Cellulase recycling method based on light-responsive group-modified lignin
By modifying lignin with photoresponsive groups to form a reversibly binding complex with cellulase, the problems of poor cellulase stability and low enzymatic hydrolysis efficiency are solved, realizing efficient recovery of cellulase and high-value utilization of lignin, and reducing the cost of enzymatic hydrolysis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANJING UNIV OF SCI & TECH
- Filing Date
- 2024-12-18
- Publication Date
- 2026-06-19
AI Technical Summary
Existing cellulases are unstable, easily inactivated, and have low hydrolysis efficiency during enzymatic hydrolysis, resulting in high hydrolysis costs and underutilization of lignin resources. Existing immobilized cellulase technologies have limitations in terms of hydrolysis and recovery efficiency.
By modifying lignin with photoresponsive groups and combining it with ligand groups to modify cellulase, a reversibly bound cellulase complex is formed. The photoresponsive properties are used to achieve periodic adsorption and desorption of cellulase, enabling enzyme recovery and reuse.
It improves the utilization efficiency of cellulase and the high-value application of lignin, reduces the cost of enzyme hydrolysis, and achieves efficient recovery and recycling of cellulase under mild conditions. The glucan conversion rate is maintained above 65%, the enzyme activity is high, and it is suitable for multiple rounds of recycling.
Smart Images
Figure CN122235120A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of cellulase recovery technology and relates to a cellulase recovery method based on photoresponsive groups modified lignin. Background Technology
[0002] With the acceleration of industrialization and the over-exploitation of fossil resources, energy crisis and environmental pollution have become two major problems that human development cannot avoid. Cellulose ethanol, which is produced from lignocellulosic biomass, is considered one of the most promising ways to solve these problems. The production of cellulose ethanol mainly utilizes hemicellulose and cellulose produced from lignocellulosic biomass after pretreatment, enzymatically hydrolyzes them to produce fermentable monosaccharides, and then produces ethanol through microbial fermentation. Although cellulose ethanol technology has been industrialized, it still faces many technical challenges in the production process. Among them, in the enzymatic hydrolysis process, the poor stability, easy inactivation, and low enzymatic hydrolysis efficiency of cellulase lead to excessively high enzymatic hydrolysis costs, which seriously limits the industrial production of cellulose ethanol (Adsul M, Sandhu SK, Singhana RR, et al. Designing acellulolytic enzyme cocktail for the efficient and economical conversion of lignocellulosic biomass to biofuels[J].Enzyme and Microbial Technology,2020(133-):133.). In addition, the production of cellulosic ethanol generates a large amount of lignin residue, estimated to be 500,000 tons per year globally. However, due to insufficient commercial application, more than 90% of lignin is used for combustion power generation or waste treatment, resulting in a large amount of lignin resources being underutilized.
[0003] Lignin is a natural high-molecular-weight polymer mainly composed of three structural units: guaiacol, syringyl, and p-hydroxyphenyl. It is characterized by its green and non-toxic nature, biodegradability, and strong biocompatibility, making it widely used in pharmaceuticals and biotechnology. With the deepening of lignin research, researchers have increasingly focused on enzyme immobilization. Lignin, with its excellent biocompatibility and multifunctionality, has become an ideal carrier for enzyme immobilization. However, due to its complex structure and unique physicochemical properties, lignin cannot be directly used for enzyme immobilization. It typically requires pretreatment methods, such as biological, chemical, or physical methods, to modify its structure and properties, increasing its affinity and availability for enzymes before application in enzyme immobilization. Park et al. reportedly successfully encapsulated lipase in biomimetic hydrogel beads composed of lignin, cellulose, and xylan. Lignin significantly improved the thermal and pH stability of the immobilized enzyme (Kim, Hee S, Choi, et al. Wood mimetic hydrogel beads for enzyme immobilization[J]. Carbohydrate Polymers: Scientific and Technological Aspects of Industrially Important Polysaccharides, 2015.). Zdarta et al. immobilized trypsin on a magnetite-lignin composite material and achieved enzyme recovery by introducing a magnetic support. It was reported that trypsin immobilized on the magnetite-lignin material retained 69% of its relative activity after 10 consecutive rounds of recovery (Zdarta J, Antecka K, J Drzak A, et al. Biopolymers conjugated with magnetite as support materials for trypsin immobilization and protein digestion[J]. Colloids & Surfaces B Biointerfaces, 2018, 169:118.).In addition, some scholars have successfully prepared lignin-based azo polymers with photoresponsive properties through chemical modification. These polymers can achieve the conversion between cis and trans isomers with only simple light treatment (Deng Y, Liu Y, Qian Y, et al. Preparation of Photoresponsive Azo Polymers Based on Lignin, a Renewable Biomass Resource[J]. Acs Sustainable Chemistry & Engineering, 2015, 3(6): 1505-08120040006.). Based on their photoresponsive properties, lignin-based azo polymers can combine with cyclodextrin to form smart molecules with photoresponsive properties. This discovery has greatly broadened the potential of lignin in high-value-added applications.
[0004] Enzyme recovery technology is one of the key applications in the field of enzyme immobilization, especially in the production of cellulosic ethanol, where it plays an important role in reducing the cost of enzyme hydrolysis. In the recovery of cellulase, researchers usually first combine cellulase with materials with specific responsive properties to form a cellulase complex with responsive properties, and then carry out enzyme hydrolysis. That is, they use the properties of these materials to improve the recovery efficiency and reuse rate of cellulase, thereby reducing the overall process cost and improving sustainability. Mackenzie et al. developed a recyclable thermoresponsive polymer-endoglucanase complex, which achieves the separation and recovery of cellulase and substrate by regulating the ambient temperature. After three rounds of recovery, the enzyme activity still exceeded 60% (Mackenzie KJ, Francis MB Recyclable thermoresponsive polymer-cellulase bioconjugates for biomass depolymerization[J]. Journal of the American Chemical Society,2013,135(1):293-300.). Furthermore, Alftren et al. successfully immobilized cellulase on silica magnetic particles via covalent bonding. After hydrolysis, the cellulase was recovered using magnetic separation technology and used in a second round of hydrolysis. Their study found that the yield of the second round of hydrolysis was 66% (Alftren J, Hobley T J. Immobilization of cellulase mixtures on magnetic particles for hydrolysis of lignocellulose and ease of recycling[J]. Biomass & Bioenergy, 2014, 65:72-78.). Although enzyme immobilization technology can improve the recovery efficiency of cellulase, it also presents some challenges. During enzyme hydrolysis, cellulase needs to bind to insoluble substrates to exert its catalytic effect. If the enzyme also becomes insoluble after immobilization, this may reduce the hydrolysis efficiency.Furthermore, previous studies have explored the immobilization of cellobiose hydrolase (CBH) on magnetic polymer nanoparticles. Adsorption and hydrolysis experiments were conducted by applying this immobilized multi-catalytic cellulose nanocomposite to cellulose (Kamat RK, Ma W, Yang Y, et al. Adsorption and hydrolytic activity of the polycatalytic cellulasenanocomplex on cellulose[J]. ACS Applied Materials & Interfaces, 2013, 5(17): 8486-8494.). However, existing immobilized cellulase multi-enzyme catalytic systems mainly bind to solid supports through physical or chemical interactions. The immobilized cellulase is confined to the support and cannot move freely in the enzymatic hydrolysis system, which greatly limits the degradation efficiency of cellulose. Connecting cellulase to an immobilization carrier using responsive groups can effectively solve this problem. Specific regulation of the responsive group's properties allows for the binding or separation of cellulase from the immobilization carrier. Specifically, during enzymatic hydrolysis, the cellulase is distributed in a free state within the hydrolysis system. After the hydrolysis reaction, the free cellulase in the liquid is adsorbed onto the immobilization carrier for enzyme recovery. Current research on enzyme immobilization using smart responsive groups mainly focuses on temperature and pH response characteristics. However, because cellulase is highly sensitive to temperature and pH, even slight changes can have a significant impact on its activity. The tolerance of cellulase must be considered during regulation, which greatly limits its application in enzyme recovery. Therefore, developing a novel cellulase recovery technology that can efficiently recover cellulase without interfering with the cellulase's co-degradation mode is of great significance for reducing the cost of enzyme hydrolysis. Summary of the Invention
[0005] The purpose of this invention is to provide a method for cellulase recovery based on photoresponsive group-modified lignin. This invention modifies lignin with photoresponsive groups and cellulase with ligand groups, enabling reversible binding between the two, thereby recovering the cellulase and improving its utilization efficiency.
[0006] The technical solution for achieving the objective of this invention is as follows:
[0007] A cellulase recovery method based on lignin modified with photoresponsive groups includes the following steps:
[0008] (1) Photoresponsive groups are attached to lignin through a substitution reaction to synthesize lignin-based compounds with photoresponsive properties. Ligand groups are attached to cellulase through a cross-linking agent to synthesize ligand-cellulase complexes.
[0009] (2) The lignin-based compound and the ligand-cellulase complex were added to the lignin-cellulose enzymatic hydrolysis system without cellulase, and enzymatic hydrolysis was carried out at 40-60℃ for 20-72h. During the enzymatic hydrolysis process, natural light or blue light was first irradiated, and then periodic light regulation was carried out as needed. Natural light or blue light and ultraviolet light were used alternately to make the free cellulase complex in the liquid periodically adsorbed and desorbed on the surface of the lignin-based compound.
[0010] (3) After the first round of enzymatic hydrolysis in step (2) is completed, the solid is recovered by solid-liquid separation method. The recovered solid is directly added to the second round of enzymatic hydrolysis. The second round of enzymatic hydrolysis does not require the addition of lignin-based compounds and ligand-cellulase complex. The recovery and enzymatic hydrolysis reactions are repeated to recover and utilize the lignin-based compounds and ligand-cellulase complex, and the enzymatic hydrolysis supernatant is collected.
[0011] Furthermore, in step (1), the photoresponsive group is a commonly used photoresponsive group, including but not limited to azobenzene, stilbene, cinnamate, and spiropyran. In a specific embodiment of the present invention, the azo group is used as a representative example of the photoresponsive group.
[0012] Furthermore, in step (1), the ligand group is a ligand group corresponding to the photoresponsive group, including but not limited to cyclodextrin, terephthalonitrile, and chiral carbon dots. In a specific embodiment of the present invention, cyclodextrin is used as a representative example of the ligand group.
[0013] Furthermore, in step (1), the lignin is a common type of lignin, including but not limited to alkali lignin, enzymatically hydrolyzed lignin, sulfate lignin, and lignin sulfonate.
[0014] Furthermore, in step (1), the cellulase is a common cellulase, including but not limited to Trichoderma cellulase, Aspergillus cellulase and Penicillium cellulase.
[0015] Furthermore, in step (2), the lignocellulose hydrolysis system without added cellulase includes substrate and buffer.
[0016] Furthermore, in step (2), the mass ratio of lignocellulose biomass to buffer solution in the lignocellulose enzymatic hydrolysis system is 1% to 15%, preferably 3% to 10%.
[0017] Furthermore, in step (2), the substrate in the lignocellulose enzymatic hydrolysis system is a common lignocellulose biomass, including but not limited to straw, bagasse, and branches.
[0018] Furthermore, in step (2), the buffer solution in the lignocellulose enzymatic hydrolysis system is a 50mM citrate buffer with pH 4.0 to 6.0, preferably a citrate buffer with pH 4.8.
[0019] Furthermore, in step (2), the enzymatic hydrolysis temperature is 50°C.
[0020] Furthermore, in step (2), the amount of lignin-based compound added is 10 mg / mL, and the amount of cellulase complex added is 10 mg cellulase complex / g dextran.
[0021] Furthermore, in step (2), natural light or blue light is first irradiated for 4±2 hours during the enzymatic hydrolysis process.
[0022] Further, in step (2), the periodic light modulation specifically involves: in one cycle, first irradiating with ultraviolet light, then irradiating with natural light or blue light, and repeating the process of irradiating with ultraviolet light, natural light, or blue light. Preferably, the total duration of the cycle is 4 hours, with irradiation with ultraviolet light for 3 hours and then irradiation with natural light or blue light for 1 hour.
[0023] Compared with the prior art, the present invention has the following advantages:
[0024] (1) The method of the present invention can promote the hydrolysis of dextran during the hydrolysis of lignocellulase, and increase the dextran conversion rate by more than 10% in the first round of enzymatic hydrolysis.
[0025] (2) The binding of cellulase to lignin is completed under mild conditions, and the enzyme is recovered. No pH adjustment, external magnetic field or temperature control is required during the recovery process. After light treatment, cellulase can be recovered by simple centrifugation. After six rounds of repetition, the dextran conversion rate can still be maintained at more than 65% of the initial enzymatic hydrolysis dextran conversion rate, achieving efficient cellulase recovery. Moreover, the recovered cellulase has high activity and can be recycled.
[0026] (3) Using lignin as an immobilization carrier to recover cellulase not only realizes the efficient utilization of cellulase, but also realizes the high-value application of lignin, thus improving the economic efficiency of lignocellulose refining. Attached Figure Description
[0027] Figure 1 The effect of light treatment on the relative activity of cellulase in the supernatant in Example 1.
[0028] Figure 2 The graph shows the enzymatic hydrolysis results of the recovery experiment in Example 2, where a is the conversion rate of dextran to xylan and b is the relative hydrolysis yield of dextran.
[0029] Figure 3The graph shows the enzymatic hydrolysis results of the recovery experiment in Example 3, where a is the conversion rate of dextran to xylan and b is the relative hydrolysis yield of dextran.
[0030] Figure 4 The graph shows the enzymatic hydrolysis results of the recovery experiment in Comparative Example 1, where a represents the conversion rate of dextran to xylan, and b represents the relative hydrolysis yield of dextran.
[0031] Figure 5 The graph shows the enzymatic hydrolysis results of the recovery experiment in Comparative Example 2, where a represents the conversion rate of dextran to xylan, and b represents the relative hydrolysis yield of dextran.
[0032] Figure 6 The graph shows the enzymatic hydrolysis results of the recovery experiment in Comparative Example 3, where a represents the conversion rate of dextran to xylan, and b represents the relative hydrolysis yield of dextran. Detailed Implementation
[0033] To facilitate understanding of the present invention, the present invention will be described in further detail below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited to the following specific embodiments.
[0034] In the following examples, the selected photoresponsive group is an azo group, and the selected ligand group is a cyclodextrin group. Under natural light or 435nm blue light irradiation, the azo group is converted to the trans isomer and combines with the cyclodextrin group; under 365nm ultraviolet light irradiation, the azo group is converted to the cis isomer and the azo group is released from the cyclodextrin lumen.
[0035] In the following examples, the lignin-based compound with photoresponsive properties used is azo lignin. Azo lignin with photoresponsive properties is synthesized by attaching azo groups to lignin through a substitution reaction. The specific synthesis process is described in reference (Borovkova VS, Malear YN, Vasilieva NY, et al. New Azo Derivatives of Ethanol Lignin:Synthesis,Structure,and Photosensitive Properties[J].Materials,2023,16:1525.).
[0036] In the following examples, the cyclodextrin and cellulase were linked via a cross-linking agent. Specifically, 40 mg of commercial cellulase (Ctec3 HS) and 100 mg of mono-6-amino-6-deoxy-β-cyclodextrin were dissolved in 10 mL of potassium phosphate buffer (50 mM, pH 6.0), and 30 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added to the reaction mixture. The mixture was stirred at room temperature for 1 h and incubated at 4 °C in a shaker for 16 h (800 rpm). The sample was then subjected to ultrafiltration to replace the solution with 50 mM sodium citrate solution (pH 4.8). After ultrafiltration, the sample was stored at 4 °C to obtain the cyclodextrin-cellulase complex.
[0037] Example 1
[0038] Azolignin and the cyclodextrin-cellulase complex were added to 50 mM citrate buffer (pH 4.8), with azolignin at a concentration of 10 mg / mL and the cyclodextrin-cellulase complex at a concentration of 1 mg / mL. The mixture was first irradiated under natural light for 3 hours, followed by alternating irradiation under ultraviolet light for 1 hour and then under natural light for 1 hour. The relative changes in cellulase activity in the supernatant were measured. A control group was used, irradiated under natural light throughout the entire process. The test results are as follows: Figure 1 As shown. From Figure 1 It can be seen that during the first 3 hours (0-3 h), when the experimental and control groups were exposed to natural light, the azo groups combined with cyclodextrin, and the cyclodextrin-cellulase complex, which was free in the liquid, was fixed on the surface of azo lignin. Therefore, at 3 h, the cellulase activity in the supernatant of the experimental and control groups decreased to 84.16% and 86.73% of the initial activity, respectively. After 3-4 h, the experimental group was exposed to ultraviolet light. The azo groups detached from the cyclodextrin, and the cyclodextrin-cellulase complex fixed on the surface of azo lignin detached and re-integrated into the liquid. Therefore, at 4 h, the cellulase activity in the experimental group increased back to 100.68% of the initial activity, while the cellulase activity in the control group was 89.38%. After three rounds of cyclic irradiation, the experimental group exhibited excellent light response characteristics, with the cellulase activity in its supernatant increasing or decreasing with different wavelengths of light.
[0039] Example 2
[0040] (1) In the first round of enzymatic hydrolysis, pretreated corn stalks (35.25% dextran and 19.12% xylan) were added to a glass bottle, along with 50 mM citrate buffer (pH 4.8), azo lignin, and a cyclodextrin-cellulase complex, making the mass ratio of corn stalks to citrate buffer 3%. The amount of cyclodextrin-cellulase complex added was 10 mg protein / g dextran, and the amount of azo lignin added was 10 mg / mL. The reaction was carried out at 50℃ and 800 rpm for 20 h. During the enzymatic hydrolysis, natural light irradiation was applied from 0 to 4 h, followed by periodic 4 h light-controlled irradiation until the end of the enzymatic hydrolysis. During the periodic light-controlled irradiation, ultraviolet light irradiation at a wavelength of 365 nm was applied for the first 3 h, followed by natural light irradiation for the last 1 h.
[0041] (2) After 20 hours of hydrolysis, the enzymatically hydrolyzed suspension was separated into solid and liquid components by centrifugation. The solid was then used in the second round of enzymatic hydrolysis. Subsequent operations were the same as those in the first round of enzymatic hydrolysis, but azo lignin and cyclodextrin-cellulase complex were no longer added. Instead, 50 mM citrate buffer at pH 4.8 and fresh corn stalks were added.
[0042] (3) After six rounds of enzymatic hydrolysis, the supernatant was collected and the sugar concentration was tested. The results of the enzymatic hydrolysis are as follows: Figure 2 As shown. The conversion rates of dextran and xylan were measured to be 87.42% and 67.10% in the first round of enzymatic hydrolysis, respectively, and 65.52% and 53.83% in the sixth round of enzymatic hydrolysis (the yield of each round of enzymatic hydrolysis was calculated based on the content of dextran and xylan added from the straw in the first round), and the relative yield of dextran in the sixth round was 65.52%.
[0043] The formula for calculating the relative yield of dextran in the nth round is:
[0044]
[0045] Example 3
[0046] This embodiment is basically the same as Example 1, except that when synthesizing the cyclodextrin-cellulase complex, the cellulase used is a compound enzyme obtained by combining cellulase obtained from Trichoderma reesei fermentation with commercial cellulase at an enzyme-protein concentration ratio of 1:1. The remaining steps are the same as in Example 1.
[0047] After six rounds of enzymatic hydrolysis, the supernatant was collected and the sugar concentration was tested. The results of the enzymatic hydrolysis are as follows: Figure 3As shown, the conversion rates of dextran and xylan were 93.32% and 65.80%, respectively, in the first round of enzymatic hydrolysis, and 63.86% and 50.23%, respectively, in the sixth round of enzymatic hydrolysis, with a relative yield of 68.43% for dextran in the sixth round. The combined results of Examples 1 and 2 demonstrate that the method of the present invention is applicable to cellulases from different sources.
[0048] Comparative Example 1
[0049] The process is basically the same as in Example 1, except that the cyclodextrin-cellulase complex is replaced with commercial cellulase during the enzymatic hydrolysis process.
[0050] After six rounds of enzymatic hydrolysis, the supernatant was collected and the sugar concentration was tested. The results of the enzymatic hydrolysis are as follows: Figure 4 As shown, the conversion rates of dextran and xylan were 80.44% and 61.96%, respectively, in the first round of enzymatic hydrolysis, and 26.11% and 42.18%, respectively, in the sixth round of enzymatic hydrolysis. Furthermore, the relative yield of dextran in the sixth round was only 32.45%. Based on the results of Example 1 and Comparative Example 1, this enzyme recovery strategy is ineffective against common commercial cellulases.
[0051] Comparative Example 2
[0052] The process was basically the same as in Example 1, except that the entire enzymatic hydrolysis process was subjected to ultraviolet light irradiation, and the cyclodextrin group and azophenyl group were not bound.
[0053] After six rounds of enzymatic hydrolysis, the supernatant was collected and the sugar concentration was tested. The results of the enzymatic hydrolysis are as follows: Figure 5 As shown, the conversion rates of dextran and xylan were 84.01% and 69.18%, respectively, in the first round of enzymatic hydrolysis, and 35.23% and 45.23%, respectively, in the sixth round of enzymatic hydrolysis. Furthermore, the relative yield of dextran in the sixth round was only 35.23%. Combining the results of Example 1 and Comparative Example 2, the enzyme recovery rate using only UV irradiation during enzymatic hydrolysis was lower than the enzyme recovery efficiency using a light-controlled strategy. Light treatment significantly affected the interaction between the azophenyl group and the cyclodextrin group, thus impacting the efficiency of cellulase.
[0054] Comparative Example 3
[0055] The process is basically the same as in Example 1, except that commercial cellulase and unmodified lignin are added directly during the enzymatic hydrolysis process.
[0056] After six rounds of enzymatic hydrolysis, the supernatant was collected and the sugar concentration was tested. The results of the enzymatic hydrolysis are as follows: Figure 6As shown, the conversion rates of dextran and xylan were 77.21% and 58.98% respectively in the first round of enzymatic hydrolysis, and 25.91% and 37.86% respectively in the sixth round of enzymatic hydrolysis, with a relative yield of only 33.55% for dextran in the sixth round. Based on the results of Example 1 and Comparative Example 3, the recovery method of the present invention can effectively promote the hydrolysis of dextran during the hydrolysis of lignocellulase.
[0057] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.
Claims
1. A method for cellulase recovery based on lignin modified with photoresponsive groups, characterized in that, Includes the following steps: (1) Photoresponsive groups are attached to lignin through a substitution reaction to synthesize lignin-based compounds with photoresponsive properties. Ligand groups are attached to cellulase through a cross-linking agent to synthesize ligand-cellulase complexes. (2) The lignin-based compound and the ligand-cellulase complex were added to the lignin-cellulose enzymatic hydrolysis system without cellulase, and enzymatic hydrolysis was carried out at 40~60℃ for 20~72 h. During the enzymatic hydrolysis process, natural light or blue light was first irradiated, and then periodic light regulation was carried out as needed. Natural light or blue light and ultraviolet light were used alternately to make the free cellulase complex in the liquid periodically adsorbed and desorbed on the surface of the lignin-based compound. (3) After the first round of enzymatic hydrolysis in step (2) is completed, the solid is recovered by solid-liquid separation method. The recovered solid is directly added to the second round of enzymatic hydrolysis. The second round of enzymatic hydrolysis does not require the addition of lignin-based compounds and ligand-cellulase complex. The recovery and enzymatic hydrolysis reactions are repeated to recover and utilize the lignin-based compounds and ligand-cellulase complex, and the enzymatic hydrolysis supernatant is collected.
2. The cellulase recovery method according to claim 1, characterized in that, In step (1), the photoresponsive group is azobenzene, stilbene, cinnamate or spiropyran, and the ligand group is cyclodextrin, terephthalonitrile or chiral carbon dots.
3. The cellulase recovery method according to claim 1, characterized in that, In step (1), the lignin is alkali lignin, enzymatic lignin, sulfate lignin or lignin sulfonate, and the cellulase is Trichoderma cellulase, Aspergillus cellulase or Penicillium cellulase.
4. The cellulase recovery method according to claim 1, characterized in that, In step (2), the lignocellulose enzymatic hydrolysis system without added cellulase includes substrate lignocellulose biomass and buffer solution. The mass ratio of lignocellulose biomass to buffer solution is 1% to 15%. The lignocellulose biomass is one or more of straw, sugarcane bagasse and branches. The buffer solution is 50 mM citrate buffer solution with pH 4.0 to 6.
0.
5. The cellulase recovery method according to claim 4, characterized in that, In the lignocellulose enzymatic hydrolysis system, the mass ratio of lignocellulose biomass to buffer solution is 3%~10%.
6. The cellulase recovery method according to claim 4, characterized in that, The buffer solution is a citrate buffer with a pH of 4.
8.
7. The cellulase recovery method according to claim 1, characterized in that, In step (2), the enzymatic hydrolysis temperature is 50℃, the amount of lignin-based compound added is 10 mg / mL, and the amount of cellulase complex added is 10 mg cellulase complex / g dextran.
8. The cellulase recovery method according to claim 1, characterized in that, In step (2), the enzyme digestion process is first irradiated with natural light or blue light for 4±2h.
9. The cellulase recovery method according to claim 1, characterized in that, In step (2), the periodic light modulation is specifically as follows: in one cycle, first irradiate with ultraviolet light, then irradiate with natural light or blue light, and repeat the process of irradiation with ultraviolet light, natural light or blue light.
10. The cellulase recovery method according to claim 9, characterized in that, The total duration of the cycle is 4 hours, first irradiating with ultraviolet light for 3 hours, and then irradiating with natural light or blue light for 1 hour.