A ucst responsive quaternary ammonium salt polymer proteolysis promoter and its preparation and method for recovering cellulase at room temperature
By using UCST-responsive quaternary ammonium salt polymer enzymatic hydrolysis promoter to form a co-precipitate with cellulase at room temperature, the problems of low enzymatic hydrolysis efficiency and high enzyme cost in cellulosic ethanol production were solved, achieving efficient recovery of cellulase and improvement of enzymatic hydrolysis efficiency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHEJIANG SCI-TECH UNIV
- Filing Date
- 2023-10-31
- Publication Date
- 2026-05-29
AI Technical Summary
In the existing technology, the enzymatic hydrolysis process in the production of cellulosic ethanol has low efficiency and high enzyme cost. The enzymatic hydrolysis process requires a large amount of acid and alkali, making it difficult to achieve efficient recovery and recycling of cellulase.
The UCST-responsive quaternary ammonium salt polymer enzymatic hydrolysis promoter is used. This promoter is prepared by free radical polymerization of amphoteric betaine monomer and quaternary ammonium salt monomer in aqueous solution. It has sensitive temperature response performance and forms a co-precipitate with cellulase at room temperature, thereby realizing the recovery of cellulase.
It significantly improves the enzymatic hydrolysis efficiency of lignocellulose, reduces the cost of enzymatic hydrolysis, and efficiently recovers cellulase at room temperature. The operation is simple and environmentally friendly, avoiding the use of acids and alkalis.
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Figure CN117467067B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a cellulose solution system and a cellulase recovery technology in a lignocellulose enzymatic hydrolysis system, specifically a UCST-responsive quaternary ammonium salt polymer enzymatic hydrolysis promoter and its preparation and a method for recovering cellulase at room temperature. Background Technology
[0002] With the increasing prominence of environmental pollution and energy crises, biomass, as a renewable resource that can replace fossil fuels, is gradually attracting attention due to its renewable advantages. Lignocellulose is the most abundant biomass renewable resource in nature. Industrially, cellulosic ethanol can be produced through lignocellulose biorefining to replace gasoline consumption. However, the production process of cellulosic ethanol faces many technical bottlenecks, among which the high cost of enzymes and low efficiency in the enzymatic hydrolysis process hinder its industrialization.
[0003] Strengthening the enzymatic hydrolysis of lignocellulose and recovering free enzymes in the enzymatic hydrolysis system is an effective way to reduce the high cost of enzymes. (1) In the process of cellulosic ethanol production, the enzymatic hydrolysis can be strengthened and cellulase can be recovered and recycled by adding pH-responsive lignin amphoteric surfactants. For example, adding 3.0 g / L of pH-responsive lignin amphoteric surfactant to the enzymatic hydrolysis system of microcrystalline cellulose and corn cob residue, and adjusting the pH of the hydrolysate to 3.2-4.0 after enzymatic hydrolysis, the lignin sulfonate quaternary ammonium salt and cellulase can form a coprecipitate, which can save 50% of the amount of cellulase used. The enzyme recovery process is simple to operate, but it requires a large amount of acid and alkali (see CN107217047B, 2020-05-22; CN115466406A, 2022-12-13). (2) Sulfobetaine homopolymer (PSPE) has a UCST response. It dissolves at higher temperatures and can form a coprecipitate with cellulase at lower temperatures. Adding 0.08 g / L of PSPE-3 to the corn cob residue enzymatic hydrolysis system can save 50% of cellulase at room temperature. The enzyme recovery process is simple to operate, green and environmentally friendly, and does not require the consumption of acid and alkali, but it has no enhancing effect on the enzymatic hydrolysis of lignocellulose (see The synthesis of a UCST-type zwitterionic polymer for the efficient recycling of cellulase at room temperature, Green Chemistry, 2021.23: 2738-2746). (3) Using sulfobetaine monomer (SPE) and isopentenyl alcohol polyoxyethylene ether (TPEG) as monomers, a UCST-responsive copolymer PPSP was synthesized by aqueous free radical polymerization. 1.5 g / L of PPSP was added to the corn cob residue (CCR) system. 10-1 (the molar ratio of SPE to TPEG is 9:1), which can improve the enzymatic hydrolysis efficiency by 1.2 times, but its temperature response performance is not sensitive and it cannot be used to recover cellulase (see Synthesis of Recyclable UCST-Type Copolymer PPSP and Its Application in Enhancing Lignocellulosic Enzymatic Hydrolysis and Recycling Cellulase, ACS Sustainable Chemistry & Engineering, 2023, 11:1355-1362). Summary of the Invention
[0004] The technical problem to be solved by the present invention is to provide a USCT-responsive quaternary ammonium salt polymer enzymatic hydrolysis promoter, its preparation method and a method for recovering cellulase at room temperature. The USCT-responsive quaternary ammonium salt polymer enzymatic hydrolysis promoter has sensitive temperature response performance, promotes the enzymatic hydrolysis of lignocellulose, and can recover cellulase at room temperature without consuming acid and alkali.
[0005] To address the above technical problems, this invention first discloses a UCST-responsive quaternary ammonium salt polymer enzymatic hydrolysis promoter, which is a polymer of amphoteric betaine monomer and quaternary ammonium salt monomer.
[0006] The amphoteric betaine monomer is a sulfonate betaine monomer represented by formula (I), a phosphate betaine monomer represented by formula (II), or a carboxylic acid betaine monomer represented by formula (III).
[0007]
[0008] In equations (Ⅰ), (Ⅰ), and (Ⅲ), m = 1 or 2; R is (CH2). n , that (CH2) n In the case where n = 2 or 3,
[0009] The quaternary ammonium salt monomer is the quaternary ammonium salt monomer shown in formula (Ⅳ).
[0010]
[0011] In equation (Ⅳ), m = 1 or 2; X is a halogen.
[0012] This invention also discloses a method for preparing a UCST-responsive quaternary ammonium salt polymer enzymatic hydrolysis promoter as described above. Amphoteric betaine monomer and quaternary ammonium salt monomer are subjected to free radical polymerization in aqueous solution at 40–70°C for 6–24 h using a persulfate compound as an initiator. Methanol is added at a volume ratio of methanol to total monomer mass of 7–21 mL / g to precipitate the target product, which is then separated by centrifugation and freeze-dried.
[0013] Furthermore, the persulfate compound is either potassium persulfate or ammonium persulfate.
[0014] Furthermore, the molar ratio of the amphoteric betaine monomer to the quaternary ammonium salt monomer is 9:1 to 1:9.
[0015] Furthermore, the mass ratio of the persulfate compound to the amphoteric betaine monomer and the quaternary ammonium salt monomer is 0.01:10 to 0.1:10.
[0016] This invention also discloses a method for recovering cellulase using the aforementioned UCST-responsive quaternary ammonium salt polymeric hydrolysis promoter at room temperature. The UCST-responsive quaternary ammonium salt hydrolysis promoter is added to a cellulase solution system or a lignocellulose hydrolysis system, dissolved at 40–60°C, and then cooled to room temperature, causing both the UCST-responsive quaternary ammonium salt hydrolysis promoter and cellulase to precipitate simultaneously. The cellulase is then recovered using a solid-liquid separation method. The cellulase is derived from *Trichoderma reesei* and *Aspergillus niger* fungi. The solid-liquid separation method is one or a combination of two or more of the following: natural sedimentation, decantation, filtration, and centrifugation.
[0017] Furthermore, the UCST-responsive quaternary ammonium salt enzymatic hydrolysis promoter is added to the lignocellulose enzymatic hydrolysis system. After dissolving at 40–60°C, the system is first enzymatically hydrolyzed for 24–72 hours. Then, the enzymatic hydrolysis residue (i.e., enzymatically hydrolyzed lignin) is removed by solid-liquid separation. Finally, the system is cooled to room temperature by natural cooling or by refrigeration equipment.
[0018] Furthermore, the room temperature is 15–30°C.
[0019] Furthermore, the pH of the cellulase solution system or the lignocellulose enzymatic hydrolysis system is 4-6, the ionic strength is 10-200 mmol / L, and the cellulase protein concentration is 40-2500 mg / L.
[0020] Furthermore, the pH of the cellulase solution system or the lignocellulose enzymatic hydrolysis system is 5, and the ionic strength is 50 mmol / L.
[0021] Furthermore, the mass ratio of UCST-responsive quaternary ammonium salt hydrolysis promoter to cellulase is 0.1–100:1.
[0022] Furthermore, the mass ratio of UCST-responsive quaternary ammonium salt hydrolysis promoter to cellulase is 1–50:1.
[0023] The quaternary ammonium salt polymer enzymatic hydrolysis promoter of the present invention, with a high upper critical solution temperature (UCST) response, is prepared by aqueous solution free radical polymerization. The synthesis method is simple, exhibits sensitive temperature response, and can significantly improve the enzymatic hydrolysis efficiency of lignocellulose. The temperature response mechanism for cellulase recovery by this hydrolysis promoter is as follows: Under enzymatic hydrolysis conditions (temperature above UCST), the zwitterionic ions in the amphoteric betaine structural units of the polymer are ionized, increasing the hydrophilicity of the polymer molecules and causing polymer dissolution; when the temperature is cooled to room temperature (temperature below UCST), the ions in the amphoteric betaine structural units associate to form an internal salt, reducing the hydrophilicity of the polymer molecules, and the polymer and cellulase co-precipitate through hydrophobic co-precipitation. The operation is simple and the process is environmentally friendly.
[0024] With PSPE and PPSP 10 Compared to -1, UCST responsive quaternary ammonium salt polymer enzymatic hydrolysis promoter can efficiently enhance enzymatic hydrolysis and efficiently recover cellulase in the enzymatic hydrolysis system at room temperature, which is of great significance for reducing the production cost of biorefining and cellulosic ethanol. Attached Figure Description
[0025] Figure 1 The graph shows the UCST response of 5 g / L PSDM1 in the cellulase solution system in Example 1. Detailed Implementation
[0026] The present invention will be further explained below with reference to the embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.
[0027] All reagents used in the following examples were commercially available. The cellulase used was the widely used Cellic CTec2, and the glucose concentration in the hydrolysate was determined using a biosensor analyzer (SBA-40E, Shandong Academy of Biological Sciences).
[0028] Synthesis of UCST-responsive quaternary ammonium salt polymer enzymatic hydrolysis promoter
[0029] PSDM was synthesized at 60°C via aqueous free radical polymerization using methacryloylethyl sulfobetaine (SPE) and methacryloyloxyethyl trimethylammonium chloride (DMC) as monomers and ammonium persulfate (APS) as an initiator. X, where x is the sample number (the molar ratio of SPE to DMC in PSDM1 and PSDM2 is 9:1, but the molecular weights are different; the molecular weights of PSDM2 and PSDM3 are basically the same, but the molar ratios of SPE to DMC are different).
[0030] The specific synthesis steps of PSDM1 are as follows: At room temperature, 9 parts by weight of SPE, 1 part by weight of DMC (i.e., a molar ratio of 9:1) and 0.01 parts by weight of APS (based on a total mass of 10 parts of monomer SPE and DMC) were rapidly added to 7 times the volume of distilled water (multiples of the total mass of monomer SPE and DMC, expressed in mL / g). The system was then heated to 60°C and stirred for 20 hours. After the reaction was completed, 14 times the volume of methanol (multiples of the total mass of monomer SPE and DMC, expressed in mL / g) was added to the reaction solution to precipitate the target product. After centrifugation, the precipitate was freeze-dried.
[0031] The specific synthesis steps of PSDM2 are as follows: At room temperature, 9 parts by weight of SPE, 1 part by weight of DMC (i.e., a molar ratio of 9:1) and 0.02 parts by weight of APS (based on a total mass of 10 parts of SPE and DMC monomers) were rapidly added to 7 times the volume of distilled water (multiples of the total mass of SPE and DMC monomers, expressed in mL / g). The system was then heated to 60°C and stirred for 20 hours. After the reaction was completed, 14 times the volume of methanol (multiples of the total mass of SPE and DMC monomers, expressed in mL / g) was added to the reaction solution to precipitate the target product. After centrifugation, the precipitate was freeze-dried.
[0032] The specific synthesis steps of PSDM3 are as follows: At room temperature, 8 parts by weight of SPE, 2 parts by weight of DMC (i.e., a molar ratio of 8:2) and 0.02 parts by weight of APS (based on a total monomer mass of 10 parts) were rapidly added to 7 times the volume of distilled water (multiples of the total mass of SPE and DMC monomers, expressed in mL / g). The system was then heated to 60°C and stirred for 20 hours. After the reaction was completed, 14 times the volume of methanol (multiples of the total mass of SPE and DMC monomers, expressed in mL / g) was added to the reaction solution to precipitate the target product. After centrifugation, the precipitate was freeze-dried.
[0033] Example 1: Recovery of cellulase from cellulase solution system
[0034] PSDM1 (molecular weight M) was added at 1.0 g / L at 50°C. w=31.6 kDa) was added to a 0.2 g / L cellulase solution (pH = 5.0, ionic strength 50 mmol / L, prepared with acetate-sodium acetate buffer). The system was cooled to room temperature (25 °C) to precipitate PSDM1 and cellulase. The solid and liquid were separated by centrifugation, and a certain amount of supernatant was filtered through an aqueous filter to remove impurities. The enzyme solution was scanned at 262 nm, and its UV absorbance at 262 nm was recorded. The protein recovery rate was calculated based on the absorbance of cellulase, and then the cellulase recovery rate in the system was calculated. The solid phase consisted of the recovered PSDM1 and cellulase. The results are shown in Table 1. The UCST response of 5 g / L PSDM1 in the cellulase solution system is shown in Table 1. Figure 1 As shown, PSDM1 completely dissolved at 50°C. Upon cooling to room temperature, the system became turbid due to the formation of internal salts from the association of ions within the amphoteric betaine structural units of the polymer molecule, leading to decreased molecular hydrophilicity, polymer aggregation, and the formation of a turbid system. The system's permeability decreased from 98.9% to 0.62%. These results demonstrate that PSDM1 exhibits a sensitive UCST response.
[0035] Example 2: Recovery of cellulase from cellulase solution system
[0036] At 50℃, PSDM2 (molecular weight M) was added at a concentration of 1.0 g / L. w PSDM2 (24.3 kDa) was added to a 0.2 g / L cellulase solution (pH 5.0, ionic strength 50 mmol / L, prepared with acetate-sodium acetate buffer). The system was then cooled to room temperature (25 °C) to precipitate PSDM2 and cellulase. The solid and liquid phases were separated by centrifugation, and a certain amount of the supernatant was filtered through an aqueous filter to remove impurities. The enzyme solution was scanned at 262 nm, and its UV absorbance was recorded. The protein recovery rate was calculated based on the absorbance of cellulase, thus representing the cellulase recovery rate in the system. The solid phase consisted of the recovered PSDM2 and cellulase. The results are shown in Table 1.
[0037] Example 3: Recovery of cellulase from cellulase solution system
[0038] At 50℃, PSDM3 (molecular weight M) was added at a concentration of 2.0 g / L. wPSDM3 (26.2 kDa) was added to a 0.2 g / L cellulase solution (pH 5.0, ionic strength 50 mmol / L, prepared with acetate-sodium acetate buffer). The system was then cooled to room temperature (25°C) to precipitate PSDM3 and cellulase. The solid and liquid phases were separated by centrifugation, and a certain amount of the supernatant was filtered through an aqueous filter to remove impurities. The enzyme solution was scanned at 262 nm, and its UV absorbance was recorded. The protein recovery rate was calculated based on the absorbance of the cellulase, thus representing the cellulase recovery rate of the system. The solid phase consisted of the recovered PSDM3 and cellulase. The results are shown in Table 1.
[0039] Comparative Example 1: Recovery of cellulase from cellulase solution system
[0040] PPSP was added at 1.2 g / L at 50℃. 10 -1 was added to a 0.2 g / L cellulase solution (pH 5.0, ionic strength 50 mmol / L, prepared with acetate-sodium acetate buffer), and the system temperature was lowered to room temperature (25°C) to allow PPSP to... 10- 1. Cellulase precipitates out, and the solid and liquid phases are separated by centrifugation. A certain amount of the supernatant is then filtered through an aqueous filter to remove impurities. The enzyme solution is scanned at 262 nm, and its UV absorbance at 262 nm is recorded. The protein recovery rate is calculated based on the absorbance of the cellulase, thus representing the cellulase recovery rate in the system. The solid phase is the recovered PPSP. 10 -1 and cellulase. The results are shown in Table 1.
[0041] Table 1. Cellulase recovery performance of UCST-responsive quaternary ammonium salt polymer hydrolysis promoter in cellulase solution system
[0042]
[0043]
[0044] As shown in Table 1, the UCST-responsive quaternary ammonium salt polymeric enzymatic hydrolysis promoter can efficiently recover cellulase from the cellulase solution system, and the enzyme recovery effect is significantly better than that of polymeric PPSP. 10 -1.
[0045] Example 4: Recovery of cellulase from a lignocellulose enzymatic hydrolysis system
[0046] PSDM1 (molecular weight M) was added at 1.0 g / L at 50°C. wPSDM1 (31.6 kDa) was added to a corn cob residue enzymatic hydrolysis system with a cellulase concentration of 0.2 g / L (pH = 5.0, ionic strength 50 mmol / L, prepared with acetate-sodium acetate buffer, temperature 50℃). After 48 h of enzymatic hydrolysis, the enzymatic residue was removed, and the system temperature was lowered to room temperature (25℃) to precipitate PSDM1 and cellulase. The solid and liquid were separated by centrifugation, and the glucose concentration of the supernatant was determined using an SBA-40E centrifuge. The enzymatic hydrolysis efficiency of the substrate after 48 hours was expressed as glucose yield. The same operation was performed three times to obtain three portions of PSDM1 and cellulase obtained after the first round of enzymatic hydrolysis and centrifugation.
[0047] The three PSDM1 samples and cellulase were further added to Cellic CTec2 at initial enzyme loads (i.e., the total amount of cellulase during the first round of enzymatic hydrolysis) of 30%, 40%, and 50%, respectively, for a second round of enzymatic hydrolysis at 50°C (other conditions were the same as the first round). After 48 hours of enzymatic hydrolysis, the enzymatic hydrolysis efficiency SED@48h was measured to calculate the cellulase recovery rate in the lignocellulose enzymatic hydrolysis system. The results are shown in Table 2.
[0048] Example 5: Recovery of cellulase from a lignocellulose enzymatic hydrolysis system
[0049] At 50℃, PSDM2 (molecular weight M) was added at a concentration of 1.0 g / L. w PSDM2 (24.3 kDa) was added to a corn cob residue enzymatic hydrolysis system with a cellulase concentration of 0.2 g / L (pH = 5.0, ionic strength 50 mmol / L, prepared with acetate-sodium acetate buffer, temperature 50℃). After 48 h of enzymatic hydrolysis, the enzymatic residue was removed, and the system temperature was lowered to room temperature (25℃) to precipitate PSDM2 and cellulase. The solid and liquid components were separated by centrifugation, and the glucose concentration of the supernatant was determined using an SBA-40E centrifuge. The enzymatic hydrolysis efficiency after 48 hours was expressed as glucose yield. The same operation was performed three times to obtain three portions of PSDM2 and cellulase obtained after the first round of enzymatic hydrolysis and centrifugation.
[0050] The three PSDM2 and cellulase samples were further added to 30%, 40%, and 50% of the initial enzyme load (i.e., the total amount of cellulase in the first round of enzymatic hydrolysis) of CTec2 for a second round of enzymatic hydrolysis (other conditions were the same as in the first round). After 48 hours of enzymatic hydrolysis, SED@48h was measured to calculate the enzyme recovery rate in the lignocellulose enzymatic hydrolysis system. The results are shown in Table 2.
[0051] Example 6: Recovery of cellulase from a lignocellulose enzymatic hydrolysis system
[0052] At 50℃, PSDM3 (molecular weight M) was added at a concentration of 2.0 g / L. wPSDM3 (26.2 kDa) was added to a corn cob residue enzymatic hydrolysis system with a cellulase concentration of 0.2 g / L (pH 5.0, ionic strength 50 mmol / L, prepared with acetate-sodium acetate buffer, temperature 50℃). After 48 h of enzymatic hydrolysis, the enzymatic residue was removed, and the system temperature was lowered to room temperature (25℃) to precipitate PSDM3 and cellulase. The solid and liquid were separated by centrifugation, and the supernatant solution (pH 6.0, prepared with 50 mmol / L acetate-sodium acetate buffer) was taken. The glucose concentration of the supernatant was determined using an SBA-40E analyzer. The enzymatic hydrolysis efficiency after 48 hours of substrate hydrolysis was expressed as glucose yield. The same operation was performed three times to obtain three portions of PSDM3 and cellulase obtained after the first round of enzymatic hydrolysis and centrifugation.
[0053] The three PSDM3 and cellulase samples were further added to 30%, 40%, and 50% of the initial enzyme load (i.e., the total amount of cellulase during the first round of enzymatic hydrolysis) of CTec2, respectively, for a second round of enzymatic hydrolysis (other conditions were the same as the first round). After 48 hours of enzymatic hydrolysis, SED@48h was measured to calculate the enzyme recovery rate in the lignocellulose enzymatic hydrolysis system. The results are shown in Table 2.
[0054] Comparative Example 2: Recovery of cellulase from the lignocellulose enzymatic hydrolysis system
[0055] The enzymatic hydrolysis conditions for corn cob residue were (pH = 5.0, ionic strength 50 mmol / L, prepared with acetate-sodium acetate buffer, temperature 50℃), with a cellulase concentration of 0.2 g / L. After 48 h of enzymatic hydrolysis, the solid and liquid phases were separated by centrifugation, and the glucose concentration in the supernatant was determined using an SBA-40E analyzer. The enzymatic hydrolysis efficiency after 48 hours was expressed as glucose yield. The hydrolysis results are shown in Table 2.
[0056] Comparative Example 3: Recovery of cellulase from the lignocellulose enzymatic hydrolysis system
[0057] PPSP at 50℃ 10 -1 was added to a corn cob residue enzymatic hydrolysis system with a cellulase concentration of 0.2 g / L (pH = 5.0, ionic strength 50 mmol / L, prepared with acetate-sodium acetate buffer, 50℃). After enzymatic hydrolysis for 48 h, the enzymatic residue was removed, and the system temperature was lowered to room temperature (25℃) to allow PPSP to... 10 -1 and cellulase precipitate out, and the solid and liquid phases are separated by centrifugation. The glucose concentration in the supernatant is determined using an SBA-40E analyzer. The enzymatic hydrolysis efficiency of the substrate after 48 hours is expressed as glucose yield. The same operation is performed three times to obtain three PPSP fractions after the first round of enzymatic hydrolysis and centrifugation. 10 -1 and cellulase.
[0058] The three PSDM3 and cellulase samples were further added to 30%, 40%, and 50% of the initial enzyme load (i.e., the total amount of cellulase during the first round of enzymatic hydrolysis) of CTec2, respectively, for a second round of enzymatic hydrolysis (other conditions were the same as the first round). After 48 hours of enzymatic hydrolysis, SED@48h was measured to calculate the enzyme recovery rate in the lignocellulose enzymatic hydrolysis system. The results are shown in Table 2.
[0059] Table 2 Enzyme recovery performance of UCST-responsive quaternary ammonium salt polymer hydrolysis promoters in lignocellulose hydrolysis systems
[0060]
[0061] In Table 2, the enzymatic hydrolysis efficiency is obtained after the first round of enzymatic hydrolysis, while the enzyme-saving efficiency is obtained after the second round of enzymatic hydrolysis. As shown in Table 2, the UCST-responsive quaternary ammonium salt polymer can efficiently recover cellulase from the lignocellulose enzymatic hydrolysis system without causing enzyme inactivation. The enzyme recovery process does not require large amounts of acid and alkali, and the recovery effect is significantly better than that of polymer PPSP. 10 -1. Meanwhile, the UCST response to quaternary ammonium salt polymer enzymatic hydrolysis efficiency was all above 60%, compared to PPSP. 10 -1, this UCST-responsive quaternary ammonium salt polymer can also enhance enzymatic hydrolysis efficiency, and the enhancing effect is significantly better than that of polymer PPSP. 10 -1.
[0062] The above examples are merely specific embodiments of the present invention. The present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.
Claims
1. A method for recovering cellulase at room temperature using a UCST-responsive quaternary ammonium salt polymer enzymatic hydrolysis promoter, characterized in that: The UCST-responsive quaternary ammonium salt polymer enzymatic hydrolysis promoter was added to the cellulase solution system or the lignocellulose enzymatic hydrolysis system and dissolved at 40-60℃. The system was then cooled to room temperature, causing the UCST-responsive quaternary ammonium salt polymer enzymatic hydrolysis promoter and cellulase to precipitate simultaneously. The cellulase was then recovered by solid-liquid separation. This enzymatic hydrolysis promoter is composed of amphoteric betaine monomers and quaternary ammonium salt monomers, obtained by aqueous free radical polymerization at 40–70 °C for 6–24 h using a persulfate compound as an initiator. The amphoteric betaine monomer is a sulfonate betaine monomer represented by formula (I), a phosphate betaine monomer represented by formula (II), or a carboxylic acid betaine monomer represented by formula (III). （Ⅰ） （Ⅱ） （Ⅲ） In equations (I), (II), and (III), m = 1 or 2; R is (CH2). n , that (CH2) n In the case where n=2 or 3, The quaternary ammonium salt monomer is the quaternary ammonium salt monomer shown in formula (Ⅳ). （Ⅳ） In equation (Ⅳ), m = 1 or 2; X is a halogen.
2. The method according to claim 1, characterized in that: The persulfate compound is either potassium persulfate or ammonium persulfate.
3. The method according to claim 1, characterized in that: The molar ratio of the amphoteric betaine monomer to the quaternary ammonium salt monomer is 9:1 to 1:
9.
4. The method according to claim 1, characterized in that: The mass ratio of the persulfate compound to the amphoteric betaine monomer and the quaternary ammonium salt monomer is 0.01:10 to 0.1:
10.
5. The method according to claim 1, characterized in that: The UCST-responsive quaternary ammonium salt polymer enzymatic hydrolysis promoter was added to the lignocellulose enzymatic hydrolysis system and dissolved at 40–60 °C. After enzymatic hydrolysis for 24–72 h, the enzymatic hydrolysis residue was removed by solid-liquid separation. The system was then cooled to room temperature to allow the UCST-responsive quaternary ammonium salt polymer enzymatic hydrolysis promoter and cellulase to precipitate simultaneously. The cellulase was then recovered by solid-liquid separation.
6. The method according to claim 5, characterized in that: The room temperature is 15–30°C.
7. The method according to claim 1, characterized in that: The pH of the cellulase solution system or the lignocellulose enzymatic hydrolysis system is 4–6; the ionic strength is 10–200 mmol / L; and the cellulase protein concentration is 40–2500 mg / L.
8. The method according to claim 7, characterized in that: The pH of the cellulase solution system or the lignocellulose enzymatic hydrolysis system is 5, and the ionic strength is 50 mmol / L.
9. The method according to claim 1, characterized in that: The mass ratio of UCST-responsive quaternary ammonium salt polymer enzymatic hydrolysis promoter to cellulase is 0.1–100:
1.
10. The method according to claim 9, characterized in that: The mass ratio of UCST-responsive quaternary ammonium salt polymer enzymatic hydrolysis promoter to cellulase is 1–50:1.