Mutants of lytic polysaccharide monooxygenase and methods of synergistic hydrolysis of cellulose

Abstract

The present application relates to a kind of lytic polysaccharide monooxygenase mutant and cellulose synergistic hydrolysis method.The present application belongs to the field of biotechnology, and traditional cellulose hydrolysis technology has the problems of low efficiency and high cost, the present application obtains mutant with higher catalytic activity and stability by site-directed mutagenesis to the 2nd and 43rd positions of the amino acid sequence shown in SEQ ID No:1, significantly improves cellulose hydrolysis efficiency, and has wide application prospect in the fields of bioenergy, papermaking, textile and the like.

Description

Technical Field

[0001] This invention relates to the field of biotechnology. More specifically, this invention relates to a cleavable polysaccharide monooxygenase mutant, a DNA molecule, a recombinant expression vector, a host cell, a method for producing the cleavable polysaccharide monooxygenase mutant, the application of the cleavable polysaccharide monooxygenase mutant in the co-hydrolysis of cellulose, and a method for the co-hydrolysis of cellulose. Background Technology

[0002] Lignocellulose, such as straw, is one of the most abundant renewable resources in nature. It can be degraded into hexoses and pentoses by cellulases, which can be further used to produce various bio-based products, including bioethanol. However, the cost of enzymatic degradation of lignocellulose accounts for about one-quarter of the cost of biofuels. Therefore, developing efficient lignocellulose-degrading enzymes and engineering their modification is crucial to promoting the benefits of lignocellulose biomass. Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes capable of oxidatively cleaving polysaccharides. LPMOs are key enzymes that work synergistically with cellulases to degrade lignocellulose, and improving their performance is crucial to the overall hydrolytic efficiency of cellulases. The main research content of this application is to accurately select mutation sites and predict the impact of mutations on enzyme function. Summary of the Invention

[0003] One object of the present invention is to provide a cleavable polysaccharide monooxygenase mutant. By performing site-directed mutations on the 2nd and 43rd positions of the amino acid sequence shown in SEQ ID No:1, the obtained LPMO mutant has higher catalytic activity and hydrolysis effect, improves catalytic reaction efficiency and product yield, is more suitable for industrial applications, and can significantly reduce the cost of the enzymatic hydrolysis process.

[0004] Another objective of this invention is to provide a DNA molecule that, through codon optimization and sequence design, solves the problem of low gene expression efficiency in heterologous hosts and enables efficient transcription and translation of LPMO mutants in engineered bacteria.

[0005] Another objective of this invention is to provide a recombinant expression vector that, by constructing an operable linker structure containing the DNA molecule and a highly efficient expression regulatory element, ensures stable transcription and induced expression of LPMO mutant genes in host cells, thereby increasing the expression level and solubility of recombinant proteins.

[0006] Another objective of this invention is to provide a host cell that, by screening suitable microbial hosts and introducing the aforementioned recombinant expression vector, solves the problems of folding errors and low secretion efficiency of natural LPMOs in traditional hosts, thereby achieving high biomass expression and functional folding of mutant enzymes.

[0007] Another objective of this invention is to provide a method for producing LPMOs that significantly improves the yield and purity of LPMO mutants, meeting the quality and cost requirements of enzyme preparations for industrial production.

[0008] Another objective of this invention is to provide an application that utilizes the synergistic mechanism of LPMO mutants with other cellulases to construct an efficient enzymatic hydrolysis system and improve cellulose degradation efficiency.

[0009] Another objective of this invention is to provide a synergistic hydrolysis method for cellulose, which solves the problems of low efficiency and high cost of traditional cellulose enzymatic hydrolysis technology by optimizing the synergistic interaction conditions of mutant enzymes and other cellulases, thereby achieving efficient conversion of lignocellulosic biomass.

[0010] To achieve these objectives and other advantages according to the present invention, a cleaving polysaccharide monooxygenase mutant is provided, wherein glycine at position 2 and phenylalanine at position 43 of the amino acid sequence shown in SEQ ID No:1 are replaced with other amino acids, said other amino acids being one of alanine, glycine, proline and tyrosine.

[0011] Preferably,

[0012] The cleaving polysaccharide monooxygenase mutant G2A has a glycine mutation at position 2 to alanine in the amino acid sequence shown in SEQ ID No:1, and the amino acid sequence of the cleaving polysaccharide monooxygenase mutant G2A is shown in SEQ ID No:3.

[0013] The cleaving polysaccharide monooxygenase mutant F43A has a phenylalanine mutation at position 43 to alanine in the amino acid sequence shown in SEQ ID No:1. The amino acid sequence of the cleaving polysaccharide monooxygenase mutant F43A is shown in SEQ ID No:5.

[0014] The cleaving polysaccharide monooxygenase mutant F43G has a phenylalanine mutation at position 43 to glycine in the amino acid sequence shown in SEQ ID No:1. The amino acid sequence of the cleaving polysaccharide monooxygenase mutant F43G is shown in SEQ ID No:7.

[0015] The cleaving polysaccharide monooxygenase mutant F43P has a phenylalanine mutation at position 43 to proline in the amino acid sequence shown in SEQ ID No:1. The amino acid sequence of the cleaving polysaccharide monooxygenase mutant F43P is shown in SEQ ID No:9.

[0016] The cleaving polysaccharide monooxygenase mutant F43Y has a phenylalanine mutation at position 43 to tyrosine in the amino acid sequence shown in SEQ ID No:1. The amino acid sequence of the cleaving polysaccharide monooxygenase mutant F43Y is shown in SEQ ID No:11.

[0017] DNA molecule, which encodes the cleavage polysaccharide monooxygenase mutant.

[0018] Preferably,

[0019] The nucleotide sequence of the DNA molecule encoding the cleavable polysaccharide monooxygenase mutant G2A is shown in SEQ ID No:4;

[0020] The nucleotide sequence of the DNA molecule encoding the cleavable polysaccharide monooxygenase mutant F43A is shown in SEQ ID No:6;

[0021] The nucleotide sequence of the DNA molecule encoding the cleavable polysaccharide monooxygenase mutant F43G is shown in SEQ ID No:8;

[0022] The nucleotide sequence of the DNA molecule encoding the cleavable polysaccharide monooxygenase mutant F43P is shown in SEQ ID No:10;

[0023] The nucleotide sequence of the DNA molecule encoding the cleavable polysaccharide monooxygenase mutant F43Y is shown in SEQ ID No:12.

[0024] A recombinant expression vector containing the DNA molecule and a regulatory sequence operatively linked to the DNA molecule for expression.

[0025] Preferably, the recombinant expression vector is pPICZαA.

[0026] The host cell contains the DNA molecule or the recombinant expression vector, and the host cell is a non-animal or plant cell.

[0027] The method for producing the cleavable polysaccharide monooxygenase mutant includes the following steps:

[0028] The host cells are cultured in a culture medium and the expression of the cleaving polysaccharide monooxygenase mutant of claim 1 or 2 is induced by the recombinant expression vector.

[0029] The application of the cleavable polysaccharide monooxygenase mutant, the DNA molecule, the recombinant expression vector, and the host cell in the synergistic hydrolysis of cellulose by cellulase.

[0030] The cellulose co-hydrolysis method is characterized by adding the cleavage polysaccharide monooxygenase mutant and cellulase to cellulose for enzymatic hydrolysis.

[0031] The present invention has at least the following beneficial effects:

[0032] The enzyme provided by this invention has excellent cellulose degradation function, and its synergistic hydrolysis effect on cellulose can be improved by up to 2.4 times. The improved hydrolysis efficiency can reduce the amount of enzyme used to hydrolyze a certain amount of reducing sugar per unit time, and enhance the role of enzyme system in balancing enzyme system to improve the overall hydrolysis rate in enzyme system compounding. It has broad application prospects.

[0033] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0034] Figure 1 This is a gel electrophoresis image of the cleaving polysaccharide monooxygenase and its mutants after expression according to the present invention;

[0035] Figure 2 This is a bar chart of the hydrolysis of microcrystalline cellulose by the mutant generated at position 2 of the cleaving polysaccharide monooxygenase of the present invention;

[0036] Figure 3 This is a bar chart showing the hydrolysis of microcrystalline cellulose by a mutant of the cleaving polysaccharide monooxygenase at position 43 of the present invention. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0038] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0039] It should be noted that, unless otherwise specified, the experimental methods described in the following embodiments are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified, and therefore should not be construed as limiting the present invention.

[0040] 1. Experimental Materials

[0041] Yeast Extract Peptone Dextrose Sorbitol (YPDS) Medium is an amino acid auxotrophic medium used for yeast hybridization and screening of genetic mutants. It was purchased from zeocin (Thermo Fisher Scientific, Runcorn, Cheshire, UK) and its formulation is: 1% yeast extract, 2% peptone, 2% glucose, 1M sorbitol, and 2% agar.

[0042] Buffered Glycerol Complex Medium is a buffered complex liquid culture medium containing glycerol, with the medium controlled at pH 6.0. It is used to optimize the expression of target proteins. Its formulation is: 1% yeast extract, 2% peptone, 100mM potassium phosphate (pH 6.0), 1.34% amino acid-free nitrogen source and 1% glycerol.

[0043] Buffered Minimal Methanol (BMMH) medium is a buffered basal medium containing methanol, used for methanol-induced protein expression in recombinant Pichia pastoris strains. Its formulation is: 100 mM potassium phosphate, pH 6.0, 1.34% amino acid-free nitrogen source, and 0.5% methanol.

[0044] <Example 1>

[0045] Using the amino acid sequence of the wild-type cleaving polysaccharide monooxygenase shown in SEQ ID No:1 as a template, sequence alignment and structural analysis identified amino acids at positions 2 and 43 as potential mutation sites that may affect substrate binding and catalytic efficiency.

[0046] Site-directed mutagenesis primers were designed based on the target mutation site. The primer sequences are as follows:

[0047] Alanine mutant primer at position 2:

[0048] G2A-F:GGTGTTGTTGGTCATGCTCATATTAATGATATTGTTATTAATGG

[0049] G2A-R: AATATCATTAATATGAGCATGACCAACAACACCAGTAGCAACA

[0050] Primer for alanine mutation at position 43:

[0051] F43A-F:GCTGATTTGGATAATGGTGCTGTTTCTCCTGATGCTTATCAAAATCCTG

[0052] F43A-R: AGCATCAGGAGAAACAGCACCATTATCCAAATCAGCAGCAGTC

[0053] Glycine mutation primer at position 43:

[0054] F43G-F:GCTGATTTGGATAATGGTGGTGTTTCTCCTGATGCTTATCAAAATCCTG

[0055] F43G-R: AGCATCAGGAGAAACACCACCATTATCCAAATCAGCAGCAGTC

[0056] Primer for proline mutation at position 43:

[0057] F43P-F:GCTGATTTGGATAATGGTCCAGTTTCTCCTGATGCTTATCAAAATCCTG

[0058] F43P-R: AGCATCAGGAGAAACTGGACCATTATCCAAATCAGCAGCAGTC

[0059] Primer with tyrosine mutation at position 43:

[0060] F43Y-F:GCTGATTTGGATAATGGTTATGTTTCTCCTGATGCTTATCAAAATCCTG

[0061] F43Y-R:AGCATCAGGAGAAACATAACCATTATCCAAATCAGCAGCAGTC

[0062] 1. Wild-type gene cloning:

[0063] Wild-type cleaving polysaccharide monooxygenase, derived from Trichoderma reesei ( Trichoderma reeseiThe secreted cleaving polysaccharide monooxygenase has the nucleotide sequence shown in SEQ ID No:2 and the encoded amino acid sequence shown in SEQ ID No:1. Using Trichoderma reesei cDNA as a template, the gene fragment of SEQ ID No:2 was amplified by PCR and cloned into the EcoRI / XbaI site of the pPICZαA vector to construct pPICZαA-LPMO-WT.

[0064] 2. Site-directed mutation:

[0065] Overlap extension PCR was used to design primers containing mutation sites (such as G2A: G2A-F, G2A-R, F43A: F43A-F, F43A-R, F43G: F43G-F, F43G-R, F43P: F43P-F, F43P-R, F43Y: F43A-Y, F43Y-R) to construct mutant plasmids such as pPICZαA-LPMO-G2A.

[0066] 3. Pichia pastoris transformation and screening:

[0067] The obtained wild-type polysaccharide monooxygenase strain and its mutant strains were plated into an amino acid auxotrophic medium containing 100 μg / mL and cultured at 30°C for 3 days. Single clones were picked and inoculated into a buffered complex liquid medium containing glycerol and cultured overnight at 30°C with shaking at 240 rpm.

[0068] 4. Induced expression:

[0069] When the bacterial concentration reaches OD 600 At 2-6, using a UNICOUV2102 UV-Vis spectrophotometer, with a buffered complex culture medium containing glycerol as a blank control, the bacterial culture was centrifuged at 4000 g, the supernatant was poured off, and the bacterial cells were resuspended in a buffered basal culture medium containing methanol. Then, 0.5% methanol was added every 12 hours, and the culture was shaken at 240 rpm and 30℃ for 3 days.

[0070] 5. Protein purification:

[0071] The crude enzyme solution was purified by Ni-NTA column chromatography. Elution was performed with 300 mM imidazole elution buffer for 10 column volumes. The target protein peak was collected, and the obtained protein was then analyzed by SDS-PAGE to determine the protein purity.

[0072] The following mutants of polysaccharide monooxygenase were obtained using the above methods: G2A (amino acid sequence as shown in SEQ ID No:3, nucleotide sequence encoding G2A as shown in SEQ ID No:4), F43A (amino acid sequence as shown in SEQ ID No:5, nucleotide sequence encoding F43A as shown in SEQ ID No:6), F43G (amino acid sequence as shown in SEQ ID No:7, nucleotide sequence encoding F43G as shown in SEQ ID No:8), F43P (amino acid sequence as shown in SEQ ID No:9, nucleotide sequence encoding F43P as shown in SEQ ID No:10), and F43Y (amino acid sequence as shown in SEQ ID No:11, nucleotide sequence encoding F43Y as shown in SEQ ID No:12).

[0073] like Figure 1 The gel electrophoresis images of the cleavable polysaccharide monooxygenase WT and its mutants G2A, F43A, F43G, F43P, and F43Y after expression show the efficient expression and purification of WT and its mutants.

[0074] Molecular weight analysis:

[0075] The target protein's main band is located at 43-55 kDa (compared to the marker, such as the WT and mutant bands which are close to 55 kDa), which is consistent with the expected molecular weight range of LPMO.

[0076] Purity and expression level:

[0077] As a control, WT showed clear main bands for G2A, F43A, F43G, F43P, and F43Y with few impurities and high purity. Among them, G2A and F43Y were the brightest, indicating high expression levels, which may enhance protein folding efficiency or resist host protease degradation.

[0078] <Example 2>

[0079] The determination of reducing sugar yield of substrates by a cleavable polysaccharide monooxygenase mutant in synergistic cellulase degradation of the substrate using microcrystalline cellulose as a substrate is as follows:

[0080] Preheat 1% (w / v) microcrystalline cellulose in a 60°C constant-temperature metal bath for 5-10 min, then add 1.33 μM enzyme dilution and cellulase (the mass ratio of cleaving polysaccharide monooxygenase to cellulase is 1:3). Place the well-mixed enzyme containing the mutant and the control group simultaneously in a 60°C constant-temperature metal bath and react for 60 h. At the end of the reaction, boil the reactants in a boiling water bath for 5 min to inactivate the enzyme.

[0081] The detection method used was the DNS method (3,5-dinitrosalicylic acid) to determine reducing sugars (converted to glucose equivalents), and the yield (mg / mL) was calculated. Glucose standard solutions with concentration gradients of 0.15, 0.30, 0.45, 0.60, 0.75, 0.90, and 1.00 mg / mL were prepared. 115 µL of each glucose standard solution and the test sample were respectively placed in 2 mL centrifuge tubes, 85 µL of DNS reagent was added, and the mixture was thoroughly mixed. The mixture was then boiled in a water bath for 5 min, cooled to room temperature, and 200 µL of distilled water was added. After mixing, 200 µL was pipetted into a 96-well plate, and the absorbance was measured at 540 nm. The results were plotted with glucose content as the x-axis and OD value as the y-axis. 540 Plot a glucose standard curve with the vertical axis as the ordinate. Substitute the absorbance value of the sample to be tested into the glucose standard curve to calculate the reducing sugar yield of the sample.

[0082] like Figure 2 The bar chart shown represents the hydrolysis of microcrystalline cellulose by a mutant generated at position 2 of the cleaving polysaccharide monooxygenase. In the chart, G2A, G2C, G2D, G2E, G2F, G2H, G2I, G2K, G2L, G2M, G2N, G2P, G2Q, G2R, G2S, G2T, G2V, G2W, and G2Y respectively refer to the components in SEQ ID NO. The amino acid sequence No:1 shows that the second position of aspartic acid is mutated to alanine (G2A), cysteine ​​(G2C), aspartic acid (G2D), glutamic acid (G2E), phenylalanine (G2F), histidine (G2H), isoleucine (G2I), lysine (G2K), leucine (G2L), methionine (G2M), asparagine (G2N), proline (G2P), glutamine (G2Q), arginine (G2R), serine (G2S), threonine (G2T), valine (G2V), tryptophan (G2W), and tyrosine (G2Y) to form a complex enzyme system with cellulase. This system was used to compare the oxidative degradation activity of the complex enzyme system on microcrystalline cellulose after mutation at the G2 site of the cleaving polysaccharide monooxygenase from Trichoderma reesei.

[0083] Control was used as an enzyme-free control, and WT was a complex enzyme system of cleaving polysaccharide monooxygenase and cellulase. G2A had the highest yield (>2.2). The mutation significantly enhances enzyme activity. In the complex enzyme system, the high activity of G2A can optimize substrate binding or catalytic efficiency. Other G2 mutations, such as G2D, G2N, and G2S, have slightly higher activities, while G2F and G2H have lower activities than WT. This indicates that the effect of G2 site mutation on activity is highly dependent on the amino acid type. Other substitutions may affect the binding of the catalytic site to the cellulose substrate. The G2A mutation introduces a methyl side chain (hydrophobic small group), which makes the N-terminal conformation of the cleaving polysaccharide monooxygenase G2 (glycine, no side chain) rigid. The tight hydrogen bond network of microcrystalline cellulose makes it difficult for traditional cellulases to penetrate. The oxidation of WT can create chain-end gaps in the crystallization region, creating sites for subsequent enzymatic hydrolysis. G2A expands the cleavage site through better substrate binding, significantly enhancing the oxidative cleavage ability of microcrystalline cellulose. The cleaving polysaccharide monooxygenase mutant and cellulase work synergistically to significantly improve the degradation efficiency of the substrate microcrystalline cellulose.

[0084] like Figure 3 The bar chart shown represents the hydrolysis of microcrystalline cellulose by a mutant generated at position 43 of the cleaving polysaccharide monooxygenase. In the chart, F43A, F43C, F43D, F43E, F43G, F43H, F43I, F43K, F43L, F43M, F43N, F43P, F43Q, F43R, F43S, F43T, F43V, and F43Y respectively refer to the components in SEQ ID NO. The amino acid sequence No:1 shows that the phenylalanine at position 43 is mutated to alanine (F43A), cysteine ​​(F43C), aspartic acid (F43D), glutamic acid (F43E), glycine (F43G), histidine (F43H), isoleucine (F43I), lysine (F43K), leucine (F43L), methionine (F43M), asparagine (F43N), proline (F43P), glutamine (F43Q), arginine (F43R), serine (F43S), threonine (F43T), valine (F43V), tyrosine (F43Y), and cellulase to form a complex enzyme system. This system was used to compare the oxidative degradation activity of the complex enzyme system on microcrystalline cellulose after mutation at position F43 of the cleaving polysaccharide monooxygenase from Trichoderma reesei.

[0085] Control was used as an enzyme-free control, while WT was a complex enzyme system of cleaving polysaccharide monooxygenase and cellulase. The yields of F43A, F43G, F43P, and F43Y were all significantly higher than those of WT. The amino acid substitution at the F43 site can significantly enhance the hydrolytic ability of cleaving polysaccharide monooxygenase on microcrystalline cellulose by optimizing enzyme-substrate interactions. F43A significantly reduces steric hindrance by removing a large aromatic ring and introducing a small hydrophobic methyl side chain; F43G reduces steric hindrance by completely eliminating the side chain, forming a random coil conformation, greatly increasing local flexibility and maximizing the conformational freedom of the N-terminus or active center region; F43P increases rigidity by forcing a rigid pyrrole ring to form a β-turn conformation; and F43Y increases polarity by introducing a polar hydroxyl group (-OH) to form hydrogen bonds with the cellulose chain, enhancing binding-oxidative coupling through a hydrophobic-polar dual-action. In the complex enzyme system, the high activity of F43A, F43G, F43P, and F43Y optimizes the balance between binding affinity and catalytic efficiency, significantly improving the hydrolysis efficiency of the cleavable polysaccharide monooxygenase mutant on microcrystalline cellulose. The cleavable polysaccharide monooxygenase mutant and cellulase work synergistically to significantly improve the degradation efficiency of the substrate microcrystalline cellulose.

[0086] As described above, according to the present invention, since enzymes and their compositions with enhanced enzyme activity are provided, the yield of reducing sugars generated by the degradation of lignocellulose per unit time can be increased, and the role of balancing enzyme systems in improving the overall hydrolysis rate can be enhanced in enzyme system compounding, which has broad application prospects.

[0087] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0088] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

[0089] WT (Tonal-Cosmetic Polysaccharide Monooxygenase):

[0090] Amino acid sequence (SEQ ID No:1):

[0091] HGHINDIVINGVWYQAYDPTTFPYESNPPIVVGWTAADLDNGFVSPDAYQNPDIICHKNATNAKGHASVKAGDTILFQWVPVPWPHPGPIVDYLANCNGDCETVDKTTLEFFKIDGVGLLSGGDPGTWASDVLISNNNTWVVKIPDNLAPGNYVLRHEIIALHSAGQANGAQNYPQCFNIAVSGSGSLQPSGVLGTDLYHATDPGVLINIYTSPLNYIIPGPTVVSGLPTSVAQGSSAAT

[0092] Nucleotide sequence (SEQ ID No: 2):

[0093] CATGGTCATATTAATGATATTGTTATTAATGGTGTTTGGTATCAAGCTTATGATCCAACTACTTTTCCTTATGAATCTAATCCACCAATTGTTGTTGGTTGGACTGCTGCTGATTTGGATAATGGTTTTGTTTCTCCTGATGCTTATCAAAATCCTGATATTATTTGTCATAAAAATGCTACTAATGCTAAAGGTCATGCTTCTGTTAAAGCTGGTGATACTATTTTGTTTCAATGGGTTCCTGTTCCTTGGCCACATCCTGGTCCAATTGTTGATTATTTGGCTAATTGTAATGGTGATTGTGAAACTGTTGATAAAACTACTTTGGAATTTTTTAAAATTGATGGTGTTGGTTTGTTGTCTGGTGGTGATCCTGGTACTTGGGCTTCTGATGTTTTGATTTCTAATAACAATACTTGGGTTGTTAAAATTCCTGATAATTTGGCTCCTGGTAATTATGTTTTGAGACATGAAATTATTGCTTTGCATTCTGCTGGTCAAGCTAATGGTGCTCAAAATTATCCACAATGTTTTAATATTGCTGTTTCTGGTTCTGGTTCTTTGCAACCATCTGGTGTTTTGGGTACTGATTTGTATCATGCTACTGATCCTGGTGTTTTGATTAATATTTATACTTCTCCATTGAATTATATTATTCCTGGTCCAACTGTTGTTTCTGGTTTGCCAACTTCTGTTGCTCAAGGTTCTTCTGCTGCTACT

[0094] Lytic polysaccharide monooxygenase mutant G2A:

[0095] Amino acid sequence (SEQ ID No:3):

[0096] HAHINDIVINGVWYQAYDPTTFPYESNPPIVVGWTAADLDNGFVSPDAYQNPDIICHKNATNAKGHASVKAGDTILFQWVPVPWPHPGPIVDYLANCNGDCETVDKTTLEFFKIDGVGLLSGGDPGTWASDVLISNNNTWVVKIPDNLAPGNYVLRHEIIALHSAGQANGAQNYPQCFNIAVSGSGSLQPSGVLGTDLYHATDPGVLINIYTSPLNYIIPGPTVVSGLPTSVAQGSSAAT

[0097] Nucleotide sequence (SEQ ID No: 4):

[0098] CATGCTCATATTAATGATATTGTTATTAATGGTGTTTGGTATCAAGCTTATGATCCAACTACTTTTCCTTATGAATCTAATCCACCAATTGTTGTTGGTTGGACTGCTGCTGATTTGGATAATGGTTTTGTTTCTCCTGATGCTTATCAAAATCCTGATATTATTTGTCATAAAAATGCTACTAATGCTAAAGGTCATGCTTCTGTTAAAGCTGGTGATACTATTTTGTTTCAATGGGTTCCTGTTCCTTGGCCACATCCTGGTCCAATTGTTGATTATTTGGCTAATTGTAATGGTGATTGTGAAACTGTTGATAAAACTACTTTGGAATTTTTTAAAATTGATGGTGTTGGTTTGTTGTCTGGTGGTGATCCTGGTACTTGGGCTTCTGATGTTTTGATTTCTAATAACAATACTTGGGTTGTTAAAATTCCTGATAATTTGGCTCCTGGTAATTATGTTTTGAGACATGAAATTATTGCTTTGCATTCTGCTGGTCAAGCTAATGGTGCTCAAAATTATCCACAATGTTTTAATATTGCTGTTTCTGGTTCTGGTTCTTTGCAACCATCTGGTGTTTTGGGTACTGATTTGTATCATGCTACTGATCCTGGTGTTTTGATTAATATTTATACTTCTCCATTGAATTATATTATTCCTGGTCCAACTGTTGTTTCTGGTTTGCCAACTTCTGTTGCTCAAGGTTCTTCTGCTGCTACT

[0099] Lytic polysaccharide monooxygenase mutant F43A:

[0100] Amino acid sequence (SEQ ID No:5):

[0101] HGHINDIVINGVWYQAYDPTTFPYESNPPIVVGWTAADLDNGAVSPDAYQNPDIICHKNATNAKGHASVKAGDTILFQWVPVPWPHPGPIVDYLANCNGDCETVDKTTLEFFKIDGVGLLSGGDPGTWASDVLISNNNTWVVKIPDNLAPGNYVLRHEIIALHSAGQANGAQNYPQCFNIAVSGSGSLQPSGVLGTDLYHATDPGVLINIYTSPLNYIIPGPTVVSGLPTSVAQGSSAAT

[0102] Nucleotide sequence (SEQ ID No: 6):

[0103] CATGGTCATATTAATGATATTGTTATTAATGGTGTTTGGTATCAAGCTTATGATCCAACTACTTTTCCTTATGAATCTAATCCACCAATTGTTGTTGGTTGGACTGCTGCTGATTTGGATAATGGTGCTGTTTCTCCTGATGCTTATCAAAATCCTGATATTATTTGTCATAAAAATGCTACTAATGCTAAAGGTCATGCTTCTGTTAAAGCTGGTGATACTATTTTGTTTCAATGGGTTCCTGTTCCTTGGCCACATCCTGGTCCAATTGTTGATTATTTGGCTAATTGTAATGGTGATTGTGAAACTGTTGATAAAACTACTTTGGAATTTTTTAAAATTGATGGTGTTGGTTTGTTGTCTGGTGGTGATCCTGGTACTTGGGCTTCTGATGTTTTGATTTCTAATAACAATACTTGGGTTGTTAAAATTCCTGATAATTTGGCTCCTGGTAATTATGTTTTGAGACATGAAATTATTGCTTTGCATTCTGCTGGTCAAGCTAATGGTGCTCAAAATTATCCACAATGTTTTAATATTGCTGTTTCTGGTTCTGGTTCTTTGCAACCATCTGGTGTTTTGGGTACTGATTTGTATCATGCTACTGATCCTGGTGTTTTGATTAATATTTATACTTCTCCATTGAATTATATTATTCCTGGTCCAACTGTTGTTTCTGGTTTGCCAACTTCTGTTGCTCAAGGTTCTTCTGCTGCTACT

[0104] Lytic polysaccharide monooxygenase mutant F43G:

[0105] Amino acid sequence (SEQ ID No:7):

[0106] HGHINDIVINGVWYQAYDPTTFPYESNPPIVVGWTAADLDNGGVSPDAYQNPDIICHKNATNAKGHASVKAGDTILFQWVPVPWPHPGPIVDYLANCNGDCETVDKTTLEFFKIDGVGLLSGGDPGTWASDVLISNNNTWVVKIPDNLAPGNYVLRHEIIALHSAGQANGAQNYPQCFNIAVSGSGSLQPSGVLGTDLYHATDPGVLINIYTSPLNYIIPGPTVVSGLPTSVAQGSSAAT

[0107] Nucleotide sequence (SEQ ID No: 8):

[0108] CATGGTCATATTAATGATATTGTTATTAATGGTGTTTGGTATCAAGCTTATGATCCAACTACTTTTCCTTATGAATCTAATCCACCAATTGTTGTTGGTTGGACTGCTGCTGATTTGGATAATGGTGGTGTTTCTCCTGATGCTTATCAAAATCCTGATATTATTTGTCATAAAAATGCTACTAATGCTAAAGGTCATGCTTCTGTTAAAGCTGGTGATACTATTTTGTTTCAATGGGTTCCTGTTCCTTGGCCACATCCTGGTCCAATTGTTGATTATTTGGCTAATTGTAATGGTGATTGTGAAACTGTTGATAAAACTACTTTGGAATTTTTTAAAATTGATGGTGTTGGTTTGTTGTCTGGTGGTGATCCTGGTACTTGGGCTTCTGATGTTTTGATTTCTAATAACAATACTTGGGTTGTTAAAATTCCTGATAATTTGGCTCCTGGTAATTATGTTTTGAGACATGAAATTATTGCTTTGCATTCTGCTGGTCAAGCTAATGGTGCTCAAAATTATCCACAATGTTTTAATATTGCTGTTTCTGGTTCTGGTTCTTTGCAACCATCTGGTGTTTTGGGTACTGATTTGTATCATGCTACTGATCCTGGTGTTTTGATTAATATTTATACTTCTCCATTGAATTATATTATTCCTGGTCCAACTGTTGTTTCTGGTTTGCCAACTTCTGTTGCTCAAGGTTCTTCTGCTGCTACT

[0109] Lytic polysaccharide monooxygenase mutant F43P:

[0110] Amino acid sequence (SEQ ID No:9):

[0111] HGHINDIVINGVWYQAYDPTTFPYESNPPIVVGWTAADLDNGPVSPDAYQNPDIICHKNATNAKGHASVKAGDTILFQWVPVPWPHPGPIVDYLANCNGDCETVDKTTLEFFKIDGVGLLSGGDPGTWASDVLISNNNTWVVKIPDNLAPGNYVLRHEIIALHSAGQANGAQNYPQCFNIAVSGSGSLQPSGVLGTDLYHATDPGVLINIYTSPLNYIIPGPTVVSGLPTSVAQGSSAAT

[0112] Nucleotide sequence (SEQ ID No: 10):

[0113] CATGGTCATATTAATGATATTGTTATTAATGGTGTTTGGTATCAAGCTTATGATCCAACTACTTTTCCTTATGAATCTAATCCACCAATTGTTGTTGGTTGGACTGCTGCTGATTTGGATAATGGTCCAGTTTCTCCTGATGCTTATCAAAATCCTGATATTATTTGTCATAAAAATGCTACTAATGCTAAAGGTCATGCTTCTGTTAAAGCTGGTGATACTATTTTGTTTCAATGGGTTCCTGTTCCTTGGCCACATCCTGGTCCAATTGTTGATTATTTGGCTAATTGTAATGGTGATTGTGAAACTGTTGATAAAACTACTTTGGAATTTTTTAAAATTGATGGTGTTGGTTTGTTGTCTGGTGGTGATCCTGGTACTTGGGCTTCTGATGTTTTGATTTCTAATAACAATACTTGGGTTGTTAAAATTCCTGATAATTTGGCTCCTGGTAATTATGTTTTGAGACATGAAATTATTGCTTTGCATTCTGCTGGTCAAGCTAATGGTGCTCAAAATTATCCACAATGTTTTAATATTGCTGTTTCTGGTTCTGGTTCTTTGCAACCATCTGGTGTTTTGGGTACTGATTTGTATCATGCTACTGATCCTGGTGTTTTGATTAATATTTATACTTCTCCATTGAATTATATTATTCCTGGTCCAACTGTTGTTTCTGGTTTGCCAACTTCTGTTGCTCAAGGTTCTTCTGCTGCTACT

[0114] Lytic polysaccharide monooxygenase mutant F43Y:

[0115] Amino acid sequence (SEQ ID No:11):

[0116] HGHINDIVINGVWYQAYDPTTFPYESNPPIVVGWTAADLDNGYVSPDAYQNPDIICHKNATNAKGHASVKAGDTILFQWVPVPWPHPGPIVDYLANCNGDCETVDKTTLEFFKIDGVGLLSGGDPGTWASDVLISNNNTWVVKIPDNLAPGNYVLRHEIIALHSAGQANGAQNYPQCFNIAVSGSGSLQPSGVLGTDLYHATDPGVLINIYTSPLNYIIPGPTVVSGLPTSVAQGSSAAT

[0117] Nucleotide sequence (SEQ ID No:12):

[0118] 。

Claims

1. A cleaving polysaccharide monooxygenase mutant, characterized in that, The cleaving polysaccharide monooxygenase mutant G2A has a glycine mutation at position 2 to alanine in the amino acid sequence shown in SEQ ID No:1, and the amino acid sequence of the cleaving polysaccharide monooxygenase mutant G2A is shown in SEQ ID No:

3.

2. A DNA molecule, characterized by, The nucleotide sequence of the DNA molecule encoding the cleavable polysaccharide monooxygenase mutant G2A is shown in SEQ ID No:

4.

3. A recombinant expression vector, characterized in that, The recombinant expression vector contains the DNA molecule of claim 2 and a regulatory sequence operatively linked to the DNA molecule for expression.

4. The recombinant expression vector as described in claim 3, characterized in that, The recombinant expression vector is pPICZαA.

5. A host cell, characterized in that, The host cell contains the DNA molecule of claim 2 or the recombinant expression vector of claim 3 or 4, and the host cell is a non-animal or plant cell.

6. The method for producing the cleavable polysaccharide monooxygenase mutant as described in claim 1, characterized in that, Includes the following steps: The host cells of claim 5 are cultured in a culture medium and expressed by the recombinant expression vector of claim 3 or 4 as described in claim 1, which induces the expression of the cleavage polysaccharide monooxygenase mutant.

7. The use of the cleavage polysaccharide monooxygenase mutant of claim 1, the DNA molecule of claim 2, the recombinant expression vector of claim 3 or 4, and the host cell of claim 5 in the synergistic hydrolysis of cellulose by cellulase.

8. A method for the co-hydrolysis of cellulose, characterized in that, The cleavage polysaccharide monooxygenase mutant of claim 1 and cellulase were added to cellulose for enzymatic hydrolysis.

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