Cracking polysaccharide monooxygenase expression system and application thereof

Abstract

The invention belongs to the technical field of biological catalysis and biological engineering, and relates to a lytic polysaccharide monooxygenase expression system and application thereof. The expression system comprises an expression vector containing a lysing polysaccharide monooxygenase gene expression cassette and a host, the host is a pyrG auxotroph type aspergillus niger strain. According to the present invention, the deltaglaA gene knockout pyrG auxotrophic aspergillus niger strain is adopted as the host, and the strong promoter GlaPr is introduced to drive the expression of the cellulose activity LPMO gene, such that the interference of the endogenous glucamylase is eliminated, and the LPMO secretion efficiency is enhanced; a dynamic fermentation process is adopted, and efficient production of the LPMO protein is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis and bioengineering, and relates to a lytic polysaccharide monooxygenase expression system and application thereof. Background Art

[0002] As the most abundant renewable organic resource in nature, the efficient conversion and utilization of cellulose is a core issue in the development of second-generation bioenergy. Although starch and cellulose are both glucose polymers, the difference in the configuration of α-1,4 glycosidic bonds and β-1,4 glycosidic bonds leads to very different physical and chemical properties: the former is easily degraded and converted by hydrolytic enzymes, while the latter forms a crystalline structure due to the strong hydrogen bonds between molecular chains and forms a complex barrier with hemicellulose and lignin. It needs to undergo physical, chemical or biological pretreatment to destroy its dense structure before it can be enzymatically degraded. Traditional cellulose degradation relies on a ternary synergistic system of endoglucanases, exoglucanases and β-glucosidases. Among them, exoglucanases account for more than 60%, but still face bottlenecks such as low hydrolysis efficiency and high cost, which restrict the industrialization process of lignocellulose biorefining.

[0003] Despite decades of optimization, the cellulase system secreted by Trichoderma reesei, a typical enzyme-producing strain, still suffers from significant drawbacks: limited room for improvement in unit enzyme activity and insufficient ability to degrade crystalline cellulose. Research has shown that supplementing auxiliary enzyme systems can overcome the current limits of enzymatic degradation efficiency. In particular, the discovery of lytic polysaccharide monooxygenases (LPMOs) in 2014 has revolutionized the crystalline cellulose degradation mechanism. These oxidoreductases, relying on copper ions and a reducing agent, disrupt the crystal surface by oxidatively cleaving glycosidic bonds, creating new hydrolysis sites and exhibiting significant synergistic effects with hydrolases. According to the CAZy database, LPMOs have been classified into nine families, AA9-AA17. Family AA9 (formerly GH61) exhibits dual activity against cellulose and some hemicelluloses, family AA10 possesses both cellulose and chitin cleavage, and family AA14 specializes in xylan oxidation, exhibiting broad substrate specificity. Experimental studies have shown that LPMOs can synergize with hydrolases, significantly improving enzymatic efficiency and reducing enzyme costs. This enzyme system has been applied to industrial enzyme preparations and has greatly promoted the pace of bioethanol industrialization. Its multi-substrate degradation characteristics are also of great value to the utilization of wood fiber resources in the feed industry.

[0004] While current research has deepened the molecular mechanisms of LPMOs and discovered many new functional enzymes, their industrialization remains limited by the following: 1. Existing cellulases require large quantities and complex pretreatment processes, resulting in high biofuel production costs; 2. As key auxiliary proteins, technologies for efficient heterologous expression, stable secretion, and low-cost purification of LPMOs are still immature; and 3. The ratio of LPMOs to cellulases and the synergistic processes in existing enzyme preparations are not optimized, limiting their potential for industrial application. These challenges provide clear technical improvement directions for the development of new complex enzyme preparations. Summary of the Invention

[0005] To address the challenges of the existing technology, the present invention provides a high-efficiency expression system for a lytic polysaccharide monooxygenase (LPMO) based on Aspergillus niger (A. niger). This expression system enables low-cost, industrialized production of LPMO. The resulting lytic polysaccharide monooxygenase significantly improves the efficiency of cellulase in degrading lignocellulose, reducing the production cost of second-generation bioenergy sources such as cellulosic ethanol.

[0006] The present invention provides a lytic polysaccharide monooxygenase expression system, which comprises an expression vector containing a lytic polysaccharide monooxygenase gene expression cassette and a host; the host is pyrG Auxotrophic Aspergillus niger strains.

[0007] Preferably, the lytic polysaccharide monooxygenase gene expression cassette includes the Aspergillus niger glucoamylase promoter GlaPr , a lytic polysaccharide monooxygenase gene retaining the wild-type signal peptide sequence, and the Aspergillus niger glucoamylase transcription terminator GlaTt .

[0008] Preferably, the expression vector contains screening markers for Escherichia coli and Aspergillus niger.

[0009] Preferably, the screening markers include: pyrG Gene, used for transformant screening; AmpR and CmR Resistance gene, used for vector amplification in E. coli.

[0010] The present invention also provides use of the system in producing lytic polysaccharide monooxygenase.

[0011] The present invention further provides a large-scale fermentation production process, which specifically comprises the following steps: (1) Propagating the lytic polysaccharide monooxygenase expression system according to any one of claims 1 to 4; (2) inoculating the seed liquid into a fermentation medium for fermentation; the fermentation medium is a bran-based medium containing 3% bran, 5% maltose, and 1% glucose by mass; a gradient speed and maltose feeding strategy are adopted during the fermentation process; (3) The fermentation broth is subjected to multi-stage filtration, purification and sterilization.

[0012] Preferably, the gradient speed is as follows: an initial speed of 100 rpm, which increases by 50 rpm to 250 rpm every 10 h.

[0013] Preferably, the maltose feeding strategy is as follows: after culturing for 48 h, a 30% maltose solution is fed at a rate of 0.4 kg / h until 10 h before the end of fermentation.

[0014] Preferably, the multi-stage filtration purification process includes: plate and frame filtration, diatomaceous earth filtration, 200 nm PP filter element filtration, ultrafiltration concentration, and 200 nm PES filter element filtration.

[0015] The present invention is ΔglaA gene knockout pyrG The auxotrophic Aspergillus niger strain was used as the host and a strong promoter was introduced GlaPr Driving the expression of cellulose-active LPMO genes eliminates the interference of endogenous glucoamylase and enhances the secretion efficiency of LPMO.

[0016] The present invention develops a dynamic fermentation process (gradient speed, maltose feeding strategy) and a multi-stage purification process (plate and frame filtration-ultrafiltration concentration-terminal sterilization) to achieve efficient production of LPMO protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the SDS-PAGE electrophoresis pattern of the LPMO protein fermentation stock solution in the embodiment of the present invention; Figure 2 Schematic diagram of the synergistic effect of LPMO protein and cellulase in degrading PCS in an embodiment of the present invention; Figure 3 This is a schematic diagram of the synergistic effect of LPMO protein and cellulase in degrading poplar pulp in an embodiment of the present invention. DETAILED DESCRIPTION

[0018] To facilitate understanding of the present invention, the present invention is described in more detail below with reference to the accompanying drawings and specific embodiments. Preferred embodiments of the present invention are shown in the accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[0019] Solvent formula: Nitrate stock solution (20×): Take 120.0 g NaNO3 (1.41 M), 10.4 g KCl (0.14 M), 16.3 g KH2PO4 (0.12 M), and 20.9 g K2HPO4 (0.12 M), dilute to 1 L with distilled water, sterilize at 121°C for 20 min, and store at room temperature.

[0020] Vitamin stock solution (100×): Add 100 mg of vitamin B1, 100 mg of vitamin B2, 100 mg of niacinamide, 50 mg of pyridoxine hydrochloride, 10 mg of pantothenic acid, and 0.2 g of biotin, dilute to 500 mL (pH 6.0) with distilled water, filter sterilize, and store at 4°C in the dark.

[0021] Trace element stock solution (1000×): Prepare 2.2 g ZnSO4•7H2O, 1.1 g H3BO3, 0.5 g FeSO4•7H2O, 0.17 g CoCl2•6H2O, 0.16 g CuSO4•5H2O, 0.5 g MnCl2•4H2O, 0.15 g Na2MoO4•2H2O, and 5.0 g EDTA. Add distilled water to 80 mL, bring to a boil for 10 min, and adjust the pH to 6.5 with 4 M KOH. Add distilled water to 100 mL, filter-sterilize, and store at 4°C in the dark.

[0022] Complete medium (CM): Mix 500 mL of 20× nitrate stock solution, 10 g D-glucose, 2 g peptone, 1 g yeast extract, 1 g casamino acids, and 1.12 g uracil, and dilute to 985 mL with distilled water. Sterilize at 121°C for 20 min. After cooling, add 1 mL of vitamin stock solution, 1 mL of trace element stock solution, and 10 mL of 100 mM uridine solution.

[0023] MgSO4 solution (0.6 M): Take 147.89 g MgSO4·7H2O, dilute to 1 L with distilled water, sterilize at 121℃ for 20 min, and store at room temperature.

[0024] NaH2PO4 solution (0.5 M): Take 6.9 g of NaH2PO4·H2O and dilute to 100 mL with distilled water. Store at room temperature.

[0025] Na2HPO4 solution (0.5 M): Take 44.5 g of Na2HPO4·2H2O and dilute to 500 mL with distilled water. Store at room temperature.

[0026] OM#1 solution: Mix 5 mL of 0.5 M NaH2PO4 solution and 26.2 mL of 0.5 M Na2HPO4 solution, dilute to 500 mL with distilled water, sterilize at 121°C for 20 min, and store at room temperature.

[0027] OM#2 solution: Take 591.55 g of MgSO4·7H2O and dilute to 1 L with distilled water. Sterilize at 121°C for 20 min and store at room temperature.

[0028] OM solution: Mix 32 mL of OM#1 solution and 50 mL of OM#2 solution, and dilute to 100 mL with sterile distilled water. Prepare immediately.

[0029] Vinoflow enzyme solution: 250 mg of Vinoflow FCE (Novozyme) was diluted to 1 mL with sterile distilled water and used immediately.

[0030] TB solution: Take 54.66 g of sorbitol and 50 mL of 1 M Tris-HCl (pH 7.5), dilute to 500 mL with distilled water, sterilize at 121°C for 15 min, and store at 4°C.

[0031] SC solution: Take 182.2 g of sorbitol and 7.35 g of CaCl2·2H2O and dilute to 1 L with distilled water. Sterilize at 121°C for 20 min and store at 4°C.

[0032] Glycerol solution (10 M): Take 92.1 g of glycerol and dilute to 100 mL with distilled water. Sterilize at 121°C for 20 min and store at 4°C.

[0033] ATA solution (0.4 M aurintricarboxylic acid): Take 1.89 g of aurintricarboxylic acid and dilute to 10 mL with distilled water. Sterilize by filtration and store at 4°C in the dark.

[0034] 60% PEG solution: Take 60 g PEG 4000, 1 mL 1 M Tris-HCl (pH 7.5), and 5 mL 1 M CaCl2, dilute to 90 mL with distilled water, dissolve in a 50°C water bath, dilute to 100 mL with distilled water, sterilize at 121°C for 15 min, and store at room temperature.

[0035] 20% PEG solution: Mix 60% PEG solution and SC solution in a volume ratio of 1:2, sterilize at 121°C for 20 min, and store at room temperature.

[0036] Sorbitol solution (1.2 M): Take 109.3 g of sorbitol and dilute to 500 mL with distilled water. Sterilize at 121°C for 20 min and store at 4°C.

[0037] Solution A (40×): Take 120 g NaNO3, 10.4 g KCl, 30.4 g KH2PO4 and 22.5 mL 4 M KOH, dilute to 500 mL with distilled water, sterilize at 121℃ for 20 min, and store at room temperature.

[0038] Solution B (40×): Take 10.4 g MgSO4·7H2O and dilute to 500 mL with distilled water. Sterilize at 121°C for 20 min and store at room temperature.

[0039] Selective Regeneration Medium Plates (SRM): Solution #1: Combine 11 mL of Solution A, 11 mL of Solution B, and 0.44 mL of trace element stock solution and bring to 40 mL with distilled water. Solution #2: Take 150 g sucrose and 8.8 g agar and dilute to 400 mL with distilled water; After sterilizing them separately, mix them at 55°C and pour them into plates.

[0040] MM "J" medium: 200 mL 20× nitrate stock solution, 4 mL trace element stock solution, and 150 g D-maltose are diluted to 980 mL with distilled water. After sterilization, cool to 50°C, add 20 mL 2.25 M MgSO4 solution, and store at 4°C.

[0041] 80% glycerol solution: Take 8 g of glycerol and dilute to 10 mL with distilled water. Sterilize at 121°C for 20 min and store at room temperature.

[0042] Note: All sterilization conditions are high-pressure steam sterilization. Unless otherwise specified, storage conditions are room temperature or 4℃ away from light.

[0043] Lytic polysaccharide monooxygenase LPMO from Thielavia terrestris ( Thielaviaterrestris ), the genome sequence ID in the JGI database is 2119040. LPMO retains the first 19 amino acids at the N-terminus of the protein (the wild-type signal peptide sequence), ensuring efficient extracellular secretion of the protein.

[0044] Example 1 Construction of LPMO expression system of Aspergillus niger and screening of high protein expression strains 1. Construction of recombinant expression strains and vectors (1) Genetic modification of host strains Using Aspergillus niger CBS 513.88 (purchased from China Industrial Microbiological Culture Collection Center) as the starting strain, a uracil auxotrophic strain was obtained by UV mutagenesis and 5-fluorouracil reverse screening. pyrG Gene inactivated strains, the strains grow in a medium containing uracil and uridine. On this basis, the glucoamylase gene in its chromosome was knocked out by homologous recombination ( glaA ) to eliminate background protein interference.

[0045] (2) Design of E. coli-fungus shuttle expression vector Aspergillus niger grows in a medium with glucose or maltose as the carbon source and secretes a large amount of protein, of which glucoamylase accounts for more than 50%. glaA ) is driven in vivo by the glucoamylase promoter (Glucoamylase promoter, GlaPr ) regulate expression.

[0046] The expression vector carries ampicillin ( Amp R ) and chloramphenicol ( Cm R ) resistance gene, used for vector amplification and screening in E. coli; carrying orotidine-5'-phosphate decarboxylase gene ( pyrG ), used for nutritional deficiency complementary screening of Aspergillus niger positive transformants; the expression cassette is driven by the Aspergillus niger glucoamylase strong promoter ( GlaPr )、target gene coding sequence (including wild-type signal peptide), glucoamylase transcription terminator ( GlaTt The expression vector was transformed into Aspergillus niger protoplasts via PEG-mediated transformation, and positive transformants were screened using selective regeneration medium.

[0047] 2. Aspergillus niger protoplast preparation and PEG-mediated transformation (1) Protoplast preparation and purification Three glass beads and 100 mL complete culture medium (CM, containing 500 μg / mL ampicillin) were added to a 500 mL sterile Erlenmeyer flask and inoculated with Aspergillus niger spores to a final concentration of 1×10 6 spores / mL, and cultured with shaking at 30°C and 150 rpm for 16-18 h. Mycelia were collected by filtration through Miracloth filter cloth, washed with 100 mL of 0.6 M MgSO4 solution, transferred to a 125 mL Erlenmeyer flask and weighed. Subsequently, the mycelia were resuspended in 10 mL of OM solution per 2 g of mycelia, and then enzyme solution was added at a ratio of 1 mL of Vinoflow hydrolase (Novozymes, Denmark) per 2 g of mycelia, and enzymatic hydrolysis was performed at 30°C and 100 rpm for 3 h.

[0048] Take 20 mL of the enzymatic hydrolysate and chromatograph it with ice-cold TB solution (20 mL of TB was covered on the upper layer of 20 mL of protoplast suspension), centrifuge it at 4°C and 5000 rpm for 10 min, and collect the protoplasts at the interface; the protoplasts were washed with SC solution (centrifuge it at 4°C and 3000 rpm for 5 min), and resuspended in pre-cooled SC solution to a final concentration of 1×10 8 / mL, and divided into a mixture of 150 μL protoplasts and 75 μL 10 M glycerol, and stored at -80℃.

[0049] (2) PEG-mediated protoplast transformation The protoplast suspension was diluted to 1×10 7 / mL, take 200 μL of protoplasts and mix with 20 μL of TA solution, 10 μL of target DNA (1-5 μg) and 100 μL of 20% PEG 4000, and let it stand at room temperature for 10 min.

[0050] Add 1.5 mL of 60% PEG 4000, mix gently, and let stand at room temperature for 20 min. Then add 5 mL of 1.2 M sorbitol solution. Centrifuge (4000 rpm, 10 min) and resuspend the pellet in 1 mL of 1.2 M sorbitol. Take 200 μL and spread it on SRM screening plates. Incubate at 30°C for 4-6 days until single colonies are formed.

[0051] 3. Screening, Verification and Preservation of High Protein Expression Strains The protein expression culture of the positive transformants was carried out in a flat-bottom 96-well cell culture plate, and the expression levels of the target proteins were compared and analyzed by SDS-PAGE electrophoresis technology to screen and obtain high-expression strains.

[0052] A single colony from the SRM plate was inoculated into a 96-well plate containing 200 μL of MM “J” medium and cultured at 30°C for 5 days.

[0053] The culture supernatant was taken for SDS-PAGE analysis, and the untransformed strain was used as a negative control to screen the clone with the highest expression of the target protein.

[0054] Short-term storage: spores of the screened strains were stored on plates at 4°C; For long-term storage: Mix the spore suspension with 80% glycerol at a ratio of 4:1 (final concentration 20%) and store at -80°C.

[0055] After genetic modification of the host strain and screening of protein expression strains, the expression level of the target protein (32 kDa) in the obtained high-protein expression strain was significantly increased. The target protein is the most abundant protein in the total protein, and the content of other background proteins is relatively small. Figure 1 .

[0056] Example 2 Production of LPMO by Aspergillus niger fermentation and product post-processing process 1. Standardized Preparation of Spore Seeds (1) Culture medium preparation (w / v) Prepare a culture medium with 5% bran, 0.5% ammonium sulfate, 0.5% glucose, 0.3% yeast extract, 0.3% potassium dihydrogen phosphate, 0.2% magnesium sulfate, and 2% agar in distilled water, pH 5.3-5.5. Sterilize at 121°C for 30 min, pour into plates (approximately 20 mL per plate), cool, and pre-incubate at 30°C for 2 days to verify sterility before use.

[0057] (2) Single colony isolation and amplification Take the bacteria stored in -80℃ glycerol tube or 4℃, streak on the plate under sterile conditions, and culture at 30℃ for 3 days to obtain black spores; pick a single colony for secondary streak amplification and culture at 30℃ for 3 days to prepare high-purity spores.

[0058] (3) Preparation of spore suspension Add 10 mL of saline / Tween solution to the plate, scrape the spores and transfer them to a sterile bottle. Store at 4°C or mix with 80% glycerol at a ratio of 4:1 and freeze at -80°C.

[0059] 2. Preparation of Seed Solution in Shake Flasks (1) Culture medium preparation (w / v) Prepare with 5% bran, 0.5% ammonium sulfate, 0.5% glucose, 0.3% yeast extract, 0.3% potassium dihydrogen phosphate, and 0.2% magnesium sulfate in tap water, pH 5.3-5.5; dispense into 150 mL / 500 mL Erlenmeyer flasks and sterilize at 121°C for 30 min.

[0060] (2) Inoculation and cultivation Aseptically inoculate the spore suspension to a final concentration of 2×10 6 spores / mL, and cultured at 30℃ and 180 rpm for 24 h to obtain mycelial seed solution.

[0061] 3. First-level seed tank expansion process (1) Culture medium formula (w / v) 3% bran, 0.5% ammonium sulfate, 0.5% maltose, 0.3% yeast extract, 0.3% potassium dihydrogen phosphate, 0.2% magnesium sulfate, tap water, pH 6.5; sterilize at 121℃ for 30 min.

[0062] (2) Inoculation and culture conditions The mycelial liquid was inoculated at a 1% (v / v) inoculum volume into the shake flask and cultured at 30°C, 3 L / min aeration, and 250 rpm stirring for 16 h.

[0063] 4. Enzyme production fermentation tank process (1) Fermentation medium (w / v) Bran 3%, ammonium sulfate 0.05%, maltose 5%, yeast extract 0.3%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.05%, glucose 1%.

[0064] (2) Fermentation control parameters Fermentation was carried out in a 500 L fermenter (containing 300 L of sterilized fermentation medium) with a 10% (v / v) inoculum. The initial rotation speed was 100 rpm and the speed was increased by 50 rpm every 10 h to a final speed of 250 rpm to ensure sufficient dissolved oxygen during the fermentation process. The temperature was 30°C, the ventilation rate was 5 L / min, and after 16 h of culture, ammonia water was passed to maintain the pH at 5.3-5.5; 30% maltose solution was added at a flow rate of 0.4 kg / h starting from 48 h of culture and continued until 10 h before the end of fermentation.

[0065] Samples were taken at regular intervals to determine the extracellular protein content in the fermentation broth at different times.

[0066] 5. Treatment process of fermentation mash The product post-processing process includes: plate and frame filtration, diatomaceous earth filtration, 200 nanometer PP filter element filtration, ultrafiltration concentration, and 200 nanometer PES filter element filtration.

[0067] (1) Plate and frame filter press: The fermentation liquid is pumped into the plate and frame filter press, and the material is pressed in quickly in the initial stage in order to press the fermentation liquid into each filter chamber to improve the filtration efficiency; when the pressure gauge of the equipment is close to 0.7 MPa, the feeding is stopped, and compressed air is introduced to squeeze the filtrate in the filter chamber, and then the material is re-fed to form a cycle operation.

[0068] (2) Diatomaceous earth pre-coating filtration: Pump the filtrate into a circulating stainless steel drum and add 20# diatomaceous earth premix to form a mixed solution. Establish a circulation system through the diatomaceous earth filter, ensuring that the pre-coat evenly covers the filter cloth surface and traps particulate matter in the filtrate. Once the filtrate is clear and transparent, open the discharge valve to discharge the material. Collect the filtrate in a dedicated collection bucket. Do not evacuate the filter during the filtration process. Keep the filter pressure ≤ 0.3 MPa.

[0069] (3) 200 nm PP filter element filtration: Open the filter drain valve, adjust the steam pressure to form an internal circulation, and sterilize at 121℃ for 30 minutes; after sterilization, introduce compressed air cooling system, press the filtrate into the filter from the feed port with pressure, maintain the feed pressure ≤0.3MPa, and discharge the filtrate through the discharge port.

[0070] (4) Ultrafiltration concentration: Turn on the water pump to allow the filtrate from the PP filter element to pass through the ultrafiltration membrane element (10 kDa MWCO ultrafiltration membrane). The large molecular liquid that cannot pass through the membrane pore size will flow back into the circulating stainless steel tank material for further circulation, filtration and concentration. The operating pressure is ≤0.3 MPa.

[0071] (5) 200 nm PES filter terminal sterilization: The ultrafiltrate was filtered through a PES filter element (the filter element was sterilized by steam for 30 min in advance and used after cooling), with an operating pressure of ≤0.3 MPa. The filtered initial liquid was returned to the system for re-filtration, and the qualified filtrate was collected in a sterile container.

[0072] Example 3 LPMO protein characteristics and verification of its synergistic effect with cellulase 1. Biochemical characteristics of LPMO proteins (1) Protein origin and structural characteristics LPMO protein is derived from Thielavia terrestris ( T. terrestris ), whose genome sequence is ID 2119040 in the JGI database; amino acid sequence analysis showed that the protein belongs to the AA9 family and has cellulose oxidation activity; the first 19 amino acids at the N-terminus of the protein are a secretion signal peptide, driving the protein to be secreted outside the cell; the C-terminal functional domain contains a cellulose binding domain (CMB1), which has a very strong binding ability to microcrystalline cellulose (it cannot be eluted by conventional buffer after binding).

[0073] (2) Molecular weight verification The theoretical molecular weight is 31.31 kDa, and the actual molecular weight determined by SDS-PAGE is 32 kDa. Figure 1 , which is consistent with the predicted value.

[0074] (3) Enzyme activity detection No endocellulase (CMCase) activity was detected using sodium carboxymethylcellulose (CMC) as substrate.

[0075] 2. Verification of the synergistic effect of LPMO and cellulase (1) Experimental system design Substrate: Pretreated corn stover (PCS) and aspen mechanical pulp pretreated with dilute acid; Main enzyme system: Trichoderma rishii cellulase ( Trichodermareesei , containing CBHI, CBHII, endonuclease and β-glucosidase, purchased from Baiyin Sino Biotechnology Co., Ltd.); Synergistic component: LPMO protein; Reaction conditions: pH 4.8, 50°C, 150 rpm shaking for 24 h.

[0076] (2) Experimental groups Experimental group: 1.5% substrate (w / w) + 0.15% cellulase (w / w substrate) + 0.1% LPMO (w / w substrate); Control group: only cellulase (0.15%) or LPMO (0.1%) was added; Blank group: no enzyme added.

[0077] The above enzymatic hydrolysis experiments of lignocellulose were all carried out in 50 mL falcon tubes with a reaction system of 10 mL.

[0078] (3) Glucose quantitative analysis The glucose concentration in the supernatant was determined using the Amplex Red Glucose Assay Kit (Invitrogen).

[0079] (4) Results analysis like Figure 2 and Figure 3 As shown in the figure, when LPMO acts alone, the amount of glucose released is equivalent to that of the blank group (background protein has no significant degradation activity).

[0080] The glucose yields of the experimental group (cellulase + LPMO) on PCS and poplar pulp were 2.3 times and 2.2 times that of cellulase alone, respectively, which were significantly higher than the sum of the two enzymes acting alone.

[0081] LPMO can significantly improve the decomposition ability of cellulose hydrolyzing enzymes on PCS and poplar pulp, showing a strong synergistic effect.

Claims

1. A lytic polysaccharide monooxygenase expression system, characterized by: The expression system comprises an expression vector containing a lytic polysaccharide monooxygenase gene expression cassette and a host; the host is pyrG Auxotrophic Aspergillus niger strains.

2. The lytic polysaccharide monooxygenase expression system according to claim 1, characterized in that: The lytic polysaccharide monooxygenase gene expression cassette includes the Aspergillus niger glucoamylase promoter GlaPr , a lytic polysaccharide monooxygenase gene retaining a wild-type signal peptide sequence, and an Aspergillus niger glucoamylase transcription terminator GlaTt .

3. The lytic polysaccharide monooxygenase expression system according to claim 1, characterized in that: The expression vector contains screening markers of Escherichia coli and Aspergillus niger.

4. The lytic polysaccharide monooxygenase expression system according to claim 3, characterized in that: The screening markers include: pyrG Gene, used for nutritional deficiency complementation screening of Aspergillus niger positive transformants; AmpR and CmR Resistance gene, used for vector amplification and screening in E. coli.

5. Use of the system according to any one of claims 1 to 4 in the production of lytic polysaccharide monooxygenase.

6. A large-scale fermentation production process for lytic polysaccharide monooxygenase, characterized in that: include: (1) Propagating the lytic polysaccharide monooxygenase expression system according to any one of claims 1 to 4; (2) inoculating the seed liquid into a fermentation medium for fermentation; the fermentation medium is a bran-based medium containing 3% bran, 5% maltose, and 1% glucose by mass; a gradient speed and maltose feeding strategy are used during the fermentation process; (3) The fermentation broth is subjected to multi-stage filtration, purification and sterilization.

7. The large-scale fermentation production process of lytic polysaccharide monooxygenase according to claim 6, characterized in that: The gradient speed is specifically as follows: the initial speed is 100 rpm, and it increases by 50 rpm every 10 h to 250 rpm.

8. The large-scale fermentation production process of lytic polysaccharide monooxygenase according to claim 6, characterized in that: The maltose feeding strategy is specifically as follows: after culturing for 48 h, a maltose solution with a concentration of 30% is fed at a rate of 0.4 kg / h until 10 h before the end of fermentation.

9. The large-scale fermentation production process of lytic polysaccharide monooxygenase according to claim 6, characterized in that: The multi-stage filtration purification process includes: plate and frame filtration, diatomaceous earth filtration, 200 nm PP filter element filtration, ultrafiltration concentration, and 200 nm PES filter element filtration.

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