A claw-shaped UCST-responsive polymer and its preparation and method for recovering cellulase at room temperature

Abstract

The present invention discloses a claw-shaped UCST responsive polymer, which is a claw-shaped polymer obtained by prepolymerizing an amphoteric betaine monomer or an amphoteric betaine monomer and an N-vinyl pyrrolidone monomer and then crosslinking with a crosslinking agent triallylamine. The amphoteric betaine monomer is a sulfobetaine monomer represented by formula (I) or a phosphobetaine monomer represented by formula (II). In formulas (I) and (II), m=1 or 2; R is (CH2) n , the (CH2) n wherein n=2 or 3. The present invention also discloses a method for preparing a claw-shaped UCST-responsive polymer and a method for recovering cellulase at room temperature. The UCST-responsive polymer of the present invention has increased molecular weight, improved structure, significantly increased hydrophobicity, increased enzyme recovery rate, improved enzymatic hydrolysis efficiency, and reduced enzyme usage.

Description

Technical Field

[0001] The present invention relates to a cellulase solution system and a cellulase recovery technology in a lignocellulose enzymatic hydrolysis system, in particular to a UCST-responsive polymer and a preparation method thereof and a method for recovering cellulase at room temperature. Background Art

[0002] With the massive consumption of fossil energy and the increasing environmental pollution worldwide, lignocellulosic biorefining has become an important path to achieving sustainable development for human society. Sugar platform is an effective tool for lignocellulosic biorefining, a technology that utilizes lignocellulose to produce biofuels and other chemicals. Saccharification is the rate-limiting step in sugar platform conversion, and the high cost of enzymatic hydrolysis is a key bottleneck in the industrialization of lignocellulosic sugar platform. Commercial cellulase production costs are generally high, and considering that its enzymatic activity does not change significantly before and after lignocellulose hydrolysis, the recovery and recycling of cellulase has become a hot research direction for reducing enzymatic hydrolysis costs.

[0003] Free enzymes from lignocellulose hydrolysates are typically recovered using methods such as membrane separation, enzyme immobilization, and the addition of pH-responsive additives. However, these methods have yet to achieve green industrialization in the short term due to mass transfer limitations, high technical and economic costs, and the consumption of acid and base during the enzyme recovery process. Sulfobetaine homopolymer (PSPE) dissolves at the lignocellulose hydrolysis temperature (e.g., 50°C) and co-precipitates with the enzyme upon cooling after hydrolysis. Compared with traditional enzyme recovery methods, this method offers simplicity and low cost, while also avoiding mass transfer limitations and the consumption of acid and base during the enzyme recovery process. Using UCST-responsive additives to recover enzymes has become an effective way to reduce enzyme dosage and hydrolysis costs, offering promising prospects for green industrialization. Currently, linear PSPE can be used to recover enzymes at room temperature, but its low binding capacity to enzyme proteins results in low enzyme recovery rates and does not enhance lignocellulose hydrolysis. For example, adding 0.4 times (mass ratio of PSPE-3 to cellulase CTec2) to the corn cob residue (CCR) enzymatic hydrolysis system (50°C) and cooling the temperature after hydrolysis (e.g., to 25°C) can only save 50% of the cellulase dosage. Although the enzyme recovery process is simple to operate, environmentally friendly, and does not require the consumption of acid and alkali, 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).

[0004] At low temperatures, the association of positive and negative ions in betaine to form salts reduces the molecular hydrophilicity, which is the inherent driving force behind the low-temperature aggregation and sedimentation of UCST-type additives such as betaines, and their co-precipitation with enzymes. The key to improving the temperature sensitivity of UCST-type additives and their binding to enzyme proteins lies in increasing their hydrophobicity. Increasing polymer molecular weight generally increases its hydrophobicity, but due to the limitations of polymer structure, linear polymers generally have poor hydrophobicity. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a claw-shaped UCST-responsive polymer, a preparation method thereof, and a method for recovering cellulase at room temperature. The claw-shaped UCST-responsive polymer has sensitive temperature response performance, promotes the enzymatic hydrolysis of lignocellulose, and can recover cellulase at room temperature without consuming acid and alkali.

[0006] To solve the above technical problems, the present invention first discloses a claw-shaped UCST responsive polymer, which is a claw-shaped polymer obtained by prepolymerizing an amphoteric betaine monomer or an amphoteric betaine monomer and an N-vinyl pyrrolidone monomer and then crosslinking with a crosslinking agent triallylamine.

[0007] The amphoteric betaine monomer is a sulfonic acid betaine monomer represented by formula (I) or a phosphobetaine monomer represented by formula (II).

[0008]

[0009] In formula (I) and (I), m=1 or 2; R is (CH2) n , the (CH2) n Where n=2 or 3.

[0010] The N-vinyl pyrrolidone monomer is N-vinyl pyrrolidone represented by formula (III),

[0011]

[0012] The cross-linking agent is triallylamine represented by formula (IV)

[0013]

[0014] Specifically, amphoteric betaine monomers or amphoteric betaine monomers and N-vinyl pyrrolidone monomers are first prepolymerized to form a linear prepolymer with a larger molecular weight. The active end of the linear prepolymer reacts with the double bond of the crosslinker triallylamine. One triallylamine has three double bonds and can be connected to three linear prepolymers, thereby forming a star-shaped polymer with three linear chains, namely a "claw-shaped" polymer.

[0015] The present invention also discloses a method for preparing the claw-shaped UCST responsive polymer as described above. The method comprises adding an amphoteric betaine monomer or an amphoteric betaine monomer and an N-vinyl pyrrolidone monomer, and a persulfate compound as an initiator to a reaction vessel containing distilled water, reacting the mixture at 40 to 70° C. for 4 to 12 hours, then adding triallylamine as a cross-linking agent, and continuing the reaction for 2 to 12 hours to obtain the UCST responsive polymer. The entire process is an aqueous solution free radical polymerization reaction, and methanol is added at a ratio of 7 to 21 mL / g of methanol volume to the total monomer mass. The target product is precipitated, centrifuged, and then freeze-dried.

[0016] Furthermore, the persulfate compound is potassium persulfate or ammonium persulfate.

[0017] Furthermore, the molar ratio of the amphoteric betaine monomer to the N-vinyl pyrrolidone monomer is 10:0 to 1:9, and the mass ratio of the crosslinking agent triallylamine to the sum of the amphoteric betaine and N-vinyl pyrrolidone monomers is 0.01 to 0.2:10.

[0018] Furthermore, the mass ratio of the persulfate compound to the sum of the amphoteric betaine monomer and the N-vinyl pyrrolidone monomer is 0.01-0.2:10.

[0019] The present invention also discloses a method for recovering cellulase at room temperature using the aforementioned claw-shaped UCST-responsive polymer. The claw-shaped UCST-responsive polymer is added to a cellulase solution system or a lignocellulose enzymatic hydrolysis system, dissolved at 40 to 60°C, and the system is cooled to room temperature so that the claw-shaped UCST-responsive polymer and cellulase are precipitated simultaneously, and the cellulase is recovered by a solid-liquid separation method.

[0020] Furthermore, when the claw-shaped UCST responsive polymer is added to the lignocellulose enzymatic hydrolysis system, it is dissolved at 40-60°C and then enzymatically hydrolyzed for 24-72 hours. Then, the enzymatic hydrolysis residue (i.e., enzymatically hydrolyzed lignin) is removed by solid-liquid separation while still hot, and then the system is cooled to room temperature.

[0021] The cellulase in the system is derived from Trichoderma reesei or Aspergillus niger.

[0022] The solid-liquid separation method is one or a combination of two or more of natural sedimentation, decantation, filtration and centrifugation.

[0023] The cooling method is not natural cooling or cooling with refrigeration equipment.

[0024] Furthermore, the room temperature is 15-30°C.

[0025] 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.

[0026] Furthermore, the pH of the cellulase solution system or the lignocellulose enzymatic hydrolysis system is 5, and the ionic strength is 50 mmol / L.

[0027] Furthermore, the mass ratio of the UCST response polymer to the cellulase is 0.1 to 100:1.

[0028] Furthermore, the mass ratio of the UCST-responsive polymer to the cellulase is preferably 1 to 50:1.

[0029] The present invention has a claw-shaped polymer with a higher critical solution temperature (UCST) response, which is prepared by aqueous solution free radical polymerization. The synthesis method is simple, has sensitive temperature responsiveness, and can significantly improve the efficiency of lignocellulose enzymatic hydrolysis. The temperature response mechanism of the UCST-responsive polymer for recovering cellulase is as follows: under enzymatic hydrolysis conditions (temperature higher than UCST), the zwitterions in the amphoteric betaine structural unit in the polymer are in an ionized state, the hydrophilicity of the polymer molecules increases, and the polymer dissolves; when the temperature is lowered to room temperature (temperature lower than UCST), the ions in the amphoteric betaine structural unit associate to form an inner salt, the hydrophilicity of the polymer molecules decreases, and the polymer and cellulase are hydrophobically co-precipitated. The operation is simple and the process is green and environmentally friendly. In addition to increasing the molecular weight to increase the hydrophobicity, the UCST-responsive polymer of the present invention has a more coiled and folded "claw-shaped" structure that is more conducive to significantly increasing the molecular hydrophobicity, thereby constructing a highly sensitive UCST-type auxiliary agent that can enhance the enzymatic hydrolysis of lignocellulose and has strong binding to cellulase.

[0030] In summary, compared with linear sulfobetaine polymers, a small amount of the UCST-responsive polymer of the present invention can significantly enhance enzymatic hydrolysis and recover cellulase in the enzymatic hydrolysis system at a high recovery rate at room temperature, which is of great significance for reducing the production cost of biorefining and cellulosic ethanol. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a diagram showing the UCST response of 0.1 g / L PSPA-0 in the cellulase solution system of Example 1. DETAILED DESCRIPTION

[0032] The present invention will be further explained below with reference to the following examples. The following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0033] All reagents used in the following examples are commercially available. The cellulase used is the widely used Cellic CTec2. The glucose concentration in the hydrolyzate was measured using a biosensor analyzer (SBA-40E, Shandong Institute of Biological Sciences).

[0034] Synthesis of claw-shaped UCST-responsive polymers of the present invention

[0035] PSPA-x was synthesized by aqueous solution free radical polymerization at 60°C using methacryloylethyl sulfobetaine (SPE) and N-vinylpyrrolidone (NVP) as monomers, triallylamine (TA) as a cross-linker, and ammonium persulfate (APS) as an initiator, where x is the sample number (PSPA-0 and PSPA-1, the molar ratios of SPE to NVP are 10:0 and 7:3, respectively).

[0036] The specific synthesis experimental steps for PSPA-0 are as follows: At room temperature, 10 parts SPE, 0 parts NVP (i.e., a molar ratio of 10:0), and 0.01 parts APS (based on a total mass of 10 parts SPE and NVP) are rapidly added to 3.5 volumes of distilled water (based on the total mass of the SPE and NVP monomers, measured in mL / g). The system is then heated to 60°C and stirred at this constant temperature for 10 hours. Then, 0.01 parts TA (based on a total mass of 10 parts SPE and NVP monomers) is added, and stirred at this constant temperature for an additional 2 hours. After the reaction is completed, 7 volumes of methanol (based on a total mass of the SPE and NVP monomers, measured in mL / g) are added to the reaction solution to precipitate the desired product. After centrifugation, the product is freeze-dried.

[0037] The specific synthesis experimental steps for PSPA-1 are as follows: 7 parts SPE, 3 parts NVP (i.e., a molar ratio of 7:3), and 0.01 parts APS (based on a total mass of 10 parts SPE and NVP) are rapidly added to 3.5 volumes of distilled water (based on the total mass of the SPE and NVP monomers, measured in mL / g) at room temperature. The system is then heated to 60°C and stirred at this constant temperature for 10 hours. 0.01 parts TA (based on a total mass of 10 parts SPE and NVP monomers) is then added and stirred at this constant temperature for 2 hours. After the reaction is completed, 7 volumes of methanol (based on a total mass of the SPE and NVP monomers, measured in mL / g) are added to the reaction solution to precipitate the desired product. After centrifugation, the product is freeze-dried.

[0038] Synthesis of linear UCST-responsive polymers

[0039] The specific synthesis experimental steps for PSPE-3 are as follows: 10 parts SPE and 0.01 parts APS (based on the total mass of the monomer SPE) are quickly added to 7 volumes of distilled water (based on the total mass of the monomer SPE, calculated in mL / g) at room temperature. The system is then heated to 50°C and stirred at this constant temperature for 18 hours. After the reaction is completed, 14 volumes of methanol (based on the total mass of the monomer SPE, calculated in mL / g) are added to the reaction solution to precipitate the desired product. After centrifugation, the product is freeze-dried.

[0040] Example 1 Recovery of Cellulase in Cellulase Solution System

[0041] PSPA-0 (molecular weight M) was diluted to 0.05 g / L at 50 °C. w =30.0kDa) was added to a 0.2g / L cellulase solution system (system pH = 5.0, ionic strength 50mmol / L, prepared with acetic acid-sodium acetate buffer), the system was cooled to room temperature 25°C, PSPA-0 and cellulase were precipitated, the solid and liquid were separated by centrifuge, a certain amount of supernatant was taken, and impurities were filtered through an aqueous phase filter. The absorbance was measured using the Coomassie Brilliant Blue method to calculate the protein recovery rate, and then the cellulase recovery rate in the system was calculated. The solid phase is the recovered PSPA-0 and cellulase. The results are shown in Table 1. The UCST response of 0.1g / L PSPA-0 in the cellulase solution system is shown in Table 1. Figure 1 As shown, PSPA-0 completely dissolved at 50°C. When the system was cooled to room temperature, the hydrophilicity of the polymer decreased due to the interionic association of the amphoteric betaine structural units within the polymer molecule, forming an internal salt. This caused the polymer to precipitate and the system to become turbid. The transmittance of the system dropped from 100.0% to 3.2%. These results demonstrate that PSPA-0 exhibits sensitive UCST responsiveness.

[0042] Example 2 Recovery of Cellulase in Cellulase Solution System

[0043] PSPA-1 (molecular weight M) was diluted to 0.05 g / L at 50 °C. w =39.7 kDa) was added to a 0.2 g / L cellulase solution (pH = 5.0, ionic strength 50 mmol / L, prepared in acetic acid-sodium acetate buffer). The system was cooled to room temperature (25°C) to precipitate PSPA-1 and cellulase. The solid and liquid were separated by centrifuge, and a certain amount of the supernatant was aspirated and filtered through an aqueous filter to filter out impurities. The absorbance was measured using the Coomassie Brilliant Blue method to calculate the protein recovery rate, and thus the cellulase recovery rate in the system. The solid phase contained the recovered PSPA-1 and cellulase. The results are shown in Table 1.

[0044] Comparative Example 1 Recovery of Cellulase in Cellulase Solution System

[0045] PSPE-3 (molecular weight M) was diluted to 0.05 g / L at 50 °C. w =355.1 kDa) was added to a 0.2 g / L cellulase solution (pH = 5.0, ionic strength 50 mmol / L, prepared in acetic acid-sodium acetate buffer). The system was cooled to room temperature (25°C) to precipitate PSPE-3 and cellulase. The solid and liquid were separated by centrifuge, and a certain amount of the supernatant was aspirated and filtered through an aqueous filter to filter out impurities. The protein recovery rate, which was then expressed as the cellulase recovery rate in the system, was calculated by measuring the absorbance using the Coomassie Brilliant Blue method. The solid phase contained the recovered PSPE-3 and cellulase. The results are shown in Table 1.

[0046] Table 1 Cellulase recovery performance of claw-shaped UCST-responsive polymers in cellulase solution system

[0047]

[0048] Example 3 Recovery of Cellulase in Lignocellulose Enzymatic Hydrolysis System

[0049] PSPA-0 was added at 0.08 g / L to a corncob residue enzymatic hydrolysis system (pH 5.0, ionic strength 50 mmol / L, prepared in acetic acid-sodium acetate buffer, temperature 50°C) containing 0.2 g / L cellulase at 50°C. After 48 hours of enzymatic hydrolysis, the residue was removed and the system temperature was lowered to room temperature (25°C) to precipitate the PSPA-0 and cellulase. The solid and liquid were separated by centrifugation, and the glucose concentration in the supernatant was measured using an SBA-40E. The enzymatic efficiency of the substrate after 48 hours of enzymatic hydrolysis was expressed as glucose yield. This procedure was repeated three times to obtain three aliquots of PSPA-0 and cellulase from the first round of enzymatic hydrolysis, followed by centrifugal separation.

[0050] The three aliquots of PSPA-0 and cellulase were further added to Cellic CTec2 at 30%, 40%, and 50% of the initial enzyme loading (i.e., the total amount of cellulase in the first round of enzymatic hydrolysis), respectively. A second round of enzymatic hydrolysis was performed at 50°C (other conditions were the same as the first round). After 48 hours of enzymatic hydrolysis, the enzymatic efficiency (SED@48h) was measured to calculate the cellulase recovery rate in the lignocellulose enzymatic hydrolysis system. The results are shown in Table 2.

[0051] Example 4 Recovery of Cellulase in Lignocellulose Enzymatic Hydrolysis System

[0052] PSPA-1 was added at 0.08 g / L to a corncob residue enzymatic hydrolysis system (pH 5.0, ionic strength 50 mmol / L, prepared in acetic acid-sodium acetate buffer, temperature 50°C) containing 0.2 g / L cellulase at 50°C. After 48 hours of enzymatic hydrolysis, the residue was removed and the system temperature was lowered to room temperature (25°C) to precipitate PSPA-1 and cellulase. The solid and liquid were separated by centrifugation, and the glucose concentration in the supernatant was measured using an SBA-40E. The enzymatic efficiency of the substrate after 48 hours of enzymatic hydrolysis was expressed as glucose yield. This procedure was repeated three times to obtain three aliquots of PSPA-1 and cellulase from the first round of enzymatic hydrolysis, followed by centrifugal separation.

[0053] The three aliquots of PSPA-1 and cellulase were further added to Cellic CTec2 at 30%, 40%, and 50% of the initial enzyme loading (i.e., the total amount of cellulase in the first round of enzymatic hydrolysis), respectively. A second round of enzymatic hydrolysis was performed at 50°C (other conditions were the same as the first round). After 48 hours of enzymatic hydrolysis, the enzymatic efficiency (SED@48h) was measured to calculate the cellulase recovery rate in the lignocellulose enzymatic hydrolysis system. The results are shown in Table 2.

[0054] Comparative Example 2 Recovery of Cellulase in Lignocellulose Enzymatic Hydrolysis System

[0055] Corncob residue was enzymatically hydrolyzed using an acetic acid-sodium acetate buffer (pH 5.0, ionic strength 50 mmol / L, temperature 50°C) with a cellulase concentration of 0.2 g / L. After 48 hours of enzymatic hydrolysis, the solid and liquid were separated by centrifuge, and the glucose concentration in the supernatant was measured using an SBA-40E. The enzymatic efficiency after 48 hours of enzymatic hydrolysis is expressed as glucose yield. The enzymatic hydrolysis results are shown in Table 2.

[0056] Comparative Example 3 Recovery of Cellulase in Lignocellulose Enzymatic Hydrolysis System

[0057] PSPE-3 was added at 0.08 g / L to a corncob residue enzymatic hydrolysis system containing 0.2 g / L cellulase at 50°C (pH 5.0, ionic strength 50 mmol / L, prepared with acetic acid-sodium acetate buffer, temperature 50°C). After 48 hours of enzymatic hydrolysis, the residue was removed and the system temperature was lowered to room temperature (25°C) to precipitate the PSPE-3 and cellulase. The solid and liquid were separated by centrifuge, and the glucose concentration in the supernatant was measured using an SBA-40E. The enzymatic efficiency of the substrate after 48 hours of enzymatic hydrolysis was expressed as glucose yield. The same procedure was repeated three times to obtain three aliquots of PSPE-3 and cellulase obtained after the first round of enzymatic hydrolysis and centrifugal separation.

[0058] The three aliquots of PSPE-3 and cellulase were further added to Cellic CTec2 at 30%, 40%, and 50% of the initial enzyme loading (i.e., the total amount of cellulase in the first round of enzymatic hydrolysis), respectively. A second round of enzymatic hydrolysis was performed at 50°C (other conditions were the same as the first round). After 48 hours of enzymatic hydrolysis, the enzymatic efficiency (SED@48h) was measured to calculate the cellulase recovery rate in the lignocellulose enzymatic hydrolysis system. The results are shown in Table 2.

[0059] Table 2 Cellulase recovery performance of claw-shaped UCST-responsive polymers in cellulase solution system

[0060]

[0061] In Table 2, the present invention uses the saved enzyme dosage to express the recovery enzyme yield. The specific calculation method is shown in the reference (see The synthesis of a UCST-type zwitterionic polymer for the efficient recycling of cellulase at room temperature, Green Chemistry, 2021.23: 2738-2746). Table 2 shows that the claw-shaped UCST responsive polymer of the present invention can efficiently recover cellulase in the lignocellulose enzymatic hydrolysis system. No large amount of acid and alkali is consumed during the enzyme recovery process, and the recovery effect is better than that of the linear polymer PSPE-3. At the same time, after NVP is introduced into the UCST responsive polymer for cross-linking, its enzymatic hydrolysis efficiency reaches 59.9%. The UCST responsive polymer of the present invention can not only efficiently recover enzymes but also improve the efficiency of lignocellulose enzymatic hydrolysis.

[0062] The above examples are merely specific embodiments of the present invention. The present invention is not limited to the above examples, and many variations are possible. All variations that can be directly derived or imagined by a person skilled in the art from the disclosure of the present invention should be considered to be within the scope of protection of the present invention.

Claims

1. A claw-shaped UCST-responsive polymer for recovering cellulase, characterized in that: The polymer is a claw-shaped polymer obtained by prepolymerizing amphoteric betaine monomers and N-vinyl pyrrolidone monomers and then crosslinking with triallylamine as a crosslinking agent. The amphoteric betaine monomer is a sulfobetaine monomer represented by formula (I) or a phosphobetaine monomer represented by formula (II). （Ⅰ） （Ⅱ） In formula (I) and (II), m = 1 or 2; R is (CH2) n , the (CH2) n n = 2 or 3; The molar ratio of the amphoteric betaine monomer to the N-vinyl pyrrolidone monomer is 7:3-1:9, and the mass ratio of the crosslinking agent triallylamine to the sum of the amphoteric betaine monomer and the N-vinyl pyrrolidone monomer is 0.01-0.2:

10.

2. A method for preparing the claw-shaped UCST responsive polymer according to claim 1, characterized in that: Amphoteric betaine monomer and N-vinyl pyrrolidone monomer, as well as a persulfate compound as an initiator, are added to a reaction container containing distilled water, and the mixture is reacted at 40-70° C. for 4-12 hours. Triallylamine is then added as a cross-linking agent, and the reaction is continued for 2-12 hours to obtain the UCST responsive polymer.

3. The method for preparing the claw-shaped UCST responsive polymer according to claim 2, characterized in that: The persulfate compound is one of potassium persulfate or ammonium persulfate.

4. The method for preparing the claw-shaped UCST responsive polymer according to claim 2, characterized in that: The mass ratio of the persulfate compound to the sum of the amphoteric betaine monomer and N-vinyl pyrrolidone is 0.01-0.2:

10.

5. A method for recovering cellulase at room temperature using the claw-shaped UCST-responsive polymer of claim 1, characterized in that: The claw-shaped UCST responsive polymer is added to a cellulase solution system or a lignocellulose enzymatic hydrolysis system, dissolved at 40-60° C., and the system is cooled to room temperature to allow the claw-shaped UCST responsive polymer and cellulase to precipitate simultaneously, and the cellulase is recovered by a solid-liquid separation method.

6. The method according to claim 5, characterized in that: When the claw-shaped UCST-responsive polymer is added to the cellulose enzymatic hydrolysis system, it is dissolved at 40-60°C and then enzymatically hydrolyzed for 24-72 hours. The enzymatic hydrolysis residue is then removed by a solid-liquid separation method. The system is then cooled to room temperature to allow the claw-shaped UCST-responsive polymer and cellulase to precipitate simultaneously, and the cellulase is recovered by a solid-liquid separation method.

7. The method according to claim 5 or 6, characterized in that: The room temperature is 15-30°C.

8. The method according to claim 5, 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.

9. The method according to claim 8, characterized in that: The pH of the cellulase solution system or the lignocellulose enzymatic hydrolysis system is 5; the ionic strength is 50 mmol / L; and the cellulase protein concentration is 40-2500 mg / L.

10. The method according to claim 5, characterized in that: The mass ratio of claw-shaped UCST responsive polymer to cellulase is 0.1-100:

1.

11. The method according to claim 10, characterized in that: The mass ratio of claw-shaped UCST responsive polymer to cellulase is 1-50:1.

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