant protein is induced to express, and purified to obtain the product.

6. The method according to claim 5, wherein The purification in step (2) is to purify the protein by nickel column affinity chromatography.

7. Use of the multi-site mutant RsLPMO9 M1 according to claim 1 in the degradation of plant polysaccharides, comprising: Using the multi-site mutant RsLPMO9 M1 as a catalytic enzyme, the polysaccharide substrate is cleaved by a monooxygenation reaction; preferably, the polysaccharide is a plant polysaccharide; more preferably, the plant polysaccharide includes cellulose or hemicellulose.

8. Use of the coding gene according to claim 2 in plant polysaccharide degradation, comprising: Using the protein encoded by the coding gene as a catalytic enzyme, the polysaccharide substrate is cleaved by a monooxygenation reaction; preferably, the polysaccharide is a plant polysaccharide; more preferably, the plant polysaccharide includes cellulose or hemicellulose.

9. Use of the expression cassette or recombinant expression vector according to claim 3 in the degradation of plant polysaccharides, comprising: Using the recombinant protein prepared from the expression cassette or recombinant expression vector as a catalytic enzyme, the polysaccharide substrate is cleaved by a monooxygenation reaction; preferably, the polysaccharide is a plant polysaccharide; more preferably, the plant polysaccharide includes cellulose or hemicellulose.

10. Use of the recombinant host cell according to claim 4 in the degradation of plant polysaccharides, comprising: Using the recombinant protein prepared from the recombinant host cell as a catalytic enzyme, the polysaccharide substrate is cleaved by a monooxygenation reaction; preferably, the polysaccharide is a plant polysaccharide; more preferably, the plant polysaccharide includes cellulose or hemicellulose.

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