A polysaccharide-cleaving monooxygenase mutant and its gene, engineering bacteria, preparation method and application

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to a lytic polysaccharide monooxygenase mutant and its gene, engineering bacteria, preparation method and application. The lytic polysaccharide monooxygenase mutant is obtained by A31Q / T39V mutation on the basis of the wild-type lytic polysaccharide monooxygenase shown in SEQ ID NO.1. Compared with the wild-type, the mutant has different degrees of improvement in enzyme activity and thermal stability at 70°C, which is conducive to expanding the application in efficient straw degradation.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a polysaccharide cleavage monooxygenase mutant and its gene, engineering bacteria, preparation method and application. Background Art

[0002] With the increase in population and the increasing consumption of resources, global resources and energy are becoming increasingly scarce, especially oil resources are facing depletion, so there is a need to seek alternative energy. Plant biomass in nature is the largest existing renewable resource. Among them, lignocellulose is widely available and renewable. It is the main structural component of wood and straw. It can be used as a raw material for biological fermentation and biochemical industries. It is considered to be a highly potential second-generation biorefining raw material and has broad application prospects in converting it into green biofuels and value-added chemical raw materials.

[0003] The main components of lignocellulose are cellulose, hemicellulose and lignin. These three interact and entangle in various forms to form a complex three-dimensional network structure, which makes it difficult to be effectively utilized. At present, the conversion of lignocellulose mainly includes three steps: pretreatment, enzymatic saccharification and fermentation. Pretreatment mainly uses physical and chemical methods to destroy the structure of lignocellulose and provide more sites for the action of later enzymes. Although pretreatment can destroy the structure of lignocellulose to a certain extent, most of the cellulose in the crystalline or microcrystalline state still exists after pretreatment. Enzymatic saccharification is the use of cellulase to convert cellulose into glucose. This step is the decisive factor for the feasibility of lignocellulose bioconversion. At present, cellulase is mainly composed of endocellulase, exocellulase and β-glucosidase. Under the joint action of the three, part of the cellulose can be degraded to obtain glucose, but the hydrolysis efficiency of crystalline cellulose is still very low.

[0004] As an auxiliary active enzyme, lytic polysaccharide monooxygenases (LPMOs) can cleave glycosidic bonds through an oxidative mechanism and effectively improve the degree of polysaccharide hydrolysis. They are lignocellulose degrading enzymes with great development potential and provide new insights into the enzymatic degradation of lignocellulose in straw. Most LPMOs are derived from fungi, such as Myceliophthora thermophila, Heterobasidiomycetes, Thermoascus ascomycetes, and Trichoderma reesei. In order to improve the production efficiency of LPMOs and promote their industrial application, heterologous expression in other hosts is an effective means. At present, the main expression hosts of LPMOs are Escherichia coli, Bacillus, and Pichia pastoris ( Shepherd's pie) and some fungal strains. In industrial applications, the thermal stability and catalytic activity of enzymes are important parameters of enzymes, which determine the economic feasibility of enzymes. For example, after processes such as steam explosion and hydrothermal treatment, the substrate usually has a high temperature. Enzymatic saccharification under high temperature conditions can reduce process costs and reduce cooling time; in addition, for enzyme reactions, the higher the temperature, the faster the enzymatic reaction rate. Therefore, the development of high temperature resistant LPMO is of great significance for the bioconversion of lignocellulose. However, the resources of high temperature resistant LPMOs are currently limited. Obtaining high temperature resistant LPMOs through protein engineering has become a focus of attention and research.

[0005] Protein engineering often uses irrational molecular design and rational and semi-rational molecular design methods to transform proteins. Irrational molecular design mainly uses error-prone PCR technology and high-throughput screening methods to obtain mutants with excellent performance. The advantage of this method is that it does not require a clear three-dimensional structure of the protein, but it has the disadvantage of cumbersome screening work. Rational design and semi-rational design are based on the known three-dimensional structure and function of the protein, and a gene sequence that is most likely to affect the function and properties of the protein is targeted for mutation, and one or two amino acid residues or modules of the protein are purposefully changed to construct a new protein molecule. Compared with irrational design, rational design has the advantages of less workload and easier to obtain effective mutants.

[0006] Therefore, in the present invention, by Rhypophila deceiving The lytic polysaccharide monooxygenase gene was rationally designed, and mutant lytic polysaccharide monooxygenase genes with improved thermal stability were obtained through mutation screening. Subsequently, the lytic polysaccharide monooxygenase mutant with improved heat resistance was expressed and prepared using the Pichia pastoris expression system, and its application in the efficient saccharification of straw lignocellulose was realized. Summary of the invention

[0007] In order to obtain a lytic polysaccharide monooxygenase with improved thermal stability, its existing properties need to be further modified. The purpose of the present invention is to provide a high temperature resistant lytic polysaccharide monooxygenase mutant. Rhytophila deceptive The lytic polysaccharide monooxygenase gene from lpmo ) were expressed in Pichia pastoris; site-directed mutagenesis was used to lpmo Mutation was performed to screen mutants with improved thermal stability, and their application in the efficient saccharification of straw lignocellulose was realized.

[0008] The technical route for achieving the purpose of the present invention is summarized as follows:

[0009] From Rhypophila deceiving Lytic polysaccharide monooxygenase gene lpmoMutation was performed and the mutant A31Q / T39V was screened using the Pichia pastoris expression system to obtain the coding gene lpmom The activity of mutant A31Q / T39V was increased to 121% of that of wild-type lytic polysaccharide monooxygenase (LPMO), and the half-life at 70 ℃ was increased to 129% of that of wild-type lytic polysaccharide monooxygenase (LPMO).

[0010] One of the technical solutions provided by the present invention is a lytic polysaccharide monooxygenase mutant, which is obtained by causing A31Q and T39V mutations on the basis of the wild-type lytic polysaccharide monooxygenase shown in SEQ ID NO.1, and the mutant is named A31Q / T39V;

[0011] Furthermore, the amino acid sequence of the lytic polysaccharide monooxygenase mutant A31Q / T39V is shown in SEQ ID NO.3;

[0012] Furthermore, the coding gene of the A31Q / T39V mutant lpmom , the nucleotide sequence is shown in SEQ ID NO.4.

[0013] The second technical solution provided by the present invention is a recombinant plasmid or recombinant strain containing the above mutant encoding gene;

[0014] Furthermore, the expression vector used was pPIC9K, and the expression host Pastor's P. GS115.

[0015] The third technical solution provided by the present invention is the use of the above-mentioned recombinant plasmid or recombinant strain in producing the lytic polysaccharide monooxygenase mutant described in the first technical solution.

[0016] The fourth technical solution provided by the present invention is the application of the lytic polysaccharide monooxygenase mutant described in the first technical solution, especially in the degradation of lignocellulose, or in the synergistic cellulase degradation of lignocellulose, more particularly in the degradation of lignocellulose raw materials such as straw, bamboo, and sugarcane bagasse, wherein the straw includes but is not limited to corn straw, rice straw, and wheat straw.

[0017] The following definitions are used in the present invention:

[0018] 1. Nomenclature of amino acid and DNA sequences

[0019] The generally accepted IUPAC nomenclature for amino acid residues is used, either as a single letter or three letter code. The generally accepted IUPAC nomenclature for DNA nucleic acid sequences is used.

[0020] 2. Identification of lytic polysaccharide monooxygenase mutants

[0021] The mutated amino acid in the LPMO mutant is represented by "original amino acid + position + replaced amino acid". For example, A31Q means that the amino acid at position 31 is replaced by Gln from Ala of the wild-type LPMO; T39V means that the amino acid at position 39 is replaced by Val from Thr of the wild-type LPMO. The position number corresponds to the amino acid sequence number of the wild-type LPMO in SEQ ID NO.1.

[0022] In the present invention, LPMO represents wild-type lytic polysaccharide monooxygenase, A31Q / T39V represents lytic polysaccharide monooxygenase mutant, lowercase italics lpmo Indicates the gene encoding the wild-type lytic polysaccharide monooxygenase LPMO, in lowercase italics lpmom It represents the coding gene of mutant A31Q / T39V. The specific information is shown in the following table.

[0023]

[0024] Beneficial Effects

[0025] The present invention utilizes overlapping PCR technology to mutate the wild-type LPMO to obtain a mutant A31Q / T39V having improved thermal stability at 70°C relative to the wild-type, the activity of the mutant A31Q / T39V being increased to 121% of the wild-type lytic polysaccharide monooxygenase (LPMO), and the half-life at 70°C being increased to 129% of the wild-type LPMO. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the electrophoresis of the PCR amplification of the wild-type lytic polysaccharide monooxygenase gene

[0027] Among them: M is DNA Marker, lane 1 is the lytic polysaccharide monooxygenase gene lpmo .

[0028] Figure 2 The curves of enzyme activity change of wild-type lytic polysaccharide monooxygenase LPMO and mutant A31Q / T39V at different times at 70 ℃ are shown. DETAILED DESCRIPTION

[0029] The technical content of the present invention is further described below in conjunction with the embodiments, but the present invention is not limited to these embodiments, and the protection scope of the present invention cannot be limited by the following embodiments.

[0030] Some solutions and culture media used in the embodiments of the present invention are as follows:

[0031] LB medium: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride (add 15 g / L agar powder to solid medium), autoclave at 121 °C for 20 min.

[0032] YPD medium: 10 g / L yeast powder, 20 g / L peptone, 20 g / L glucose (add 15 g / L agar powder to solid medium), autoclave at 115 ℃ for 20 min.

[0033] 10×YNB (containing ammonium sulfate): Dissolve 13.4 g of YNB in ​​100 mL of filtered water, filter and sterilize, and store at 4°C.

[0034] 500× Biotin: Dissolve 20 mg of biotin in 100 mL of filtered water, filter sterilize, and store at 4°C.

[0035] 1 mol / L, pH 6.0 potassium phosphate buffer: Prepare 1 mol / L dipotassium hydrogen phosphate and 1 mol / L potassium dihydrogen phosphate and mix to a pH of 6.0.

[0036] MD medium: Prepare 100 mL of medium, consisting of 2 g glucose and 1.5 g agar powder dissolved in 90 mL of filtered water, and sterilize at 115 °C for 20 min. Before use, cool the medium to about 50 °C after dissolving it, and add 10 mL of the above-mentioned filtered and sterilized 10×YNB and 200 μL of 500×biotin on an ultra-clean workbench.

[0037] BMGY medium: Prepare 50 mL of medium, the components of which are 1 g of tryptone, 0.5 g of yeast powder and 1 mL of glycerol dissolved in 40 mL of membrane water, sterilize at 121 °C for 20 min, and add 5 mL of the above-mentioned filtration-sterilized 10×YNB, 100 μL of 500×biotin, and 5 mL of 1 mol / L, pH 6.0 potassium phosphate buffer on the clean bench before use. Prepare other volumes of medium according to the above proportions.

[0038] BMMY medium: Prepare 50 mL of medium, consisting of 1 g of tryptone and 0.5 g of yeast powder dissolved in 40 mL of membrane water, sterilize at 121 °C for 20 min, and add 5 mL of the above-mentioned filtration-sterilized 10×YNB, 100 μL of 500×biotin, 5 mL of 1 mol / L, pH 6.0 potassium phosphate buffer, and 250 μL of methanol on a clean bench before use. Prepare other volumes of medium according to the above ratios.

[0039] In the present invention, the wild-type lytic polysaccharide monooxygenase LPMO sequence is shown in SEQ ID NO.1:

[0040] MKYLSALALAALLPSATVAHYFFPHFVINGAVTPEWQYTRKHDNFLNPSWEDGAFLKSNELRCNKGASNHMREPQTAKVTAGKDTIGFQTYAGTDGLYHPGPVQIYMSKAPGDVRDYDGSGDW FKVAQWGHRRGSTADSNWITWKMRQFTFKMPAEIPAGQYLIRIEQAATHQPYTHREFYVACAHIEVASSYNGAKPGPTFKIPGVYNRDQPFFKYDSWAKPQATECPLPGPAMWPNNNNLKNLT

[0041] In the present invention, the sequence of the A31Q / T39V mutant is shown in SEQ ID NO.3:

[0042] MKYLSALALAALLPSATVAHYFFPHFVINGQVTPEWQYVRKHDNFLNPSWEDGAFLKSNELRCNKGASNHMREPQTAKVTAGKDTIGFQTYAGTDGLYHPGPVQIYMSKAPGDVRDYDGSGDW FKVAQWGHRRGSTADSNWITWKMRQFTFKMPAEIPAGQYLIRIEQAATHQPYTHREFYVACAHIEVASSYNGAKPGPTFKIPGVYNRDQPFFKYDSWAKPQATECPLPGPAMWPNNNNLKNLT

[0043] The present invention will be further explained below through specific examples.

[0044] Example 1 Obtaining the wild-type lytic polysaccharide monooxygenase gene

[0045] From Rhypophila deceiving The gene sequence of LPMO was optimized by comparing the codon preference of Pichia pastoris GS115 host and synthesized by GeneWeiZ (shown in SEQ ID NO.2). Echo RI and Not I two restriction sites and plasmid pPIC9K connected. The ligation product is first transferred into the cloning host E. coli JM109, coated on LB solid medium, extract the plasmid from the transformant for PCR and double enzyme digestion verification, and sequence verification for successful verification. Restriction endonuclease was used to verify the correct plasmid Salt I linearization, purification and recovery, and electrotransfer Pastor's P.GS115 was integrated into the GS115 genome (0.2 mm electroporation cuvette, 1500 V, 5 ms), and plated on MD plates. After 2-3 days, the transformants were picked and plated on G418-resistant YPD solid medium. The transformants were sequenced and pPIC9K- lpmo Plasmid.

[0046] Example 2 Construction of lytic polysaccharide monooxygenase mutants

[0047] According to the results of bioinformatics analysis (according to the molecular dynamics simulation of the enzyme molecule at high temperature, the amino acids with large changes in the B-factor were obtained, and the mutation sites were determined to be A31Q and T39V, and they were mutated.), the vector pPIC9K- lpmo As a template, overlapping PCR was used to construct different mutants.

[0048] 1. Overlap PCR: In the wild type lpmo Overlap PCR was performed on the basis of the gene, and the mutation primers (upstream primer F, downstream primer R) and reaction system were designed as follows:

[0049] F1: CCG GAATTC ATGAAATATTTGTCTGCTTTGGC

[0050] R1: CCATTCAGG AGTAACTTGACCAT

[0051] F2: ATGGTCAAGTTACTCCTGAATGG

[0052] R2: CATG CCATGG AGTCAAATTTTTCAAATTATTGTTATTTGG

[0053] In the first step of overlapping PCR reaction system, F1 and R1 were used as upstream and downstream primers, and F2 and R2 were used as upstream and downstream primers. lpmo Using the PCR reaction as a template, the upstream fragment and the downstream fragment were obtained respectively.

[0054] The reaction system for upstream fragment amplification is:

[0055]

[0056] The reaction system for downstream fragment amplification is:

[0057]

[0058] The amplification program was as follows: pre-denaturation at 98 °C for 30 min; 30 cycles of denaturation at 98 °C for 10 s, annealing at 54 °C for 20 s, and extension at 72 °C for 7 s; and extension at 72 °C for 10 min.

[0059] After gel excision and recovery of upstream and downstream fragments, PCR reaction was performed. The reaction system was:

[0060]

[0061] The amplification program was as follows: pre-denaturation at 98 °C for 30 s; denaturation at 98 °C for 10 s, annealing at 54 °C for 20 s, and extension at 72 °C for 7 s, for 5 cycles; and extension at 72 °C for 10 min.

[0062] After the PCR was completed, 2 μL of upstream primer F1 and downstream primer R2 were added to the system for PCR reaction. The amplification program was as follows: 98 °C pre-denaturation for 30 s; 98 °C denaturation for 10 s, 54 °C annealing for 20 s, 72 °C extension for 10 s, 30 cycles; 72 °C extension for 10 min. The PCR amplification products were subjected to agarose gel electrophoresis and recovered using a small amount of DNA recovery kit.

[0063]

[0064] Gene Echo RI and Not I two restriction sites and plasmid pPIC9K connected. The ligation product is first transferred into the cloning host E. coli JM109 was plated on LB solid medium, and the plasmids in the transformants were extracted for PCR and double enzyme digestion verification. The successful ones were sequenced and verified to obtain pPIC9K- A31Q Plasmid.

[0065] pPIC9K- A31Q Overlapping PCR was performed using the plasmid as a template, and the mutation primers (upstream primer F3, downstream primer R3) and reaction system were designed as follows:

[0066] F3: TGAATGGCAATATGTTAGAAAACAT

[0067] R3:ATGTTTTCTAACATATTGCCATTCA

[0068] F1 and R3 were used as upstream and downstream primers, and F3 and R2 were used as upstream and downstream primers. A31Q Using the PCR reaction as a template, the upstream fragment and the downstream fragment were obtained respectively.

[0069] The reaction system for upstream fragment amplification is:

[0070]

[0071] The reaction system for downstream fragment amplification is:

[0072]

[0073] The amplification program was as follows: pre-denaturation at 98 °C for 30 min; 30 cycles of denaturation at 98 °C for 10 s, annealing at 54 °C for 20 s, and extension at 72 °C for 7 s; and extension at 72 °C for 10 min.

[0074] After gel excision and recovery of upstream and downstream fragments, PCR reaction was performed. The reaction system was:

[0075]

[0076] The amplification program was as follows: pre-denaturation at 98 °C for 30 s; denaturation at 98 °C for 10 s, annealing at 54 °C for 20 s, and extension at 72 °C for 7 s, for 5 cycles; and extension at 72 °C for 10 min.

[0077] After the PCR was completed, 2 μL of upstream primer F1 and downstream primer R2 were added to the system for PCR reaction. The amplification program was as follows: 98 °C pre-denaturation for 30 s; 98 °C denaturation for 10 s, 54 °C annealing for 20 s, 72 °C extension for 10 s, 30 cycles; 72 °C extension for 10 min. The PCR amplification products were subjected to agarose gel electrophoresis and recovered using a small amount of DNA recovery kit.

[0078]

[0079] Gene Echo RI and Not I two restriction sites and plasmid pPIC9K connected. The ligation product is first transferred into the cloning host E. coli JM109 was plated on LB solid medium, and the plasmids in the transformants were extracted for PCR and double enzyme digestion verification. The successful ones were sequenced and verified to obtain pPIC9K- A31Q / T39V Plasmid.

[0080] Plasmid pPIC9K- A31Q / T39V and plasmid pPIC9K- lpmo Restriction endonucleases Salt I was linearized, purified and recovered, and then electrotransferred into Pastor's P. GS115 was integrated into the genome of GS115 (0.2 mm electroporation cuvette, 1500 V, 5 ms), and plated on MD plates. After 2-3 days, the transformants were picked and plated on G418-resistant YPD solid medium. The positive transformants were named Pastor's P. GS115 / pPIC9K- A31Q / T39V and Pastor's P. GS115 / pPIC9K- lpmo .

[0081] Example 3 Expression and purification of lytic polysaccharide monooxygenase

[0082] 1. Expression of lytic polysaccharide monooxygenase

[0083] Take transformants separately Pastor's P. GS115 / pPIC9K- A31Q / T39V and Pastor's P. GS115 / pPIC9K- lpmo Incubate in 200 mL BMGY medium containing glycerol for 16-18 h at 30 °C and 200 r / min. Then inoculate all transformants into 200 mL BMMY medium and add methanol (0.5% v / v) for induction. 2+ LPMO, BMMY medium was supplemented with Cu at a final concentration of 1 mmol / L 2+ Fermentation was performed at 30 °C and 200 r / min for 6 days, with 0.5% v / v methanol added every day. After fermentation, the fermentation broth was centrifuged at 4 °C and 12,000 r / min for 30 min, and the supernatant was filtered through a 0.45-µm filter to obtain the A31Q / T39V mutant crude enzyme solution and the LPMO wild-type crude enzyme solution for enzyme activity determination.

[0084] The substrate 2,6-dimethoxyphenol (2,6-DMP) can be converted into 2,6-DMP free radicals and water in the presence of hydrogen peroxide (H2O2), and then the formed 2,6-DMP free radicals rapidly dimerize to form hydrogenated woody acetoquinone, which can be rapidly oxidized by LPMO to form the color-forming product woody acetoquinone. Therefore, the enzyme activity of LPMO can be determined using 2,6-DMP and H2O2 as co-substrates.

[0085] The reaction system was 1 mL, containing 380 μL 116 mmol / L sodium phosphate buffer (pH 7.5), 100 μL 10 mmol / L 2,6-DMP, 20 μL 5 mmol / L H2O2 and 500 μL LPMO or A31Q / T39V crude enzyme solution, and the reaction was carried out at 70 ° C. The change in absorbance was measured at 469 nm for 300 s.

[0086] The unit enzyme activity is defined as: under certain conditions (unless otherwise specified, the conditions are 70 °C, pH 7.5), 1 µmol of oxidation product (ε469 = 53,200 M) is catalyzed per minute.−1 cm −1 ) The enzyme activity calculation formula is as follows:

[0087]

[0088] Wherein, ΔOD: change in absorbance before and after reaction; V1: total reaction volume; Δt: reaction time; V2: volume of enzyme; ε: molar extinction coefficient of product at 469 nm ε469 = 53,200 M −1 cm −1 .

[0089] The results showed that the fermentation activity of wild-type LPMO was 955.6 U / L, and that of mutant A31Q / T39V was 1156.3 U / L.

[0090] 2. Purification of lytic polysaccharide monooxygenase

[0091] The fermentation broth was purified using Ni-NTA affinity chromatography column. The column was first equilibrated with solution A (0.02 mol / L Tris-HCl, pH 7.4, 0.5 mol / L NaCl, 0.02 mol / L imidazole, 1 mmol / L DTT), then combined with LPMO or A31Q / T39V crude enzyme solution, and then eluted with solution B (0.02 mol / LTris-HCl, pH 7.4, 0.5 mol / L NaCl, 0.03mol / Limidazole, 1 mmol / L DTT). The recombinant protein with (His)6 tag was eluted with solution C (0.02 mol / LTris-HCl, pH 8.0, 0.5 mol / L NaCl, 0.5 mol / L imidazole, 1 mmol / LDTT). The eluted protein was dialyzed against 20 mmol / L, pH 6.0 phosphate buffer at 4 °C to obtain pure enzyme solution. The protein concentration of the pure enzyme solution was determined using a Nanodrop 2000 protein and nucleic acid quantifier.

[0092] The enzyme activity of pure enzyme solution was determined using 2,6-dimethoxyphenol (2,6-DMP) as substrate and hydrogen peroxide as co-substrate. The reaction system was 1 mL, containing 860 μL 116 mmol / L sodium phosphate buffer (pH 6.0-8.5) or acetate buffer (pH 3.5-5.5), 100 μL 10 mmol / L 2,6-DMP, 20 μL 5 mmol / L H2O2 and 20 μL purified LPMO. The change in absorbance after 300 s of reaction was measured at 469 nm. The unit enzyme activity was catalyzed to generate 1 µmol of oxidation product per minute (ε469= 53,200 M −1 cm −1 ) The amount of enzyme required.

[0093] Enzyme specific activity (U / mg) = enzyme activity / protein concentration.

[0094] The final calculated activity ratio of wild-type LPMO and mutant in pure enzyme solution is as follows:

[0095]

[0096] Example 4 Determination of the half-life of lytic polysaccharide monooxygenase

[0097] The half-life of the purified wild-type LPMO and mutant A31Q / T39V at 70°C (pH 7.5) was determined, i.e., the purified wild-type LPMO and mutant A31Q / T39V were placed at 70°C (pH 7.5) for a certain period of time, taken out, and the enzyme activity was determined. The enzyme activity detection method and reaction system were the same as the detection of the pure enzyme solution in Example 3 (the measurement temperature was set to 70°C and the pH was 7.5).

[0098] After testing, the mutant A31Q / T39V has a higher thermal stability than the wild-type LPMO at 70 ℃. The enzyme activity change curves of the wild-type LPMO and the mutant A31Q / T39V are shown in Figure 2 The final calculated half-life of wild-type LPMO and mutant A31Q / T39V at 70 °C is as follows:

[0099]

[0100] Example 5 Application of lytic polysaccharide monooxygenase mutants to degrade straw

[0101] Corn straw, rice straw and wheat straw were used as substrates and cellulase was used as a synergistic catalyst to detect the ability of LPMO and its mutants to degrade different types of straw.

[0102] The reaction system was 1 mL, 20 mmol / L, pH 7.5 sodium phosphate buffer, including 40 mg / mL substrate (substrate corn straw, rice straw, wheat straw treated with 0.4% alkali), 0.08 mg / mL LPMO or mutant, and 1 mmol / L ascorbic acid. The reaction system without LPMO was used as the control. The reaction was carried out in a 2 mL EP tube. After reacting at 50 ℃ and 900 r / min for 48 h, commercial cellulase (3 U / mg) was added to the system at a final concentration of 0.1 mg / mL. After continuing the reaction at 50 ℃ for different time periods, the reaction was terminated by boiling for 10 min, and the supernatant was obtained by centrifugation at 12,000 r / min for 10 min. The reducing sugar concentration was determined by the dinitrosalicylic acid (DNS) method, that is, 150 μL DNS reagent was added to the reaction solution and mixed thoroughly and boiled for 5 min for color development. 200 μL was added to a 96-well plate and the absorbance at 540 nm was measured using an ELISA reader.

[0103] 1. Saccharification results in corn stover

[0104] Compared with the treatment with cellulase alone, the reducing sugar content in the treatment with mutant A31Q / T39V and cellulase was 1.7 times that in the treatment with cellulase alone.

[0105]

[0106] 2. Saccharification results in rice straw

[0107] Compared with the treatment with cellulase alone, the reducing sugar content in the treatment with mutant A31Q / T39V and cellulase was 1.64 times that in the treatment with cellulase alone.

[0108]

[0109] 3. Saccharification results in wheat straw

[0110] Compared with the treatment with cellulase alone, the reducing sugar content in the treatment with mutant A31Q / T39V and cellulase was 1.67 times that in the treatment with cellulase alone.

[0111]

[0112] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A lytic polysaccharide monooxygenase mutant, characterized in that: The lytic polysaccharide monooxygenase mutant is obtained by causing A31Q and T39V mutations on the basis of the wild-type lytic polysaccharide monooxygenase shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

3.

2. A gene encoding the lytic polysaccharide monooxygenase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the gene encoding the lytic polysaccharide monooxygenase mutant is shown in SEQ ID NO.

4.

4. A recombinant plasmid or recombinant strain comprising the mutant encoding gene according to claim 2.

5. Use of the recombinant plasmid or recombinant strain according to claim 4 in producing the lytic polysaccharide monooxygenase mutant according to claim 1.

6. Use of the lytic polysaccharide monooxygenase mutant according to claim 1 in degrading lignocellulose.

7. The use according to claim 6, characterized in that It is used in the degradation of straw, bagasse or bamboo.

8. The use according to claim 6, characterized in that It is used in the synergistic cellulase degradation of lignocellulose.

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