Cracking polysaccharide monooxygenase mutant RsLPMO9M2 as well as preparation method and application thereof

Abstract

The invention discloses a cleavable polysaccharide monooxygenase mutant RsLPMO9 M2 as well as a preparation method and application of the cleavable polysaccharide monooxygenase mutant RsLPMO9 M2. 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 subjected to multi-site mutation to obtain a multi-site mutant with an amino acid sequence as shown in SEQ ID NO.3, and the expression quantity of the multi-site mutant is obviously higher than that of a wild type protein RsLPMO9. Thermal stability analysis shows that the thermal stability of the multi-site mutant is obviously improved compared with that of a wild protein RsLPMO9, and the cleavage polysaccharide monooxygenase mutant provided by the invention has an application prospect in the aspects of degrading plant polysaccharide or biomass and the like.

Description

Technical Field

[0001] The invention relates to a lytic polysaccharide monooxygenase mutant, in particular to a lytic polysaccharide monooxygenase mutant and application thereof in degrading biomass or plant polysaccharides, belonging to the field of lytic polysaccharide monooxygenase mutants and applications thereof. 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, and unlike traditional hydrolases, they break glycosidic bonds through oxidation reactions, thereby accelerating the decomposition of polysaccharides.

[0003] LPMOs are a class of enzymes that rely on copper ions and usually contain 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 polysaccharide molecules to break. The specific mechanism is that after LPMOs bind to the substrate, they use oxygen to introduce oxygen atoms into the non-reducing end of the sugar molecule through copper ion catalysis, 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 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 position to generate oligosaccharides, disaccharides or smaller sugar molecules, and finally produce small molecule products 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 the large-scale biorefining industry. The expression level of LPMOs in microbial expression systems is low, and LPMOs with high activity, excellent thermal stability and high expression level are still scarce. Summary of the invention

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

[0006] The second object of the present invention is to provide a gene encoding a 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: 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.

[0010] 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.

[0011] Another aspect of the present invention is to provide a gene encoding a multi-site mutant RsLPMO9 M2 of the lytic polysaccharide monooxygenase RsLPMO9.

[0012] Another aspect of the present invention is an expression cassette, a recombinant expression vector or a recombinant host cell containing the recombinant expression vector containing the gene encoding 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.

[0013] The present invention further provides a method for preparing the multi-site mutant RsLPMO9 M2 of the lytic polysaccharide monooxygenase RsLPMO9, comprising: (1) The coding gene of the multi-site mutant RsLPMO9 M2 of the lytic polysaccharide monooxygenase RsLPMO9 is operably connected to an expression regulatory element to construct a recombinant expression vector; (2) Transforming the recombinant expression vector into host cells, culturing the host cells, inducing the expression of the recombinant protein, and purifying the recombinant protein.

[0014] In a preferred embodiment of the present invention, the purification in step (2) is to purify the protein using nickel column affinity chromatography.

[0015] Another aspect of the present invention is to apply the multi-site mutant RsLPMO9 M2 of the lytic polysaccharide monooxygenase RsLPMO9, its encoding gene, an expression cassette containing the encoding 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 polysaccharide substrates or biomass through a monooxygenation reaction; or using the protein encoded by the encoding gene as a catalytic enzyme to catalyze the cleavage of polysaccharide substrates or biomass through a monooxygenation reaction; or using the recombinant protein prepared by the expression cassette or recombinant expression vector as a catalytic enzyme to catalyze the cleavage of polysaccharide substrates or biomass 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 or biomass through a monooxygenation reaction.

[0016] In a preferred embodiment of the present invention, the polysaccharide is a plant polysaccharide; the plant polysaccharide includes cellulose or hemicellulose.

[0017] In view of the problems of low expression level and poor stability of the current lytic polysaccharide monooxygenase in the microbial expression system, the present invention obtains three multi-site mutants by mutating the lytic polysaccharide monooxygenase RsLPMO9, namely mutant RsLPMO9 M1, mutant RsLPMO9 M2 and mutant RsLPMO9 M3. After protein purification and electrophoresis, it was found that the mutant RsLPMO9 M3 had no bands, while the expression level of the mutant RsLPMO9 M2 was significantly higher than that of the wild-type protein RsLPMO9. Through the analysis of the thermal stability of the mutant RsLPMO9 M2, it was found that the thermal stability of the mutant RsLPMO9 M2 was significantly improved compared with the wild-type protein RsLPMO9: after treatment at 70°C for 30 minutes, the residual enzyme activity of the wild-type protein RsLPMO9 was 70%, while the residual enzyme activity of the mutant RsLPMO9 M2 was about 79%; after treatment at 70°C for 1 hour, the residual enzyme activity of the wild-type protein RsLPMO9 was only 53%, while the residual enzyme activity of the mutant RsLPMO9 M2 was about 75%. The lytic polysaccharide monooxygenase mutant provided by the present invention has application prospects in the degradation of plant polysaccharides or biomass.

[0018] Definitions of terms used in this invention 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 the 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, preferred methods, devices and materials are now described.

[0019] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.). Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. In particular, degenerate codon substitutions ( Mol Cell. Probes 8:91-98 (1994)).

[0020] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description directed to a polypeptide is equally applicable to describing a peptide and describing a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as 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.

[0021] The terms "mutation" and "mutant" have their ordinary meanings herein and refer to genetic, naturally occurring or introduced changes in nucleic acid or polypeptide sequences, and their meanings are the same as those generally known to those skilled in the art.

[0022] The term "recombinant host cell strain" or "host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used for insertion to produce a 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.

[0023] The term "transformation" refers to the genetic transfer of a polynucleotide or polypeptide into a host cell in such a way that the encoding gene is introduced into the interior of the host cell.

[0024] The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgene in a host cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 for RsLPMO9 A three-dimensional structural model of the protein encoded by a gene.

[0026] Figure 2 The figures are the electrophoresis results of wild-type protein RsLPMO9 and mutant RsLPMO9 M2; wherein, 1 is the 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 RsLPMO9M2 expression cassette; 4 is the fermentation supernatant of Trichoderma reesei SUS7 strain.

[0027] Figure 3 The figure is the electrophoresis result of mutant RsLPMO9 M3; wherein, 1 is the protein electrophoresis molecular weight standard Marker; 2-10 are the fermentation supernatant of the recombinant strain containing the RsLPMO9 M3 expression cassette; 11 is the fermentation supernatant of Trichoderma reesei SUS7 strain.

[0028] Figure 4This is the relative enzyme activity test results of the wild-type protein RsLPMO9 and the mutant RsLPMO9 M2. DETAILED DESCRIPTION

[0029] 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 embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0030] Reagents and culture media Sorbitol and PEG6000 were purchased from Sigma; 2,6-dimethylphenol (2,6-DMP) was purchased from Aladdin; recombinant enzyme was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; DNA gel recovery kit was purchased from Beijing Tiangen Biotechnology Co., Ltd.; restriction endonuclease Not I was purchased from TaKaRa Co., Ltd.; all other reagents were domestic analytical grade reagents.

[0031] 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, carbon source is 2% glucose (volume fraction), natural pH.

[0032] Cellulose induction medium: replace the carbon source in the basal medium with 2% microcrystalline cellulose, natural pH.

[0033] LB liquid medium: 0.5% yeast extract, 1% NaCl, 1% peptone; LB solid medium is supplemented with 2% agar powder.

[0034] Experimental Example 1 Bioinformatics Analysis of Lytic Polysaccharide Monooxygenase RsLPMO9 RsLPMO9 The gene encodes a total of 253 amino acids. RsLPMO9The protein encoded by the gene has a conserved domain of the AA9 superfamily (positions 27-243). This enzyme family was originally classified as glycoside hydrolases (GH61), but 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) prediction RsLPMO9 The signal peptide of the gene encoding protein has the first 25 amino acids at the N-terminus as its possible signal peptide sequence, and the theoretical molecular weight of its mature protein is 24.3 kDa. RsLPMO9 The three-dimensional structure modeling of the gene's coding protein is predicted, and the prediction results are as follows Figure 1 shown.

[0035] RsLPMO9 The amino acid sequence of the gene-encoded protein RsLPMO9 is shown in SEQ ID NO.1: MLSSTTRTLAFTGLAGLLSAPLVKAHGFVQGIVIGDQFYSGYIVNSFPYESNPPPVIGWATTATDLGFVDGTGYQGPDIICHRNATPAPLTAPVAAGGTVELQWTPWPDSHHGPVITYLAPCNGNCSTVDKTTLEFFKIDQQGLIDDTSPPGTWASDNLIANNNSWTVTIPNSVAPGNYVLRHEIIALHSANNKDGAQNYPQCINIEVTGGGSDAPEGTLGEDLYHDTDPGILVDIYEPIATYTIPGPPEPTF (SEQ ID NO.1).

[0036] RsLPMO9The nucleotide sequence of the gene such as SEQ ID Shown in NO.5: ATGCTGTCTTCGACGACTCGCACCCTCGCCTTTACAGGCCTTGCGGGCCTTCTGTCCGCTCCCCTGGTCAAGGCCCATGGCTTTGTCCAGGGCATTGTCATCGGTGACCAATTGTAAGTCCCTCTCTTGCAGTTCTGTCGATTAACTGCTGGACTGCTTGCTTGACTCCCTGCTGACTCCCAACAGCTACAGCGGGTACATCGTCAAC TCGTTCCCCTACGAATCCAACCCACCCCCCGTCATCGGCTGGGCCACGACCGCCACCGACCTGGGCTTCGTCGACGGCACAGGATACCAAGGCCCGGACATCATCTGCCACCGGAATGCGACGCCCGCGCCGCTGACAGCCCCCGTGGCCGCCGGCGGCACCGTCGAGCTGCAGTGGACGCCGTGGCCGGACAGCCACCACGGACCCGTCATCAC CTACCTGGCGCCGTGCAACGGCAACTGCTCGACCGTCGACAAGACGACGCTGGAGTTCTTCAAGATCGACCAGCAGGGCCTGATCGACGACACGAGCCCGCCGGGCACCTGGGCGTCGGACAACCTCATCGCCAACAACAAATAGCTGGACCGTCACCATTCCCAACAGCGTCGCCCCCGGCAACTACGTCCTGCGCCACGAGATCATCGCCCTGC ACTCGGCCAACAACAAGGACGGCGCCCAGAACTACCCCCAGTGCATCAACATCGAGGTCACGGGCGGCGGCTCCGACGCGCCTGAGGGTACTCTGGGCGAGGATCTCTACCATGACACCGACCCGGGCATTCTGGTCGACATTTACGAGCCCATTGCGACGTATACCATTCCGGGGCCGCCTGAGCCGACGTTCCATCACCACCACCACCACTAG (SEQ ID NO. 5).

[0037] Experimental Example 2 Design, screening, expression and enzymatic performance determination of lytic polysaccharide monooxygenase mutants 1 Design of lytic polysaccharide monooxygenase mutants With the help of the protein expression prediction model MPB-EXP fine-tuned by MP-TRANS and the protein multi-point mutant generation model MPB-MUT, three multi-point mutants of the protein RsLPMO9, RsLPMO9 M1, RsLPMO9 M2 and RsLPMO9M3, were designed and screened.

[0038] The multiple mutation positions of the mutant RsLPMO9 M1 are: D36G, S46Q, F47Y, N84G, P121N, N125D, S149T and D227S.

[0039] The multiple mutation positions of mutant RsLPMO9 M2 are T12A, F38T, I43L, S51P, N125D, N193Q, D235N and P249A.

[0040] The multiple mutation positions of mutant RsLPMO9 M3 are: F11L, D36G, S40G, P55A, T91S and T133Q.

[0041] The amino acid sequence of mutant RsLPMO9 M1 is as SEQ ID Shown in NO.2: MLSSTRTLAFTGLAGLSAPLVKAHGFVQGIVIGGQFYSGYIVNQYPYESNPPPVIGWATTATDLGFVDGTGYQGPDIICHRGATPAPLTAPVAAGGTVELQWTPWPDSHHGPVITYLANCNGD CSTVDKTTLEFFKIDQQGLIDDTTPPGTWASDNLIANNNSWTVTIPNSVAPGNYVLRHEIIALHSANNKDGAQNYPQCINIEVTGGGSDAPEGTLGEDLYHSTDPGILVDIYEPIATYTIPGPPEPTF (SEQ ID NO.2).

[0042] The nucleotide sequence of the coding gene of mutant RsLPMO9 M1 is shown in SEQ ID NO.6: ATGCTGTCTTCGACGACTCGCACCCTCGCCTTTACAGGCCTTGCGGGCCTTCTGTCCGCTCCCCTGGTCAAGGCCCATGGCTTTGTCCAGGGCATTGTCATCGGTGGCCAATTGTAAGTCCCTCTCTTGCAGTTCTGTCGATTAACTGCTGGACTGCTTGCTTGACTCCCTGCTGACTCCCAACAGCTACAGCGGGTACATCGTCAACCAGTACCCCTACGAATCCAACCCACCCCCCGTCATCGGCTGGGCCACGACCGCCACCGACCTGGGCTTCGTCGACGGCACAGGATACCAAGGCCCGGACATCATCTGCCACCGGGGCGCGACGCCCGCGCCGCTGACAGCCCCCGTGGCCGCCGGCGGCACCGTCGAGCTGCAGTGGACGCCGTGGCCGGACAGCCACCACGGACCCGTCATCACCTACCTGGCGAACTGCAACGGCGACTGCTCGACCGTCGACAAGACGACGCTGGAGTTCTTCAAGATCGACCAGCAGGGCCTGATCGACGACACGACCCCGCCGGGCACCTGGGCGTCGGACAACCTCATCGCCAACAACAATAGCTGGACCGTCACCATTCCCAACAGCGTCGCCCCCGGCAACTACGTCCTGCGCCACGAGATCATCGCCCTGCACTCGGCCAACAACAAGGACGGCGCCCAGAACTACCCCCAGTGCATCAACATCGAGGTCACGGGCGGCGGCTCCGACGCGCCTGAGGGTACTCTGGGCGAGGATCTCTACCATTCCACCGACCCGGGCATTCTGGTCGACATTTACGAGCCCATTGCGACGTATACCATTCCGGGGCCGCCTGAGCCGACGTTCCATCACCACCACCACCACTAG (SEQ ID NO.6).

[0043] The amino acid sequence of mutant RsLPMO9 M2 is shown in SEQ ID NO.3: MLSSTTRTLAFAGLAGLLSAPLVKAHGFVQGIVIGDQTYSGYLVNSFPYEPNPPPVIGWATTATDLGFVDGTGYQGPDIICHRNATPAPLTAPVAAGGTVELQWTPWPDSHHGPVITYLAPCNGDCSTVDKTTLEFFKIDQQGLIDDTSPPGTWASDNLIANNNSWTVTIPNSVAPGNYVLRHEIIALHSANQKDGAQNYPQCINIEVTGGGSDAPEGTLGEDLYHDTDPGILVNIYEPIATYTIPGPAEPTF (SEQ ID NO.3).

[0044] The nucleotide sequence of the coding gene of mutant RsLPMO9 M2 is shown in SEQ ID NO.7: ATGCTGTCTTCGACGACTCGCACCCTCGCCTTTGCCGGCCTTGCGGGCCTTCTGTCCGCTCCCCTGGTCAAGGCCCATGGCTTTGTCCAGGGCATTGTCATCGGTGACCAAACGTAAGTCCCTCTCTTGCAGTTCTGTCGATTAACTGCTGGACTGCTTGCTTGACTCCCTGCTGACTCCCAACAGCTACAGCGGGTACCTGGTCAACTCGTTCCCCTACGAACCCAACCCACCCCCCGTCATCGGCTGGGCCACGACCGCCACCGACCTGGGCTTCGTCGACGGCACAGGATACCAAGGCCCGGACATCATCTGCCACCGGAATGCGACGCCCGCGCCGCTGACAGCCCCCGTGGCCGCCGGCGGCACCGTCGAGCTGCAGTGGACGCCGTGGCCGGACAGCCACCACGGACCCGTCATCACCTACCTGGCGCCGTGCAACGGCGACTGCTCGACCGTCGACAAGACGACGCTGGAGTTCTTCAAGATCGACCAGCAGGGCCTGATCGACGACACGAGCCCGCCGGGCACCTGGGCGTCGGACAACCTCATCGCCAACAACAATAGCTGGACCGTCACCATTCCCAACAGCGTCGCCCCCGGCAACTACGTCCTGCGCCACGAGATCATCGCCCTGCACTCGGCCAACCAGAAGGACGGCGCCCAGAACTACCCCCAGTGCATCAACATCGAGGTCACGGGCGGCGGCTCCGACGCGCCTGAGGGTACTCTGGGCGAGGATCTCTACCATGACACCGACCCGGGCATTCTGGTCAACATTTACGAGCCCATTGCGACGTATACCATTCCGGGGCCGGCCGAGCCGACGTTCCATCACCACCACCACCACTAG (SEQ ID NO.7).

[0045] The amino acid sequence of mutant RsLPMO9 M3 is shown in SEQ ID NO.4: MLSSTTRTLALTGLAGLLSAPLVKAHGFVQGIVIGGQFYGGYIVNSFPYESNPPAVIGWATTATDLGFVDGTGYQGPDIICHRNATPAPLSAPVAAGGTVELQWTPWPDSHHGPVITYLAPCNGNCSTVDKTQLEFFKIDQQGLIDDTSPPGTWASDNLIANNNSWTVTIPNSVAPGNYVLRHEIIALHSANNKDGAQNYPQCINIEVTGGGSDAPEGTLGEDLYHDTDPGILVDIYEPIATYTIPGPPEPTF (SEQ ID NO.4).

[0046] The nucleotide sequence of the coding gene of mutant RsLPMO9 M3 is shown in SEQ ID NO.8: ATGCTGTCTTCGACGACTCGCACCCTCGCCCTGACAGGCCTTGCGGGCCTTCTGTCCGCTCCCCTGGTCAAGGCCCATGGCTTTGTCCAGGGCATTGTCATCGGTGGCCAATTGTAAGTCCCTCTCTTGCAGTTCTGTCGATTAACTGCTGGACTGCTTGCTTGACTCCCTGCTGACTCCCAACAGCTACGGCGGGTACATCGTCAACTCGTTCCCCTACGAATCCAACCCACCCGCCGTCATCGGCTGGGCCACGACCGCCACCGACCTGGGCTTCGTCGACGGCACAGGATACCAAGGCCCGGACATCATCTGCCACCGGAATGCGACGCCCGCGCCGCTGTCCGCCCCCGTGGCCGCCGGCGGCACCGTCGAGCTGCAGTGGACGCCGTGGCCGGACAGCCACCACGGACCCGTCATCACCTACCTGGCGCCGTGCAACGGCAACTGCTCGACCGTCGACAAGACGCAGCTGGAGTTCTTCAAGATCGACCAGCAGGGCCTGATCGACGACACGAGCCCGCCGGGCACCTGGGCGTCGGACAACCTCATCGCCAACAACAATAGCTGGACCGTCACCATTCCCAACAGCGTCGCCCCCGGCAACTACGTCCTGCGCCACGAGATCATCGCCCTGCACTCGGCCAACAACAAGGACGGCGCCCAGAACTACCCCCAGTGCATCAACATCGAGGTCACGGGCGGCGGCTCCGACGCGCCTGAGGGTACTCTGGGCGAGGATCTCTACCATGACACCGACCCGGGCATTCTGGTCGACATTTACGAGCCCATTGCGACGTATACCATTCCGGGGCCGCCTGAGCCGACGTTCCATCACCACCACCACCACTAG (SEQ ID NO.8).

[0047] 2 Construction, expression and purification of mutants Transformation method of Trichoderma reesei: The spores of Trichoderma reesei SUS7 were inoculated on PDA plates and cultured at 28°C. Fresh spores were collected after 5 days. The spore suspension was inoculated into 50 mL PDB medium and cultured at 28°C and 200 rpm for 12 h. The germinated mycelium was collected by filtration with a 200-mesh sieve, washed with sterile water and 0.8 mol / L MgSO4 solution, and then resuspended in 20 mL bacterial cell wall lysis solution. The protoplasts were collected after digestion at 28°C and 90 rpm for 3 h. PEG6000 was mixed with protoplasts, ice-bathed for 30 min, and incubated at room temperature for 20 min. The linearized DNA fragment was transferred into Trichoderma reesei protoplasts by PEG6000-mediated protoplast transformation.

[0048] The coding genes of wild-type protein RsLPMO9 and its mutants were synthesized, and their expression plasmids Ppcbh-RsLPMO9, Ppcbh-RsLPMO9 M1, Ppcbh-RsLPMO9 M2 and Ppcbh-RsLPMO9 M3 in Trichoderma reesei were constructed respectively. CBHI The expression cassettes of wild-type protein RsLPMO9 and its mutants encoding gene promoter and terminator sequences were transferred into Trichoderma reesei strains. The single clones grown on the screening plate were transferred to a 24-well plate, and the cells after 48 h of culture were treated with fungal lysis solution to extract the genomic DNA of the transformants. Colony PCR was used to verify whether the expression cassettes of wild-type protein RsLPMO9 and its mutants had been transferred into Trichoderma reesei strains.

[0049] The electrophoresis results showed that the transformant had a single band at 1 kb, and there was no band in the negative control strain, confirming that the expression cassettes of the wild-type protein RsLPMO9 and its mutants had been integrated into the T. reesei genome.

[0050] The transformants of Trichoderma reesei were scraped from the 24-well plate with a sterile cotton swab and inoculated into the basal medium. After culturing at 28°C and 200 rpm for 48 h, they were inoculated into the cellulose induction medium at 10% and then cultured for another 120 h. After centrifugation at 12000 rpm for 10 min, the supernatant crude enzyme solution was collected.

[0051] The wild-type protein RsLPMO9 and its mutants both have 6×His tags at the ends, so Ni + The protein was purified by -NTA nickel column affinity chromatography, identified by SDS-PAGE electrophoresis, and stored in a 4°C refrigerator.

[0052] Protein electrophoresis results Figure 2 , Figure 3As shown, a clear band at around 35 kDa appeared in the fermentation supernatant of the wild-type protein RsLPMO9 and the mutant RsLPMO9 M2 recombinant strain ( 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 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 spectrum identification. The above results show that the wild-type protein RsLPMO9 and mutant RsLPMO9 M2 were successfully heterologously expressed in Trichoderma reesei, and the expression level of mutant RsLPMO9M2 was significantly higher than that of the wild-type protein RsLPMO9. The theoretical molecular weight of the wild-type protein RsLPMO9 is about 25kDa. The electrophoretic bands of the purified recombinant wild-type protein RsLPMO9 and mutant RsLPMO9 M2 are larger than the theoretical molecular weight and have differences, which may be due to the different degrees of glycosylation modification of the target protein in the Trichoderma reesei host.

[0053] 3. Thermal stability analysis of mutants The purified wild-type protein RsLPMO9 and mutant RsLPMO9 M2 were concentrated by ultrafiltration tube. 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 at 4°C for 1 h to allow the wild-type protein RsLPMO9 and mutant RsLPMO9 M2 to fully bind to CuSO4. 2+ , forming Cu 2+ Saturated protein. The fully bound protein was dialyzed to remove excess CuSO4 in the solution: phosphate buffer with a pH of 7 was used as the dialysate and dialyzed three times at 4°C. After the dialysis, the enzyme solution was collected for subsequent enzyme activity and application research.

[0054] The 2,6-DMP rapid enzyme activity assay was used to determine the enzyme activity of the wild-type protein RsLPMO9 and the mutant RsLPMO9 M2 using 2,6-DMP and H2O2 as co-substrates: (1) Preparation of 116 mmol·L -1 , phosphate buffer with a pH of 7; 10 mmol·L -1 2,6-DMP solution; 5mmol·L -1 All solutions should be used within 12 hours after preparation.

[0055] (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 thoroughly, and incubate at the corresponding temperature.

[0056] (3) Add 20 μL of the wild-type protein RsLPMO9 and mutant RsLPMO9 M2 samples and mix well. Measure the increase in absorbance at a wavelength of 469 nm within 5 min (ε469 = 53200 L·mol -1 cm -1 ) was used to calculate the activity of LPMO. One unit of enzyme activity was defined as the amount of 1 μmol of oxidation product produced per minute under the reaction conditions.

[0057] The diluted enzyme solution was treated at 70°C for 5 min, 10 min, 30 min, and 60 min, and then immediately placed on ice. 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 samples under different temperature conditions and after treatment for different time periods was calculated to determine the stability of the enzyme at different temperatures.

[0058] The test results are as follows Figure 4 As shown in the data, the thermal stability of the mutant RsLPMO9 M2 is significantly improved compared with the wild-type protein RsLPMO9: after treatment at 70°C for 30 minutes, the residual enzyme activity of the wild-type protein RsLPMO9 is 70%, and the residual enzyme activity of the mutant RsLPMO9M2 is about 79%; after treatment at 70°C for 1 hour, the residual enzyme activity of the wild-type protein RsLPMO9 is only 53%, and the residual enzyme activity of the mutant RsLPMO9 M2 is about 75%.

Claims

1. A multi-site mutant RsLPMO9 M2 of a 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 the multi-site mutant RsLPMO9M2 of the lytic polysaccharide monooxygenase RsLPMO9 according to claim 1, characterized in that: include: (1) The coding gene described in claim 2 is operably connected to an expression control element to construct a recombinant expression vector; (2) Transforming the recombinant expression vector into host cells, culturing the host cells, inducing the expression of the recombinant protein, and purifying the recombinant protein.

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

7. Use of the multi-site mutant RsLPMO9 M2 according to claim 1 in the degradation of plant polysaccharides, comprising: The multi-site mutant RsLPMO9 M2 is used as a catalytic enzyme to catalyze the cleavage of polysaccharide substrates through a monooxygenation reaction; wherein the polysaccharide includes cellulose or hemicellulose.

8. Use of the encoding gene according to claim 2 in the degradation of plant polysaccharides, comprising: The protein encoded by the encoding gene is used as a catalytic enzyme to catalyze the cleavage of a polysaccharide substrate through 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: The recombinant protein prepared by the expression cassette or the recombinant expression vector is used as a catalytic enzyme to catalyze the cleavage of a polysaccharide substrate through a monooxygenation reaction; wherein the polysaccharide includes cellulose or hemicellulose.

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

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