Lytic polysaccharide monooxygenase mutant RsLPMO9 M2, its preparation method and application

Abstract

The present invention discloses a lytic polysaccharide monooxygenase mutant RsLPMO9 M2, its preparation method and applications. Aiming at the problems of low expression level and poor stability of lytic polysaccharide monooxygenase in microbial expression systems, the present invention obtained a multi-site mutant with the amino acid sequence shown in SEQ ID NO.3 by performing multi-site mutations on lytic polysaccharide monooxygenase RsLPMO9. The expression level of this multi-site mutant is significantly higher than that of the wild-type protein RsLPMO9. Thermal stability analysis found that the thermal stability of this multi-site mutant is significantly improved compared with the wild-type protein RsLPMO9. The lytic polysaccharide monooxygenase mutant provided by the present invention has application prospects in aspects such as degrading plant polysaccharides or biomass.

Description

Technical Field

[0001] The present invention relates to mutants of lytic polysaccharide monooxygenase, in particular to mutants of lytic polysaccharide monooxygenase and their applications in degrading biomass or plant polysaccharides, 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 monooxygenation reactions. 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 oxidation reactions, 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 oxidation reactions, causing the breakage of polysaccharide molecules. Specifically, after LPMOs bind to the substrate, they use oxygen to catalyze the reaction through copper ions, introducing an oxygen atom into the non-reducing end of the sugar molecule, thereby triggering the cleavage of glycosidic bonds and producing 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 site 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, and improving energy production efficiency. 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. Summary of the Invention

[0005] One object of the present invention is to provide a mutant RsLPMO9 M2 of a fungal-derived lytic polysaccharide monooxygenase RsLPMO9.

[0006] Another object of the present invention is to provide a coding gene for the mutant RsLPMO9 M2 of the fungal-derived lytic polysaccharide monooxygenase RsLPMO9.

[0007] The third object of the present invention is to provide an expression cassette containing the gene encoding the mutant RsLPMO9 M2, 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 RsLPMO9 M2 of the fungal 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 RsLPMO9M2 of the lytic polysaccharide monooxygenase RsLPMO9. The multi-site mutant RsLPMO9 M2 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 T12A, F38T, I43L, S51P, N125D, N193Q, D235N and P249A, and its amino acid sequence is shown in SEQID NO.3.

[0011] In a preferred embodiment of the present invention, the multi-site mutant "T12A, F38T, I43L, S51P, N125D, N193Q, D235N, P249A" means that the amino acid sequence is SEQ ID The 12th amino acid of the lytic polysaccharide monooxygenase RsLPMO9 shown in NO.1 mutates from threonine (T) to alanine (A), the 38th amino acid mutates from phenylalanine (F) to threonine (T), the 43rd amino acid mutates from isoleucine (I) to leucine (L), the 51st amino acid mutates from serine (S) to proline (P), the 125th amino acid mutates from asparagine (N) to aspartic acid (D), the 193rd amino acid mutates from asparagine (N) to glutamine (Q), the 235th amino acid mutates from aspartic acid (D) to asparagine (N), and the 249th amino acid mutates from proline (P) to alanine (A); 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 M2 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 coding gene of the multi-site mutant RsLPMO9 M2 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 M2 of the lytic polysaccharide monooxygenase RsLPMO9, comprising:

[0015] (1) operably linking the coding gene of the multi-site mutant RsLPMO9 M2 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 M2 of the lytic polysaccharide monooxygenase RsLPMO9, its coding gene, an expression cassette containing the coding gene of the multi-site mutant, a recombinant expression vector or a recombinant host cell containing the recombinant expression vector, etc. to the degradation of plant polysaccharides or biomass, including: using the multi-site mutant as a catalytic enzyme to catalyze the cleavage of a polysaccharide substrate or biomass through a monooxygenation reaction; or using the protein encoded by the coding gene as a catalytic enzyme to catalyze the cleavage of a polysaccharide substrate or biomass 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 or biomass 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 or biomass through a monooxygenation reaction.

[0019] In a preferred specific embodiment of the present invention, the polysaccharide is a plant polysaccharide; 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 at present, three multi-site mutants, namely mutant RsLPMO9 M1, mutant RsLPMO9 M2, and mutant RsLPMO9 M3, were obtained by mutating the lytic polysaccharide monooxygenase RsLPMO9. After protein purification and electrophoresis, it was found that there was no band for mutant RsLPMO9 M3, while the expression level of mutant RsLPMO9 M2 was significantly higher than that of the wild-type protein RsLPMO9. Through the analysis of the thermal stability of mutant RsLPMO9 M2, it was found that the thermal stability of mutant RsLPMO9 M2 was significantly improved compared with that of the wild-type protein RsLPMO9: after treatment at 70 °C for 30 minutes, the remaining enzyme activity of the wild-type protein RsLPMO9 was 70%, while the remaining enzyme activity of mutant RsLPMO9 M2 was about 79%; after treatment at 70 °C for 1 hour, the remaining enzyme activity of the wild-type protein RsLPMO9 was only 53%, while the remaining enzyme activity of mutant RsLPMO9 M2 was about 75%. The lytic polysaccharide monooxygenase mutants provided by the present invention have application prospects in degrading plant polysaccharides or biomass and the like.

[0021] Term definitions involved in 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 limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to those of reference nucleic acids and are metabolized in a manner similar to that of naturally occurring nucleotides. Unless otherwise specifically limited, 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 sequences. 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 bases and / or 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 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 via covalent peptide bonds.

[0025] The terms "mutation" and "mutant" have their ordinary meanings herein, referring to a genetic, naturally occurring, or introduced change in a nucleic acid or polypeptide sequence, the meaning of which is the same as that commonly known to those skilled in the art.

[0026] The term "recombinant host cell line" or "host cell" means a cell that contains the 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 remain as a non-integrated 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 of introducing a coding gene into the interior of a host cell to genetically transform a polynucleotide or polypeptide 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 THE DRAWINGS

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

[0030] Figure 2 is the electrophoretic result diagram of wild-type protein RsLPMO9 and mutant RsLPMO9 M2; 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 RsLPMO9M2 expression cassette; 4 is the fermentation supernatant of Trichoderma reesei SUS7 strain.

[0031] Figure 3 is the electrophoretic 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 4This is the graph showing the relative enzyme activity assay results of wild-type protein RsLPMO9 and mutant RsLPMO9 M2. 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 as the description proceeds. However, it should be understood that the described embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements 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; recombinant enzymes were purchased from Beijing TransGen Biotech Co., Ltd.; DNA gel extraction kits were purchased from Beijing Tiangen Biotech Co., Ltd.; restriction endonuclease Not I was purchased from TaKaRa Co., Ltd.; all other reagents were domestic analytical pure reagents.

[0036] Basic medium (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), natural pH value.

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

[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] RsLPMO9 The gene encodes a total of 253 amino acids, RsLPMO9The encoded protein of the 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) predicts RsLPMO9 the signal peptide of the encoded protein of the 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 / ) with A0A0F4YK14 as the template for RsLPMO9 the three-dimensional structure modeling prediction of the encoded protein of the gene, the prediction results are as Figure 1 shown.

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

[0042] RsLPMO9The nucleotide sequence of the gene is shown in SEQ ID NO.5: ATGCTGTCTTCGACGACTCGCACCCTCGCCTTTACAGGCCTTGCGGGCCTTCTGTCCGCTCCCCTGGTCAAGGCCCATGGCTTTGTCCAGGGCATTGTCATCGGTGACCAATTGTAAGTCCCTCTCTTGCAGTTCTGTCGATTAACTGCTGGACTGCTTGCTTGACTCCCTGCTGACTCCCAACAGCTACAGCGGGTACATCGTCAACTCGTTCCCCTACGAATCCAACCCACCCCCCGTCATCGGCTGGGCCACGACCGCCACCGACCTGGGCTTCGTCGACGGCACAGGATACCAAGGCCCGGACATCATCTGCCACCGGAATGCGACGCCCGCGCCGCTGACAGCCCCCGTGGCCGCCGGCGGCACCGTCGAGCTGCAGTGGACGCCGTGGCCGGACAGCCACCACGGACCCGTCATCACCTACCTGGCGCCGTGCAACGGCAACTGCTCGACCGTCGACAAGACGACGCTGGAGTTCTTCAAGATCGACCAGCAGGGCCTGATCGACGACACGAGCCCGCCGGGCACCTGGGCGTCGGACAACCTCATCGCCAACAACAATAGCTGGACCGTCACCATTCCCAACAGCGTCGCCCCCGGCAACTACGTCCTGCGCCACGAGATCATCGCCCTGCACTCGGCCAACAACAAGGACGGCGCCCAGAACTACCCCCAGTGCATCAACATCGAGGTCACGGGCGGCGGCTCCGACGCGCCTGAGGGTACTCTGGGCGAGGATCTCTACCATGACACCGACCCGGGCATTCTGGTCGACATTTACGAGCCCATTGCGACGTATACCATTCCGGGGCCGCCTGAGCCGACGTTCCATCACCACCACCACCACTAG (SEQ ID NO.5).

[0043] Experimental Example 2 Design, Screening, Expression and Enzymatic Property Determination of Lytic Polysaccharide Monooxygenase Mutants

[0044] 1 Design of Lytic Polysaccharide Monooxygenase Mutants

[0045] Three multi-site mutants of the protein RsLPMO9, namely RsLPMO9 M1, RsLPMO9 M2, and RsLPMO9 M3, were designed and screened using the protein expression level prediction model MPB-EXP and the protein multi-site mutant generation model MPB-MUT fine-tuned by MP-TRANS.

[0046] The multi-site mutation positions of the mutant RsLPMO9 M1 are: D36G, S46Q, F47Y, N84G, P121N, N125D, S149T, and D227S.

[0047] The multi-site mutation positions of the mutant RsLPMO9 M2 are: T12A, F38T, I43L, S51P, N125D, N193Q, D235N, and P249A.

[0048] The multi-site mutation positions of the mutant RsLPMO9 M3 are: F11L, D36G, S40G, P55A, T91S, and T133Q.

[0049] The amino acid sequence of the mutant RsLPMO9 M1 is shown in SEQ ID NO.2: MLSSTTRTLAFTGLAGLLSAPLVKAHGFVQGIVIGGQFYSGYIVNQYPYESNPPPVIGWATTATDLGFVDGTGYQGPDIICHRGATPAPLTAPVAAGGTVELQWTPWPDSHHGPVITYLANCNGDCSTVDKTTLEFFKIDQQGLIDDTTPPGTWASDNLIANNNSWTVTIPNSVAPGNYVLRHEIIALHSANNKDGAQNYPQCINIEVTGGGSDAPEGTLGEDLYHSTDPGILVDIYEPIATYTIPGPPEPTF (SEQ ID NO.2).

[0050] The nucleotide sequence of the coding gene of mutant RsLPMO9 M1 is shown in SEQ ID NO.6: ATGCTGTCTTCGACGACTCGCACCCTCGCCTTTACAGGCCTTGCGGGCCTTCTGTCCGCTCCCCTGGTCAAGGCCCATGGCTTTGTCCAGGGCATTGTCATCGGTGGCCAATTGTAAGTCCCTCTCTTGCAGTTCTGTCGATTAACTGCTGGACTGCTTGCTTGACTCCCTGCTGACTCCCAACAGCTACAGCGGGTACATCGTCAACCAGTACCCCTACGAATCCAACCCACCCCCCGTCATCGGCTGGGCCACGACCGCCACCGACCTGGGCTTCGTCGACGGCACAGGATACCAAGGCCCGGACATCATCTGCCACCGGGGCGCGACGCCCGCGCCGCTGACAGCCCCCGTGGCCGCCGGCGGCACCGTCGAGCTGCAGTGGACGCCGTGGCCGGACAGCCACCACGGACCCGTCATCACCTACCTGGCGAACTGCAACGGCGACTGCTCGACCGTCGACAAGACGACGCTGGAGTTCTTCAAGATCGACCAGCAGGGCCTGATCGACGACACGACCCCGCCGGGCACCTGGGCGTCGGACAACCTCATCGCCAACAACAATAGCTGGACCGTCACCATTCCCAACAGCGTCGCCCCCGGCAACTACGTCCTGCGCCACGAGATCATCGCCCTGCACTCGGCCAACAACAAGGACGGCGCCCAGAACTACCCCCAGTGCATCAACATCGAGGTCACGGGCGGCGGCTCCGACGCGCCTGAGGGTACTCTGGGCGAGGATCTCTACCATTCCACCGACCCGGGCATTCTGGTCGACATTTACGAGCCCATTGCGACGTATACCATTCCGGGGCCGCCTGAGCCGACGTTCCATCACCACCACCACCACTAG (SEQ ID NO.6).

[0051] The amino acid sequence of mutant RsLPMO9 M2 is shown in SEQ ID NO.3: MLSSTTRTLAFAGLAGLLSAPLVKAHGFVQGIVIGDQTYSGYLVNSFPYEPNPPPVIGWATTATDLGFVDGTGYQGPDIICHRNATPAPLTAPVAAGGTVELQWTPWPDSHHGPVITYLAPCNGDCSTVDKTTLEFFKIDQQGLIDDTSPPGTWASDNLIANNNSWTVTIPNSVAPGNYVLRHEIIALHSANQKDGAQNYPQCINIEVTGGGSDAPEGTLGEDLYHDTDPGILVNIYEPIATYTIPGPAEPTF (SEQ ID NO.3).

[0052] The nucleotide sequence of the coding gene of mutant RsLPMO9 M2 is shown in SEQ ID NO.7: ATGCTGTCTTCGACGACTCGCACCCTCGCCTTTGCCGGCCTTGCGGGCCTTCTGTCCGCTCCCCTGGTCAAGGCCCATGGCTTTGTCCAGGGCATTGTCATCGGTGACCAAACGTAAGTCCCTCTCTTGCAGTTCTGTCGATTAACTGCTGGACTGCTTGCTTGACTCCCTGCTGACTCCCAACAGCTACAGCGGGTACCTGGTCAACTCGTTCCCCTACGAACCCAACCCACCCCCCGTCATCGGCTGGGCCACGACCGCCACCGACCTGGGCTTCGTCGACGGCACAGGATACCAAGGCCCGGACATCATCTGCCACCGGAATGCGACGCCCGCGCCGCTGACAGCCCCCGTGGCCGCCGGCGGCACCGTCGAGCTGCAGTGGACGCCGTGGCCGGACAGCCACCACGGACCCGTCATCACCTACCTGGCGCCGTGCAACGGCGACTGCTCGACCGTCGACAAGACGACGCTGGAGTTCTTCAAGATCGACCAGCAGGGCCTGATCGACGACACGAGCCCGCCGGGCACCTGGGCGTCGGACAACCTCATCGCCAACAACAATAGCTGGACCGTCACCATTCCCAACAGCGTCGCCCCCGGCAACTACGTCCTGCGCCACGAGATCATCGCCCTGCACTCGGCCAACCAGAAGGACGGCGCCCAGAACTACCCCCAGTGCATCAACATCGAGGTCACGGGCGGCGGCTCCGACGCGCCTGAGGGTACTCTGGGCGAGGATCTCTACCATGACACCGACCCGGGCATTCTGGTCAACATTTACGAGCCCATTGCGACGTATACCATTCCGGGGCCGGCCGAGCCGACGTTCCATCACCACCACCACCACTAG (SEQ ID NO.7).

[0053] The amino acid sequence of mutant RsLPMO9 M3 is shown in SEQ ID NO.4: MLSSTTRTLALTGLAGLLSAPLVKAHGFVQGIVIGGQFYGGYIVNSFPYESNPPAVIGWATTATDLGFVDGTGYQGPDIICHRNATPAPLSAPVAAGGTVELQWTPWPDSHHGPVITYLAPCNGNCSTVDKTQLEFFKIDQQGLIDDTSPPGTWASDNLIANNNSWTVTIPNSVAPGNYVLRHEIIALHSANNKDGAQNYPQCINIEVTGGGSDAPEGTLGEDLYHDTDPGILVDIYEPIATYTIPGPPEPTF (SEQ ID NO.4).

[0054] The nucleotide sequence of the encoding gene of mutant RsLPMO9 M3 is shown in SEQ ID NO.8: ATGCTGTCTTCGACGACTCGCACCCTCGCCCTGACAGGCCTTGCGGGCCTTCTGTCCGCTCCCCTGGTCAAGGCCCATGGCTTTGTCCAGGGCATTGTCATCGGTGGCCAATTGTAAGTCCCTCTCTTGCAGTTCTGTCGATTAACTGCTGGACTGCTTGCTTGACTCCCTGCTGACTCCCAACAGCTACGGCGGGTACATCGTCAACTCGTTCCCCTACGAATCCAACCCACCCGCCGTCATCGGCTGGGCCACGACCGCCACCGACCTGGGCTTCGTCGACGGCACAGGATACCAAGGCCCGGACATCATCTGCCACCGGAATGCGACGCCCGCGCCGCTGTCCGCCCCCGTGGCCGCCGGCGGCACCGTCGAGCTGCAGTGGACGCCGTGGCCGGACAGCCACCACGGACCCGTCATCACCTACCTGGCGCCGTGCAACGGCAACTGCTCGACCGTCGACAAGACGCAGCTGGAGTTCTTCAAGATCGACCAGCAGGGCCTGATCGACGACACGAGCCCGCCGGGCACCTGGGCGTCGGACAACCTCATCGCCAACAACAATAGCTGGACCGTCACCATTCCCAACAGCGTCGCCCCCGGCAACTACGTCCTGCGCCACGAGATCATCGCCCTGCACTCGGCCAACAACAAGGACGGCGCCCAGAACTACCCCCAGTGCATCAACATCGAGGTCACGGGCGGCGGCTCCGACGCGCCTGAGGGTACTCTGGGCGAGGATCTCTACCATGACACCGACCCGGGCATTCTGGTCGACATTTACGAGCCCATTGCGACGTATACCATTCCGGGGCCGCCTGAGCCGACGTTCCATCACCACCACCACCACTAG (SEQ ID NO.8).

[0055] 2 Construction, Expression and Purification of Mutants

[0056] Transformation method of Trichoderma reesei: Inoculate the spores of Trichoderma reesei SUS7 on a PDA plate and incubate statically at 28 °C. Collect fresh spores after 5 d. Inoculate the spore suspension into 50 mL of PDB medium and incubate with shaking at 28 °C and 200 rpm for 12 h. Filter and collect the germinated hyphae through a 200-mesh sieve, 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 for 3 h at 28 °C and 90 rpm to collect protoplasts. Mix PEG6000 and 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 PEG6000-mediated protoplast transformation method.

[0057] Synthesize the coding genes of 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 wild-type protein RsLPMO9 and its mutants containing the promoter and terminator sequences of the coding genes into the Trichoderma reesei strain. Transfer the monoclonal colonies grown on the screening plate into a 24-well plate, and extract the genomic DNA of the transformants after culturing the cells for 48 h with fungal lysis solution. Verify whether the expression cassettes of wild-type protein RsLPMO9 and its mutants have been transferred into the Trichoderma reesei strain by colony PCR. CBHI 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 wild-type protein RsLPMO9 and its mutants have been integrated into the genome of Trichoderma reesei.

[0058] Scrape the Trichoderma reesei transformants from the 24-well plate with a sterilized cotton swab and inoculate them into the medium containing basal medium. Culture at 28 °C and 200 rpm for 48 h, then inoculate at 10% into the cellulose induction medium and culture for another 120 h. Centrifuge at 12000 rpm for 10 min to collect the crude enzyme solution in the supernatant.

[0059] Both the wild-type protein RsLPMO9 and its mutants have a 6×His tag at the end, so Ni

[0060] -NTA nickel column affinity chromatography is used to purify the protein. The purified protein is identified by SDS-PAGE electrophoresis, and the purified protein is stored in a 4 °C refrigerator. + The protein electrophoresis results are as

[0061] shown in Figure 2 and Figure 3As shown, an obvious band appeared around 35 kDa in the fermentation supernatant of the wild-type protein RsLPMO9 and the recombinant strain of the mutant RsLPMO9 M2 ( Figure 2 ), while the target band did not appear in the fermentation supernatant of the recombinant strain of the mutant RsLPMO9 M3 ( Figure 3 ). The target protein was purified by nickel column affinity chromatography. The purified wild-type protein RsLPMO9 and mutant RsLPMO9 M2 had LPMO enzyme activity, and the amino acid sequence of the target band was confirmed to be consistent with the reference sequence by protein mass spectrometry. The above results showed that the wild-type protein RsLPMO9 and the mutant RsLPMO9 M2 were successfully heterologously expressed in Trichoderma reesei, and the expression level of the mutant RsLPMO9 M2 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. The electrophoretic bands of the purified recombinant wild-type protein RsLPMO9 and mutant RsLPMO9 M2 were larger than the theoretical molecular weight and were different, probably due to different degrees of glycosylation modification of the target protein in the Trichoderma reesei host.

[0062] 3 Thermal stability analysis of mutants

[0063] The purified wild-type protein RsLPMO9 and mutant RsLPMO9 M2 were concentrated by ultrafiltration tubes. Before use, the concentrated wild-type protein RsLPMO9 or mutant RsLPMO9 M2 was mixed with CuSO4 at a molar ratio of 1:3 and incubated with shaking at 4 °C for 1 h to allow the wild-type protein RsLPMO9 and mutant RsLPMO9 M2 to fully bind to Cu 2+ to form Cu 2+ -saturated protein. The excess CuSO4 in the solution was removed from the bound protein by dialysis: a phosphate buffer with a pH of 7 was used as the dialysis solution, and dialysis was performed 3 times at 4 °C. After dialysis, the enzyme solution was collected for subsequent enzyme activity and application studies.

[0064] The 2,6-DMP rapid enzyme activity assay method was used to measure the enzyme activities of the wild-type protein RsLPMO9 and mutant RsLPMO9 M2 with 2,6-DMP and H2O2 as co-substrates:

[0065] (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 ; and an H2O2 solution with a concentration of 5 mmol·L -1 . All solutions should be used within 12 h after preparation.

[0066] (2) Add 860 μL of phosphate buffer, 100 μL of 2,6-DMP solution, and 20 μL of H2O2 solution into a centrifuge tube, mix well, and incubate at the corresponding temperature.

[0067] (3) After adding 20 μL of the samples of wild-type protein RsLPMO9 and mutant RsLPMO9 M2, mix well, and measure the increase in absorbance at 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.

[0068] 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 test the remaining enzyme activity of the treated enzyme solution at room temperature. 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.

[0069] The test results are as Figure 4 shown. The thermal stability of mutant RsLPMO9 M2 is significantly improved compared to wild-type protein RsLPMO9: after treatment at 70 °C for 30 minutes, the remaining enzyme activity of wild-type protein RsLPMO9 is 70%, and the remaining enzyme activity of mutant RsLPMO9 M2 is about 79%; after treatment at 70 °C for 1 hour, the remaining enzyme activity of wild-type protein RsLPMO9 is only 53%, and the remaining enzyme activity of mutant RsLPMO9 M2 is about 75%.

Claims

1. The multi-site mutant RsLPMO9 M2 of the lytic polysaccharide monooxygenase RsLPMO9, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

3.

2. The coding gene of the multi-site mutant RsLPMO9 M2 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 RsLPMO9M2 of the lytic polysaccharide monooxygenase RsLPMO9 as claimed in claim 1, characterized in that, Comprising: (1) A recombinant expression vector is constructed by operably linking 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 be expressed, and purified to obtain the product.

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

7. Use of the multi-site mutant RsLPMO9 M2 according to claim 1 in the degradation of plant polysaccharides, including: Using the multi-site mutant RsLPMO9 M2 as a catalytic enzyme, the polysaccharide substrate is cleaved by a monooxygenation reaction; wherein, the 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; wherein, the 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; wherein, the polysaccharide includes cellulose or hemicellulose.

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

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