A method for improving the production of hydrolysis sugars from cellulosic feedstocks

Abstract

The application discloses a method for improving hydrolysis of fibrous material, and belongs to the field of agricultural and forestry biomass material resource utilization and biotechnology. In order to solve the problem of low cellulose hydrolysis efficiency, the application adds thermophilic lytic polysaccharide monooxygenase to synergize with cellulase in the process of hydrolyzing fibrous material, so that the hydrolysis efficiency is effectively improved. On this basis, by adding thermophilic lytic polysaccharide monooxygenase, thermophilic endoglucanase and thermophilic xylanase, the fibrous material is hydrolyzed in the mode of liquefaction and saccharification, so that the hydrolysis efficiency of high-concentration fibrous material substrate can be significantly improved, and the utilization rate of cellulase is improved.

Description

Technical Field

[0001] This invention relates to a method for improving the sugar production from the hydrolysis of fibrous raw materials, belonging to the fields of agricultural and forestry biomass resource utilization and biotechnology. Background Technology

[0002] Environmental challenges and energy security concerns are forcing countries worldwide to seek sustainable, renewable, and carbon-neutral resources. Lignocellulose is one of the most promising renewable resources; as early as 2008, global production of lignocellulose exceeded 200 billion tons. Its main raw materials include various trees and large amounts of agricultural and forestry waste, such as bagasse, corn cobs, and straw. As a friendly alternative to fossil resources, lignocellulose can be used to produce second-generation biofuels such as ethanol, as well as other chemicals. Therefore, the effective utilization of lignocellulose is of great significance in alleviating the increasingly serious resource, energy, and ecological crises. However, lignocellulose has a complex composition. In addition to cellulose (40-60%), hemicellulose (20-35%), and lignin (15-25%), it also contains small amounts of pectin, ash, lipids, and other components. Cellulose acts as a "skeleton," cross-linking with the surrounding hemicellulose and lignin; lignin acts as a "binder" and "filler" filling the intercellular layer and between microfibers. These complex components form the complex and dense multi-level structure of natural lignocellulose, making it difficult to biodegrade effectively.

[0003] In the hydrolysis of fibrous substrates, a single cellulase protein cannot achieve good hydrolytic efficiency; often, complex enzyme preparations made from multiple cellulase proteins play a crucial role. Substrate hydrolysis generally consists of three types of glycosidic hydrolases: endoglucanase, exoglucanase, and β-glucosidase. These three enzymes work synergistically, playing complementary roles in the hydrolysis of cellulose. Nevertheless, cellulase hydrolysis remains the rate-limiting step in the utilization of lignocellulose, and improving its efficiency has always been a key focus for researchers. A recently discovered copper-dependent lytic polysaccharide monooxygenase (LPMO) significantly enhances the hydrolytic efficiency of glycosidic hydrolases by breaking glycosidic bonds in the crystalline region of cellulose through redox reactions. This offers a potential solution to the current problem of low efficiency in the hydrolysis of lignocellulose by cellulase. Hu et al. studied the effect of Staphylococcus aurantiacus TaAA9 on cellulase Cellullast, finding that adding only 1 mg / g of cellulase to corn stalks pretreated with organic solvents significantly improved the efficiency of glycosidic hydrolysis. -1 TaAA9 and cellulase (20 mg / g) of cellulose -1 When cellulose is added, the cellulose hydrolysis rate can be increased by 25%. In subsequent studies, they added AA9 to a complex cellulase (54 mg / g).-1 Cellulase Cellulase Cellluclast + 5 mg / g -1 Hydrolysis of poplar pretreated with high solid-phase loading (20% substrate concentration) using LPMO (a cellulase) was performed, and the results showed that the addition of LPMO could increase the degradation rate by about 30%. Müler et al. showed that when hydrolyzing birch pretreated with steam explosion under aerobic conditions, the use of cellulase Cellic CTec2 containing 15% LPMO increased glucose production by about 40%. The article "LPMO AfAA9_B and Cellobiohydrolase AfCel6A from A. fumigatus Boost Enzymatic Saccharification Activity of Cellulase Cocktail; DOI.org / 10.3390 / ijms22010276" disclosed the use of LPMO in combination with cellulase to degrade bagasse, increasing the hydrolysis rate of bagasse by 70% and 95% after 24 and 48 hours, respectively. However, this technology has problems such as low enzymatic concentration in bagasse, a still low hydrolysis rate (2%) after synergistic enzymatic treatment, and the need for additional oxygen. In summary, existing research indicates that LPMO can effectively enhance cellulase activity and has great potential for efficient hydrolysis of lignocellulose raw materials. Summary of the Invention

[0004] To address the technical bottlenecks of high-concentration enzymatic hydrolysis of fibrous raw materials, such as high viscosity leading to hydrodynamic problems and low enzymatic hydrolysis efficiency, this invention proposes a two-step hydrolysis method for starchy raw materials, involving liquefaction followed by saccharification, aiming to provide a method to improve sugar production from fibrous raw material hydrolysis. This invention employs a two-step enzymatic hydrolysis of fibrous raw materials. First, the fibrous raw material is liquefied at high temperature (pre-hydrolysis) using a cleaving polysaccharide monooxygenase (mgLPMO). This catalyzes the C1 or C4 hydroxyl groups in the glucosyl units of cellulose, disrupting the cellulose structure and forming an amorphous and loose cellulose structure. The generation of reducing and non-reducing sugar chain ends improves the accessibility and efficiency of subsequent cellulase hydrolysis. After liquefaction and cooling, cellulase is added for further hydrolysis and saccharification, completely generating fermentable sugars such as glucose. Furthermore, thermophilic endoglucanase and thermophilic xylanase are added during the initial enzymatic hydrolysis for synergistic hydrolysis, further disrupting the cellulose structure. Even further, increasing the reaction temperature during the initial enzymatic hydrolysis improves the enzymatic hydrolysis rate of pretreated sugarcane bagasse.

[0005] The first objective of this invention is to provide a method for improving the sugar production from the hydrolysis of cellulose raw materials, the method comprising the following steps:

[0006] (1) The first step hydrolysis product was obtained by reacting the fibrous raw material with a lysin monooxygenase in conjunction with a thermophilic enzyme at 60℃~100℃.

[0007] (2) Cool the hydrolysate obtained in step (1) and add cellulase to hydrolyze and produce sugar.

[0008] In one embodiment, the cleaving polysaccharide monooxygenase can be obtained by heterologous expression in Escherichia coli.

[0009] In one embodiment, the cleaving polysaccharide monooxygenase described in step (1) is mgLPMO10, with the amino acid sequence shown in SEQ ID NO.1. Alternatively, it can be a cleaving polysaccharide monooxygenase mutant obtained by mutating the 17th aspartic acid to tryptophan based on the cleaving polysaccharide monooxygenase shown in SEQ ID NO.1, abbreviated as D17W.

[0010] In one embodiment, step (1) is a reaction at 60°C to 80°C, or optionally, at 65°C to 75°C.

[0011] In one embodiment, the thermophilic enzyme in step (1) is either a thermophilic endoglucanase or a thermophilic xylanase, or a combination of both.

[0012] Optionally, the amino acid sequences of the thermophilic endoglucanase and the thermophilic xylanase are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0013] In one embodiment, the fibrous raw material in step (1) is any one or a combination of cellulose, pulp, straw-based lignocellulose, agricultural product processing fiber residues, and forest product processing residues.

[0014] In one embodiment, the fibrous raw material is pretreated bagasse, microcrystalline cellulose, phosphate-swellable cellulose, etc.

[0015] In one embodiment, the pretreated bagasse is prepared by grinding the bagasse into fine particles, sieving and drying them; adding glycerol and sodium hydroxide, and heating to react; cooling after the reaction is completed, washing with water multiple times and filtering to remove the glycerol solution; collecting the bagasse and drying it to obtain the pretreated bagasse.

[0016] In one embodiment, the concentration of the lignocellulose substrate in step (1) is 0.5% to 40% w / v. Optionally, the concentration of the pretreated bagasse is 3% w / v to 15% w / v; further, the concentration is 6% w / v to 15% w / v, or further, 10% w / v to 15% w / v.

[0017] Optionally, the concentration of microcrystalline cellulose is 0.5% w / v; the concentration of phosphate-swellable cellulose is 0.5% w / v; and the concentration of pretreated bagasse is 0.5% w / v or 15% w / v.

[0018] In one embodiment, the ratio of thermophilic endoglucanase, polysaccharide lysin monooxygenase and thermophilic xylanase in step (1) is (1-3):(0-3):(0-3).

[0019] Optionally, the ratio of thermophilic endoglucanase, polysaccharide-lysing monooxygenase and thermophilic xylanase is (0.5–2):(0.5–2):(1–2).

[0020] Preferably, the ratio of endoglucanase, cleaving polysaccharide monooxygenase and thermophilic xylanase is 1:1:(0.5-2).

[0021] In one embodiment, the amount of each enzyme added in step (1) is 3–60 μmol·L. -1 Alternatively, the dosage may be 0.01–10 U / g dry substrate. Optionally, the dosage of endoglucanase may be 0.3–8 U / g dry substrate, the dosage of polysaccharide monooxygenase may be 0.01–0.4 U / g dry substrate, and the dosage of xylanase may be 0.2–5 U / g dry substrate.

[0022] In one embodiment, the amount of mg LPMO10 and thermophilic enzyme added in step (1) is 15 μmol·L⁻¹. -1 .

[0023] In one embodiment, an electron donor is added in step (1), which may be cellobiose dehydrogenase, lignin derivatives, or reducing agents such as ascorbic acid or gallic acid.

[0024] In one embodiment, 0.5–1.5 mmol·L⁻¹ is added in step (1). -1 ascorbic acid.

[0025] Optionally, in step (1), add 1 mmol·L -1 ascorbic acid.

[0026] In one implementation, the pretreatment time for step (1) is 1 to 3 hours.

[0027] In one embodiment, the temperature of enzymatic hydrolysis in step (2) is 45-55°C.

[0028] Optionally, the enzymatic hydrolysis temperature in step (2) is 50°C.

[0029] The beneficial effects of this invention are:

[0030] Unlike traditional enzymatic hydrolysis of cellulose, this invention employs a two-step enzymatic hydrolysis method for cellulose raw materials. First, a cleaving polysaccharide monooxygenase, in conjunction with thermophilic endoglucanase and thermophilic xylanase, performs high-temperature preliminary enzymatic hydrolysis of the cellulose raw material. After cooling, cellulase is added for further enzymatic hydrolysis and saccharification. The enzymatic hydrolysis rate of 15% w / v cellulose raw material (pretreated sugarcane bagasse) reaches over 25%. This invention is highly practical and can be widely applied to agricultural and forestry biomass raw materials from various sources. The thermophilic cleaving polysaccharide monooxygenase is easy to prepare, and using biological enzymatic hydrolysis of cellulose is more in line with environmental protection, greenness, renewability, and biodegradability.

[0031] Specifically, under improved conditions:

[0032] (1) This invention uses a two-step method, adding thermophilic enzymes and synergistic polysaccharide monooxygenases to pretreat sugarcane bagasse at 70°C, followed by cellulase hydrolysis, which significantly improves the hydrolysis rate of sugarcane bagasse. The hydrolysis rate can reach up to 40% in 24 hours, which is 39% higher than the traditional one-step method of adding only cellulase to hydrolyze sugarcane bagasse; and 26% higher than the method of using thermophilic enzymes and synergistic polysaccharide monooxygenases to pretreat sugarcane bagasse at 50°C and then adding cellulase hydrolysis.

[0033] (2) In the first step of the two-step method, when pretreating sugarcane bagasse, adding mgLPMO, thermophilic endoglucanase and thermophilic xylanase can further improve the enzymatic hydrolysis efficiency of sugarcane bagasse. The hydrolysis rate is 31.6% higher than that of adding only thermophilic endoglucanase, and 13.74% higher than that of adding mgLPMO and thermophilic endoglucanase but not adding thermophilic xylanase.

[0034] (3) The high temperature of 70°C during the initial enzymatic hydrolysis can reduce the risk of microbial infection during subsequent cellulase saccharification. At the same time, the high temperature of 70°C can reduce the viscosity of the substrate, increase the solubility and diffusion rate of the substrate, and further improve the hydrolysis efficiency of sugarcane bagasse.

[0035] (4) The present invention improves the D17W mutant of cleavable polysaccharide monooxygenase, which has a better two-step enzymatic hydrolysis effect on cellulose substrate. Attached Figure Description

[0036] Figure 1 Determination of enzymatic hydrolysis rate of pretreated sugarcane bagasse;

[0037] Figure 2 : Detection of optimal temperature for mgLPMO10;

[0038] Figure 3 Optimization of the ratio of mgLPMO10 to thermophilic enzymes;

[0039] Figure 4 Optimization of the concentration of mg LPMO10 and thermophilic enzymes;

[0040] Figure 5 Two-step enzymatic hydrolysis of common lignocellulose;

[0041] Figure 6 Comparison of the effects of mgLPMO10 or its mutant D17W on the synergistic degradation of fibrous raw materials. Detailed Implementation

[0042] The technical solutions described below will be clearly and completely described with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0043] 1. Experimental materials

[0044] The *E. coli* strains (E. coli DH5α, E. coli BL21(DE3)) were all preserved in our laboratory; the plasmid pET-20b(Amp+) was also preserved in our laboratory; the thermophilic endoglucanase TnCelB and thermophilic xylanase Xyn10A used were expressed in the *E. coli* strain preserved in our laboratory in the previous stage, and the amino acid sequences of thermophilic endoglucanase TnCelB and thermophilic xylanase Xyn10A are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0045] The enzymes used in this invention have the following activity and molecular weight (mg / L): LPMO10: 25.5 U / g, molecular weight 34821.14; TnCelB: 571.3 U / g, molecular weight 31752.04; Xyn10A: 78.7 U / g, molecular weight 131243.65. The enzyme addition amounts in the embodiments of this invention are all expressed in μmol·L⁻¹. -1 Add the units.

[0046] 2. The purification method for recombinant cellulase protein is as follows:

[0047] Heterogeneously expressed *E. coli* cells were collected, washed with PBS buffer, and resuspended in PBS buffer at half the volume of the fermentation broth. The cells were then completely lysed using an ultrasonic cell disruptor. The cell disruption buffer was centrifuged at 4°C and 12,000 rpm, and the supernatant was collected as the crude protein enzyme solution. Ultrasonic disruption parameters: power 70%, on for 5 seconds, off for 5 seconds, total time 45 min.

[0048] The recombinant protein has a 6×His tag at its end, therefore, Ni+-NTA nickel column affinity chromatography was used for protein purification. The specific procedure is as follows:

[0049] (1) Flow out the 20% ethanol preservation solution in the nickel resin and equilibrate the column with Binding Buffer. Add the cell lysis supernatant to bind with the nickel resin and pour it into a centrifuge tube. Incubate the tube at 4°C with a molecular hybridization apparatus and shake vertically for 1 hour to allow mg LPMO10 protein to fully bind with the nickel resin.

[0050] (2) Pour the binding solution into the column. After the resin settles, open the stopper to let the liquid flow out through the column.

[0051] (3) Take 10 mL of binding buffer and wash away any unbound or poorly bound proteins.

[0052] (4) Add 10 mL of imidazole at concentrations of 50, 100, 200, and 400 mmol·L⁻¹ respectively. -1 Elution buffer was slowly added to the column to elute the recombinant protein from the nickel resin. The eluent was collected to obtain the purified target protein. The purified protein was stored at 4°C.

[0053] 3. The enzymatic hydrolysis rate determination method (DNS method) involved in this invention is as follows:

[0054] During the enzymatic hydrolysis process, 0.4 mL of the hydrolysate was taken and the supernatant was obtained by centrifugation at 8000–10000 rpm for 5–15 min. The supernatant was diluted 10–100 times and the reducing sugar content was determined using high-performance liquid chromatography (HPLC), and the enzymatic hydrolysis rate of reducing sugar was calculated accordingly.

[0055] 4. Preparation of phosphoric acid swollen cellulose (PASC), the specific steps are as follows:

[0056] (1) Add 0.2g MCC to a 50mL centrifuge tube and add 0.6mL ddH2O to wet it to form a suspension.

[0057] (2) Slowly add 10 mL of cold 86.2% phosphoric acid and stir thoroughly until the final concentration is 83.2%. Before adding the last 2 mL, mix the cellulose suspension thoroughly.

[0058] (3) Place on ice for 1 hour, stirring every 10 minutes.

[0059] (4) Centrifuge at 4℃ and 5000rpm for 20min, suspend the particles in cold water, add 10mL of cold water each time, for a total of 40mL, and stir thoroughly after each addition. Finally, a white turbid precipitate is produced, and centrifuge at 4℃ and 6500rpm for 25min.

[0060] (5) Wash the precipitate with 50 mL of cold water, repeating four times.

[0061] (6) Add 0.5 mL of 2 mol·L⁻¹ -1 The phosphoric acid was neutralized with Na2CO3 solution, resuspended in 45 mL of cold water, and centrifuged at 4 °C and 6500 rpm for 25 min.

[0062] (7) Add 50 mL of cold water to resuspend and centrifuge twice to make the pH about 5-7.

[0063] (8) Storage: Store in a refrigerator at 4℃ or -20℃.

[0064] 5. The preparation method of pretreated sugarcane bagasse, the specific steps are as follows:

[0065] (1) Grind the sugarcane bagasse into fine particles using a grinder, and then sieve it through a 60-mesh sieve and dry it in a 60℃ oven until constant weight.

[0066] (2) Place 10g of dried sugarcane bagasse in a three-necked flask, add 140g of glycerol and 0.3g of NaOH;

[0067] (3) Place the three-necked flask in a heating mantle and heat it to 240°C. At the same time, stir it with a stir paddle to make it mix and heat evenly, and react for 30 minutes.

[0068] (4) After the reaction is complete, allow it to cool down naturally, and then use 4500 mL of H2O to filter and wash it three times to remove the glycerol solution adhering to the sugarcane bagasse, and collect the sugarcane bagasse.

[0069] (5) Place the processed sugarcane bagasse in a 60℃ oven and dry it to constant weight. Collect it, dry it, and store it for later use.

[0070] 6. The method for inducing expression in Escherichia coli, the specific steps are as follows:

[0071] Recombinant Escherichia coli was inoculated at a rate of 1% (v / v) into an Erlenmeyer flask containing 200 mL of LB broth and fermented for 3 h. A final concentration of 0.5 mmol·L⁻¹ was then added. -1 IPTG was added, and then fermented at 37°C for 6 hours. After collecting the cells, they were broken up and purified to obtain the target protein. mgLPMO10, thermophilic endoglucanase TnCelB, and thermophilic xylanase Xyn10A were all prepared by this method.

[0072] Example 1: Two-step enzymatic hydrolysis of sugarcane bagasse, pretreatment at 70℃, enzymatic hydrolysis at 50℃

[0073] mg LPMO10, thermophilic endoglucanase TnCelB, and thermophilic xylanase Xyn10A were prepared. First, mg LPMO10, along with thermophilic endoglucanase TnCelB and thermophilic xylanase Xyn10A, was pretreated with sugarcane bagasse at 70℃. Then, the mixture was cooled to 50℃ and cellulase Celluclast was added for enzymatic hydrolysis. The specific experimental steps are as follows:

[0074] (1) Using pretreated sugarcane bagasse as a substrate, 15% w / v sugarcane bagasse was hydrolyzed for 3 h at 70 °C with mg LPMO10, thermophilic endoglucanase TnCelB, and thermophilic xylanase Xyn10A. The enzyme reaction was carried out at 50 mmol·L⁻¹. -1 The reaction was carried out in a phosphate buffer solution at pH 6, and the reaction solution contained 3 μmol·L⁻¹ -1 TnCelB, 3 μmol·L -1 mgLPMO10, 3μmol·L -1 Xyn10A, 1 mmol·L -1 AscA, 15% w / v sugarcane bagasse, was placed in a molecular hybridization apparatus and reacted by vertical rotation.

[0075] (2) Cool the reaction solution obtained in step (1) to 50°C, and add cellulase Celluclast (10 mg·g). -1 The substrate was reacted at 50°C for 72 h. The enzymatic hydrolysis rate was measured during the hydrolysis.

[0076] The results are as follows Figure 1 As shown in the figure, the high-temperature liquefaction in the figure is the two-step enzymatic hydrolysis of sugarcane bagasse in this embodiment. The first step is to pretreat the sugarcane bagasse at 70°C. As can be seen from the figure, the enzymatic hydrolysis rate reaches 35% after 24 hours.

[0077] Example 2: Effect of temperature on the activity of mgLPMO10

[0078] mg LPMO10 was prepared, and its relative activity was determined at different temperatures using the 2,6-DMP method. The specific steps are as follows:

[0079] (1) Preparation of 116 mmol·L -1 Phosphate buffer at pH 7.5; 10 mmol·L⁻¹ -1 2,6-DMP solution; 5 mmol·L -1 The H2O2 solution. All solutions should be used within 12 hours after preparation.

[0080] (2) Add 860 μL of phosphate buffer, 100 μL of 2,6-DMP solution and 20 μL of H2O2 solution to a centrifuge tube and mix thoroughly. Incubate at the appropriate temperature for 15 min.

[0081] (3) After adding 20 μL of mg LPMO10 protein sample and mixing well, measure the increase in absorbance within 5 min at a wavelength of 469 nm (ε469=53200 L·mol⁻¹). -1 ·cm -1 The activity of LPMO is calculated using [a specific method / method]. One unit of enzyme activity is defined as the conversion of 2 μmol of 2,6-DMP per minute under the reaction conditions.

[0082] The results are as follows Figure 2 It can be seen that the enzyme activity of mgLPMO10 increases with increasing temperature in the range of 40-70℃, reaches the highest enzyme activity at 70℃, and still has high enzyme activity in the range of 65-75℃.

[0083] Example 3: Effect of the ratio and concentration of mg LPMO to thermophilic enzyme on enzymatic hydrolysis efficiency

[0084] (1) Optimization of the ratio of mgLPMO to thermophilic enzyme

[0085] Based on Example 1, the addition ratios of mgLPMO, TnCelB, and Xyn10A were optimized. Specifically, the addition ratios of TnCelB, mgLPMO, and Xyn10A were 1:0:0, 1:1:0, 1:1:0.1, 1:1:1, and 1:1:2, respectively. The remaining steps were the same as in Example 1, and the concentration of reducing sugars in the enzymatic hydrolysate obtained after treatment at 70°C was measured.

[0086] The results are as follows Figure 3 As shown, the enzymatic hydrolysis efficiency was higher when the ratio of TnCelB, mgLPMO, and Xyn10A was 1:1:1 and 1:1:2 compared to other ratios. When the ratio of TnCelB, mgLPMO, and Xyn10A was 1:1:1, the reducing sugar concentration was significantly increased, by 31.6% compared to adding only TnCelB and by 13.74% compared to not adding Xyn10A. However, when the ratio of Xyn10A was greater than 1, the increase in reducing sugar concentration was less significant. This demonstrates that appropriate addition of xylanase has a positive effect on the hydrolysis pretreatment of sugarcane bagasse, and the effect of adding TnCelB, mgLPMO, and Xyn10A is better than adding only TnCelB or adding only TnCelB and mgLPMO. Therefore, a ratio of 1:1:1 for the addition of TnCelB, mgLPMO, and Xyn10A was selected for subsequent optimization.

[0087] (2) Optimization of mgLPMO and thermophilic enzyme dosage

[0088] Based on Example 1, while maintaining the ratio of the three thermophilic enzymes at 1:1:1, the addition concentrations of mg LPMO and thermophilic enzymes were optimized. Specifically, the addition concentrations of both mg LPMO and thermophilic enzymes were 0 μmol·L⁻¹. -1 3 μmol·L -1 6 μmol·L -1 15 μmol·L -1 30 μmol·L -1 60 μmol·L -1 The effect of enzyme addition on the two-step hydrolysis of 15% sugarcane bagasse was investigated. The concentration of reducing sugar in the enzymatic hydrolysate obtained after the two-step enzymatic hydrolysis was measured. The remaining steps were the same as in Example 1.

[0089] The results are as follows Figure 4 As shown, when the enzyme concentration is 15 μmol·L⁻¹ -1 The two-step hydrolysis method yielded the best hydrolysis efficiency for 15% sugarcane bagasse, with a specific addition amount of 3 μmol·L⁻¹ at 24, 48, and 36 h. -1 The enzymatic hydrolysis rates increased by 15%, 20%, and 24% respectively, and were 61%, 43%, and 42% higher than the blank control group (without TnCelB, mgLPMO, and Xyn10A). Therefore, when hydrolyzing sugarcane bagasse with a substrate concentration of 15%, a two-step method is used, first adding 15 μmol·L⁻¹. -1 mgLPMO10, 15μmol·L - 1 TnCelB, 15 μmol·L -1 The best enzymatic hydrolysis results were obtained by pretreating Xyn10A at 70℃ for 3 hours, cooling it to 50℃, and then adding cellulase Celluclast for saccharification. The hydrolysis rate reached 40% at 24 hours and 46% at 72 hours.

[0090] Example 4: Two-step enzymatic hydrolysis of other lignocellulose substrates

[0091] Based on Example 1, the enzymatic hydrolysis of other cellulose substrates by mgLPMO10 in conjunction with thermophilic enzymes was investigated. Specifically, the cellulose substrates were swollen cellulose sulfate (PASC), microcrystalline cellulose (MCC), sodium carboxymethyl cellulose (CMC-Na), and filter paper, with the remaining reaction steps consistent with Example 1.

[0092] The results are as follows Figure 5 As shown, mgLPMO10, in synergy with thermophilic enzymes, exhibited good enzymatic hydrolysis activity on different substrates. Among them, the addition of mgLPMO10 increased the enzymatic hydrolysis rate of PASC, MCC and bagasse by 19.3%, 22.4% and 16.8% respectively compared with the absence of mgLPMO10.

[0093] Example 5: Two-step enzymatic hydrolysis of cellulose substrate using mgLPMO10 and its mutants.

[0094] The pretreated sugarcane bagasse at a concentration of 15% w / v was hydrolyzed using mgLPMO10 wild-type and its mutant, synergistic with thermophilic endoglucanase TnCelB and thermophilic endoxylanase Xyn10A, and the hydrolysis rate was determined.

[0095] The amino acid sequence of wild-type mgLPMO10 is shown in SEQ ID NO.1.

[0096] The mgLPMO10 mutant, a single mutant named D17W, is obtained by mutating the 17th aspartic acid to tryptophan (abbreviated as D17W) on the cleaving polysaccharide monooxygenase shown in SEQ ID NO.1. The mutant can be constructed using conventional methods.

[0097] The specific steps of hydrolysis are as follows:

[0098] 15% w / v pretreated sugarcane bagasse was hydrolyzed with mg LPMO10 (wild WT or its mutant D17W), thermophilic endoglucanase TnCelB, and thermophilic xylanase Xyn10A at 70 °C for 3 h. The enzyme reaction was carried out at 50 mmol·L⁻¹. -1 The reaction was carried out in a phosphate buffer solution at pH 6, and the reaction solution contained 3 μmol·L⁻¹ -1 TnCelB, 3 μmol·L -1 mgLPMO10, 3μmol·L - 1 Xyn10A, 1 mmol·L -1 AscA, 15% w / v pretreated sugarcane bagasse, was placed in a molecular hybridization apparatus and rotated vertically for reaction; the reaction solution was cooled to 50°C, and cellulase Celluclast (10 mg·g) was added. -1 The substrate was reacted at 50°C for 72 hours.

[0099] The results are as follows Figure 6 As shown, the mutant D17W, in synergistic enzymatic hydrolysis with two high-temperature enzymes on pretreated sugarcane bagasse, achieved a higher hydrolysis rate than the wild type, increasing by 29%, 16%, and 12% at 6, 12, and 24 hours, respectively.

[0100] Comparative Example 1: One-step enzymatic hydrolysis of sugarcane bagasse using mg LPMO10 in conjunction with thermophilic endoglucanase TnCelB, thermophilic xylanase Xyn10A, and cellulase Celluclast.

[0101] First, sugarcane bagasse was pretreated with mg LPMO10 in conjunction with thermophilic endoglucanase TnCelB, thermophilic xylanase Xyn10A, and cellulase Celluclast at 50℃. The specific experimental steps are as follows:

[0102] (1) Prepare mg LPMO10 protein, thermophilic endoglucanase TnCelB and thermophilic xylanase Xyn10A. The method and steps are the same as steps (1) in Example 1.

[0103] (2) Using 15% pretreated sugarcane bagasse as a substrate, mg LPMO10, thermophilic endoglucanase TnCelB, and thermophilic xylanase Xyn10A were added to the 15% sugarcane bagasse for hydrolysis at 50℃ for 72 h. The enzyme reaction was carried out at 50 mmol·L⁻¹. -1 The reaction was carried out in a phosphate buffer solution at pH 6, and the reaction solution contained 3 μmol·L⁻¹ -1 TnCelB, 3 μmol·L -1 mgLPMO10, 3μmol·L - 1 Xyn10A, 1 mmol·L -1 AscA, cellulase Cellulase (10 mg·g) -1 The substrate was 15% sugarcane bagasse, and its enzymatic hydrolysis rate was measured during hydrolysis.

[0104] The results are as follows Figure 1 As shown in the figure, liquefaction at 50℃ is the control example 1. Sugarcane bagasse was treated with thermophilic endoglucanase TnCelB and thermophilic xylanase Xyn10A at 50℃, and cellulase Celluclast was added for enzymatic hydrolysis. As can be seen from the figure, the hydrolysis rate reached 26% after 24 hours.

[0105] Comparative Example 2: One-step enzymatic hydrolysis of sugarcane bagasse using only cellulase Celluclast

[0106] The sugarcane bagasse was enzymatically hydrolyzed using cellulase Celluclast at 50°C. The specific steps are as follows:

[0107] Using 15% pretreated sugarcane bagasse as a substrate, cellulase Celluclast (10 mg·g) -1 The substrate was reacted with 15% sugarcane bagasse at 50°C for 72 h. The enzyme reaction was carried out at 50 mmol·L⁻¹. -1 The reaction was carried out in a phosphate buffer solution at pH 6, and the reaction solution included cellulase Celluclast (10 mg / g). -1 substrate), 1 mmol·L -1 AscA, 15% sugarcane bagasse. Its enzymatic hydrolysis rate was measured during hydrolysis, and the results... Figure 1As shown in the figure, the blank area represents Comparative Example 2, where only cellulase Celluclast was used to enzymatically hydrolyze sugarcane bagasse. As can be seen from the figure, the hydrolysis rate reached 23%.

[0108] Combination Figure 1 It was found that the two-step method of pretreating sugarcane bagasse at 70℃ with mgLPMO10, TnCelB, and Xyn10A before adding cellulase Celluclast for enzymatic hydrolysis (high-temperature liquefaction group) was more effective than the one-step method of adding mgLPMO10, TnCelB, Xyn10A, and cellulase Celluclast at 50℃ (50℃ liquefaction group), and also more effective than the one-step method of using only cellulase for enzymatic hydrolysis (blank group). At 12 hours, the hydrolysis rate in the high-temperature liquefaction group was 49% and 93% higher than that in the 50℃ liquefaction group and the blank control group, respectively; at 24 hours, the hydrolysis rate in the high-temperature liquefaction group was 26% and 39% higher than that in the 50℃ liquefaction group and the blank control group, respectively. In conclusion, pretreating sugarcane bagasse at 70℃ with thermophilic enzymes in combination with mgLPMO10, followed by cooling to 50℃ and then adding cellulase, can significantly improve the enzymatic hydrolysis efficiency of sugarcane bagasse.

[0109] The amino acid sequence of mgLPMO10 is SEQ ID NO.1:

[0110] HGAAMVPGSRTYLCWRDGLSPTGQIIPNNPACAAAVAVSGANSLYNWFSVLRSDAGGRTVGYIPDGQLCSGGNPGFLGYDLARDDWPLTHLTAGATIEFRYSNWAHHPGTFYFYVTRDSWSPTRPLAWSDLESEPFLTVTNPPQRGAVGTNDGHYYFTGRLPNKSG RHIIYSRWVRSDSQENFFGCSDVVFDGGNGEVTGIGSGSGPTTPPTTPPTTPPTTPPTTPPTTPGGSTGCAATYQVVGSWTGGFQAEVTVRNTGTAPLNGWTIQWTFANGETVGSLWNGQHSQSGSTVTVRNVDHNGSLAPGASTSFGFVGSGSTGATPTPTCTSA

[0111] The amino acid sequence of TnCelB is SEQ ID NO.2:

[0112] MRLVVSFLLVVSAFLFSAEVVLTDIGATDITFKGFPVTMELNFWNVKSYEGETWLKFDGEKVQFYADIYNIVLQNPDSWVHGYPEIYYGYKPWAAHNSGTEILPVKVKDLPDFYVTLDYSIWYENDLPINLAMETWITRKPDQTSVSSGDVEIMVWFYNNILMPGGQKVDEFTTTIEINGSPVETKWDVYFAPWGWDYLAFRLTTPMKDGRVKFNVKDFVEKAAEVIKKHSTRVENFDEMYFCVWEIGTEFGDPNTTAAKFGWTFKDFSVEIGE

[0113] The amino acid sequence of Xyn10A SEQ ID NO. 3:

[0114]

[0115] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for improving sugar production from the hydrolysis of cellulose raw materials, characterized in that, The method includes the following steps: (1) The first step hydrolysis product was obtained by reacting the fibrous raw material with a cleaving polysaccharide monooxygenase in conjunction with a thermophilic enzyme at 60℃~100℃; The amino acid composition of the cleaving polysaccharide monooxygenase is based on SEQ ID NO.1, with aspartic acid at position 17 mutated to tryptophan; The fibrous raw material is pretreated bagasse, and the preparation method is as follows: the bagasse is ground into fine particles, dried through a sieve; glycerol and sodium hydroxide are added and heated to react; after the reaction is completed, the mixture is cooled, washed with water multiple times, and filtered to remove the glycerol solution; the bagasse is collected and dried to obtain pretreated bagasse. The thermophilic enzyme is any one or a combination of thermophilic endoglucanase and thermophilic xylanase, the amino acid sequence of which is shown in SEQ ID NO.2 and the amino acid sequence of which is shown in SEQ ID NO.

3. (2) Cool the hydrolysate obtained in step (1) and add cellulase to hydrolyze and produce sugar.

2. The method according to claim 1, characterized in that, Step (1) The concentration of the fibrous raw material is 0.5%~40% w / v.

3. The method according to claim 1, characterized in that, In step (1), the amount of both the polysaccharide-cleaving monooxygenase and the thermophilic enzyme added is 3~60 μmol·L. -1 Or 0.01~10 U / g dry substrate.

4. The method according to claim 1, characterized in that, Add 0.5~1.5 mmol·L in step (1) -1 ascorbic acid.

5. The method according to claim 1, characterized in that, Step (1) is to process for 0.5~10 h.

6. The method according to claim 1, characterized in that, The hydrolysis temperature in step (2) is 45~55℃.

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