Thermo-sensitive lignin-based copolymer, preparation method thereof and application of thermo-sensitive lignin-based copolymer in enzyme immobilization

Abstract

The invention discloses a thermo-sensitive lignin-based copolymer as well as a preparation method and application thereof in enzyme immobilization. According to the invention, lignin and acryloyl glycinamide are copolymerized to synthesize a thermo-sensitive lignin-based copolymer with UCST characteristics, and the thermo-sensitive lignin-based copolymer is used as a carrier for enzyme immobilization. According to the method, the renewability of lignin and the temperature-sensitive characteristic of pNAGA are combined, the catalytic efficiency and stability of the immobilized enzyme are remarkably improved, the thermal stability and pH stability of the immobilized enzyme are remarkably enhanced, the immobilized enzyme still keeps 60% of enzyme activity after five times of circulation, the service life of the immobilized enzyme is prolonged, the production cost is reduced, and the method is suitable for industrial production and application.

Description

Technical Field

[0001] The invention belongs to the technical field of high-value utilization of biomass and biocatalysis, and relates to a temperature-sensitive lignin-based copolymer, a preparation method thereof, and application thereof in enzyme immobilization. Background Art

[0002] In the enzymatic hydrolysis of lignocellulose, β-glucosidase (βG) plays a key role in the degradation of cellulose into glucose. The cellulose hydrolysis process usually involves endoglucanases and exoglucanases first degrading cellulose into oligosaccharides and cellobiose, while βG catalyzes the further hydrolysis of cellobiose into glucose, and its catalytic activity directly affects the conversion efficiency of cellulose and the final glucose yield. However, in industrial applications, the use of βG faces problems such as high consumption, high cost, and low recovery rate. Since βG is mainly dissolved in the aqueous phase, traditional methods such as centrifugation and filtration are difficult to effectively recover, resulting in low reuse rate and high enzyme cost. In addition, free βG is prone to inactivation during use and is greatly affected by factors such as pH changes, temperature fluctuations, and long-term storage. Therefore, developing an efficient βG immobilization strategy to improve the recovery rate, stability, and catalytic activity of the enzyme has become a key challenge in enzyme catalysis technology.

[0003] At present, the methods for immobilizing βG are mainly divided into the following categories: (1) Physical adsorption method uses electrostatic or hydrophobic interaction to adsorb βG on the carrier. The method is simple, but the adsorption force is weak, the enzyme is easy to fall off, and the catalytic stability is affected; (2) Chemical crosslinking method uses covalent bonds to fix βG on the carrier to improve stability, but chemical crosslinking may affect the active center of the enzyme and reduce the catalytic efficiency; (3) Encapsulation method uses polymer networks to encapsulate βG to prevent its loss, but it is easy to cause substrate diffusion restriction and reduce reaction efficiency. Although these methods have improved the recovery rate and stability of βG to a certain extent, the catalytic activity loss of the immobilized enzyme is still large, the number of reuses is limited, and especially the recovery efficiency of the enzyme is still low.

[0004] In recent years, thermosensitive polymers have attracted wide attention in the field of enzyme immobilization due to their reversible dissolution-precipitation behavior. Among them, upper critical solution temperature (UCST) type thermosensitive polymers precipitate at low temperatures and dissolve at high temperatures. They can be used for low-temperature enzyme immobilization and high-temperature enzyme recovery, and have important application value in biocatalytic systems. However, existing UCST type thermosensitive polymer carriers are mainly based on synthetic polymers, such as polyacrylamide (PAM), poly N-isopropylacrylamide (PNIPAM) and its derivatives (Mól, Paula Chequer Gouveia, Lizzy Ayra Veríssimo, Luis Antonio Minim. Adsorption and immobilization of β-glucosidase from thermoascusaurantiacus on macroporous cryogel by hydrophobic interaction [J]. Preparative Biochemistry & Biotechnology, 2023, 53: 297-307.). Although these synthetic polymer materials have thermosensitive properties, they have significant defects such as non-renewable, poor biocompatibility, and poor degradability, which makes it difficult to meet the needs of green biocatalysis. In contrast, natural lignin (Lignin) is a rich, renewable, and environmentally friendly biomass material with far superior biocompatibility, degradability, and sustainability than synthetic polymer materials. Therefore, the development of UCST-type thermosensitive copolymers based on lignin, combined with the environmental friendliness of natural lignin and the intelligent response characteristics of UCST materials, can provide a greener and more sustainable enzyme immobilization carrier.

[0005] Jiang et al. successfully immobilized His-tagged adenylate cyclase (HaAC) by surface functionalized lignin-based sub-micro hybrid particles (LS-G-NTA-Ni) (Jiang, Dahai, Ting Xu, Honggang Xiao. Fabrication of lignin-based sub-micro hybrid particle as a novel support for adenylate cyclase immobilization [J]. Colloids and Surfaces B: Biointerfaces, 2024, 233: 113658.). The covalent binding method used in the literature requires multiple steps of chemical modification, including APTES treatment, the introduction of glutamate (GA) and NTA, and nickel ions (Ni 2 +). These steps not only increase the complexity of the operation, but also increase the cost of the immobilization process, limiting its feasibility in large-scale industrial applications. At the same time, although the immobilized HaAC maintained a high activity under optimized conditions, its activity decreased significantly (54.15%) after repeated use, indicating that the immobilization process may have a certain impact on the structure and activity of the enzyme.

[0006] Acryloyl glycinamide (NAGA) grafted enzyme immobilization carrier utilizes its unique thermosensitive properties (UCST) to immobilize the enzyme on the polymer by physical or chemical methods, achieving reversible dissolution and precipitation of the enzyme, thereby simplifying the enzyme recovery and reuse process. Chen et al. designed and synthesized a NAGA-based UCST-type thermosensitive polymer p (NAGA-b-VBA), immobilized horseradish peroxidase (HRP) through affinity reaction, and used it to degrade phenol in simulated wastewater (Chen, Zhaohui, Jiacong Wu, Wenrui Huang. A recyclable UCST-type biocatalyst to catalyze H2O2degradation of phenol [J]. Journal of Environmental Chemical Engineering, 2023, 11: 109072.). After multiple cycles of use, the activity of the immobilized enzyme of the thermosensitive polymer P (NAGA-b-VBA) in the literature decreased significantly, indicating that it still has shortcomings in terms of the number of cycles and stability. After five cycles of use, the activity of the immobilized enzyme was only 74%, and after being stored at 4°C for 5 weeks, the relative activity dropped to 41.3%. This may be because the binding force between the enzyme and the polymer is not sufficient to resist the mechanical and chemical effects in multiple cycles, resulting in reduced enzyme activity. Summary of the invention

[0007] The purpose of the present invention is to provide a thermosensitive lignin-based copolymer and a preparation method thereof and an application thereof in enzyme immobilization. The present invention synthesizes a thermosensitive lignin-based copolymer by grafting NAGA having UCST properties onto lignin molecules, and successfully uses the thermosensitive lignin-based copolymer as a carrier to immobilize enzymes, enhances the binding force between the enzyme and the carrier through covalent bonds, exhibits good stability and reusability under mild conditions, and improves the service life of the immobilized enzyme.

[0008] The technical solution for achieving the purpose of the present invention is as follows:

[0009] The preparation method of the temperature-sensitive lignin-based copolymer comprises the following steps:

[0010] (1) dissolving lignin and NAGA in dimethyl sulfoxide (DMSO) to form a mixed solution, and then subjecting the solution to ultrasonic-vacuum treatment to remove gas;

[0011] (2) dissolving the initiator azobisisobutyronitrile (AIBN) in DMSO and performing ultrasonic-vacuum treatment to remove gas;

[0012] (3) AIBN solution was added to the mixed solution in a ratio of lignin, NAGA, AIBN and DMSO of 60-120 mg: 480-540 mg: 6.22 mg: 20 mL, and the polymerization reaction was carried out in a water bath at 70°C for 1 to 12 h. After the reaction, the mixture was precipitated with cold methanol, centrifuged, washed and dried to obtain a thermosensitive lignin-based copolymer with UCST properties.

[0013] Furthermore, in step (3), the water bath temperature is 70° C. and the reaction time is 3 h.

[0014] Furthermore, in step (3), the drying temperature is 40 to 50° C., and the drying time is 12 to 24 hours.

[0015] The present invention provides a temperature-sensitive lignin-based copolymer prepared by the above preparation method.

[0016] Furthermore, the present invention provides the use of the above-mentioned temperature-sensitive lignin-based copolymer in enzyme immobilization.

[0017] Furthermore, the enzyme includes but is not limited to β-glucosidase, cellulase, xylanase and the like enzymes.

[0018] Specifically, the application method is as follows: dissolving the thermosensitive lignin-based copolymer in a buffer solution, adding epichlorohydrin (ECH) for activation, after complete activation, removing the supernatant by centrifugation, washing the precipitate with water, adding enzyme and buffer solution, carrying out an immobilization reaction, and obtaining an immobilized enzyme.

[0019] Furthermore, the buffer is a citrate buffer or a phosphate buffer.

[0020] Furthermore, the activation reaction conditions are as follows: the mass ratio of the thermosensitive lignin-based copolymer to ECH is 1:2, and the buffer solution pH=8.

[0021] Furthermore, the enzyme is β-glucosidase, and the immobilization reaction conditions are: buffer pH = 5, immobilization reaction temperature is 4°C, immobilization time is 3h, the mass ratio of the activated thermosensitive lignin-based copolymer, buffer, and enzyme is 10:1000:2-10, and the β-glucosidase concentration is 4mg / mL.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) Based on lignin, the present invention introduces NAGA with UCST thermosensitive properties, and obtains a thermosensitive lignin-based copolymer through copolymerization.

[0024] (2) The present invention uses a thermosensitive lignin-based copolymer as a carrier material. Since lignin is a natural, renewable and low-cost bio-based material with abundant functional groups (such as hydroxyl, carboxyl and methoxy), it can firmly fix the enzyme by covalent bonds, has good physical and chemical stability, can provide protection for the enzyme molecules, and enhance the stability of the enzyme at different temperatures and pH. In addition, its thermosensitive properties can realize temperature-responsive enzyme recovery, improving the existing problems of low enzyme utilization, high cost, and poor enzyme stability.

[0025] (3) The thermosensitive lignin-based copolymer immobilized β-glucosidase prepared by the present invention exhibits good durability in a complex reaction environment, reduces enzyme inactivation, has good recycling performance, and still maintains 60% of the enzyme activity after 5 cycles, thereby increasing the service life of the immobilized enzyme, reducing production costs, and is suitable for industrial production applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the Fourier transform infrared spectroscopy (FT-IR) analysis of the temperature-sensitive lignin-based copolymer in Example 2.

[0027] Figure 2 This is the dynamic light scattering (DLS) analysis of the thermosensitive lignin-based copolymer in Example 2.

[0028] Figure 3 ] are the temperature response performance curves of the five thermosensitive lignin-based copolymers in Example 2.

[0029] Figure 4 The following are the effects of the five immobilization conditions on the immobilization effect in Example 3: (a) the effect of the amount of ECH added on the immobilization effect, (b) the effect of the activation pH on the immobilization effect, (c) the effect of the immobilization time on the immobilization effect, (d) the effect of the fixation temperature on the immobilization effect, and (e) the effect of the β-glucosidase concentration on the immobilization effect.

[0030] Figure 5 (a) pH stability and (b) thermal stability of the immobilized enzyme in Example 4.

[0031] Figure 6 This is the recyclability of the immobilized enzyme in Example 4.

[0032] Figure 7 (a) Optimal pH and (b) Optimal temperature of the immobilized enzyme and free enzyme in Example 5. DETAILED DESCRIPTION

[0033] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and in detail below in conjunction with specific embodiments and drawings, but the protection scope of the present invention is not limited to the following specific embodiments.

[0034] All technical terms used below have the same meanings as those commonly understood by those skilled in the art unless otherwise defined.

[0035] Unless otherwise specified, the reagents, raw materials, instruments and equipment used in the present invention can be purchased or prepared by existing methods.

[0036] Example 1: Preparation of thermosensitive lignin-based copolymers

[0037] In this example, different types of thermosensitive lignin-based copolymers were prepared under different synthesis conditions using an aqueous solution free radical polymerization method, which were labeled as LN-1, LN-2, LN-3, LN-4 and LN-5, respectively.

[0038] 1. Experimental Methods

[0039] In a 25mL flask, 60mg of lignin and 540mg of NAGA were dissolved in 19mL of DMSO; in another 10mL flask, 6.22mg of AIBN was dissolved in 1mL of DMSO. The liquids in the two flasks were first ultrasonicated for 10min, and then the escaping gas was extracted with a vacuum pump, and then ultrasonicated again for 10min, and the ultrasonic-vacuum was repeated 3 times until the excess gas was removed. Finally, the AIBN solution in the 10mL reaction bottle was injected into the 25mL flask with a syringe, and placed in a 70℃ water bath for polymerization, and the reaction time was controlled at 3h. The reaction mixture was cooled in an ice bath, precipitated in cold methanol, and centrifuged. After washing with methanol three times, it was dried to obtain the thermosensitive lignin-based copolymer LN-1.

[0040] The above method was used to adjust the addition amounts of lignin and NAGA. The specific experimental parameters are shown in Table 1. Thermosensitive lignin-based copolymers LN-2, LN-3, LN-4 and LN-5 were prepared respectively.

[0041] 2. Results Analysis

[0042] Table 1 shows the synthesis conditions and properties of five different types of thermosensitive lignin-based copolymers, LN-1, LN-2, LN-3, LN-4 and LN-5. According to Table 1, when the mass ratio of lignin to NAGA is 60:540, the yield of the copolymer is the highest, reaching 65.7%. After the temperature is reduced from 50°C to 4°C and stored for 24 hours, the self-recovery rates of copolymers with different ratios are 98.4%, 90.2%, 44.6%, 26.3% and 5.1%, respectively. Among them, when the mass ratio of lignin to NAGA is 60:540, the self-recovery rate of the copolymer is the highest. Further analysis shows that when the composition of the copolymer is fixed, as the molecular weight of NAGA increases, its temperature response performance becomes more sensitive, and the recovery rate of the copolymer also increases accordingly.

[0043] Table 1 Synthesis conditions and properties of thermosensitive lignin

[0044]

[0045] 3. Conclusion

[0046] According to the results in Table 1, the optimal synthesis mass ratio is lignin:NAGA=60:540.

[0047] Example 2: Structural characterization and thermosensitive performance analysis of thermosensitive lignin-based copolymers

[0048] In this example, the thermosensitive lignin-based copolymers LN-1, LN-2, LN-3, LN-4 and LN-5 were characterized and their thermosensitive properties were analyzed. The functional group composition was analyzed by Fourier transform infrared spectroscopy, the change of the hydration kinetic diameter (Rh) at different temperatures was determined by dynamic light scattering, and the temperature response performance was determined by ultraviolet visible spectroscopy (UV-Vis).

[0049] 1. Experimental Methods

[0050] (1) Fourier transform infrared spectroscopy: The structural properties of five thermosensitive lignin-based copolymers (LN-1, LN-2, LN-3, LN-4, and LN-5) were determined by infrared spectrometry using an infrared spectrometer (Shimadzu IRTracer 100, Japan). The infrared spectrometer was scanned with a scanning range of 400–4000 cm -1 , scanned 32 times.

[0051] (2) Dynamic light scattering: Using a nanoparticle size analyzer, the dynamic light scattering technique was used to measure the changes in the hydration kinetic diameter of the thermosensitive lignin-based copolymer at different temperatures to explore its temperature response behavior. First, the thermosensitive lignin-based copolymer was dissolved in deionized water to prepare a 20 mg / mL polymer solution, and the solution was ensured to be evenly dispersed. The experiment set two temperature conditions of 4°C and 50°C. Each sample was scanned three times at the corresponding temperature, and the average particle size change was recorded.

[0052] (3) Temperature response performance: The temperature response behavior of the thermosensitive lignin-based copolymer was measured using a UV-visible spectrophotometer. Five thermosensitive lignin-based copolymers LN-1, LN-2, LN-3, LN-4 and LN-5 were prepared into 10 mg / mL aqueous solutions. The absorbance of the five thermosensitive lignin-based copolymer aqueous solutions at a wavelength of 590 nm was measured in a temperature range of 0 to 50 °C with a temperature gradient of 5 °C. Repeat the steps three times and take the average absorbance.

[0053] 2. Results Analysis

[0054] Figure 1 Fourier transform infrared spectroscopy analysis of thermosensitive lignin-based copolymers. Figure 1 It can be seen that the peaks of each characteristic group of the synthesized thermosensitive lignin-based copolymer, such as 1021cm -1 The peak at 1420cm is the stretching vibration peak of CO bond. -1 The peak at is the amino group (-NH 2 ) stretching vibration peak. 1568cm -1 The peak at 1658cm is the stretching vibration peak of the C=N bond. -1 The C=O bond stretching vibration peak of the amide group appeared at 2930cm. This peak was not observed in lignin, but was found in other synthesized thermosensitive lignin-based copolymers, indicating that lignin may react with NAGA. -1 The peak at 3424cm is the CH stretching vibration peak of the methyl group. -1 The peak at is the stretching vibration peak of the hydroxyl (-OH) in lignin. Infrared spectroscopy analysis shows that chemical interactions occur between lignin and NAGA, which may involve amide bonds or other covalent bonds.

[0055] Figure 2 In order to analyze the dynamic light scattering of thermosensitive lignin-based copolymers, this paper uses DLS technology to measure the changes in the hydration kinetic diameter of thermosensitive lignin-based copolymers at different temperatures to explore their thermosensitive behavior. Figure 2 It can be seen that when the polymer is at 4°C, the thermosensitive lignin-based copolymer exhibits a larger Rh (1901nm). When the temperature of the thermosensitive lignin-based copolymer solution is higher than its upper critical solution temperature (UCST, 30°C), the polymer gradually dissolves in the aqueous solution to form smaller monodisperse particles. This is because at temperatures above the UCST, the hydrogen bonding between polymer chains is weakened, resulting in enhanced particle dispersibility and a decrease in Rh.

[0056] Figure 3 The temperature response performance curves of five thermosensitive lignin-based copolymers, LN-1, LN-2, LN-3, LN-4 and LN-5, are shown in Figure 2. Figure 3Analysis shows that at low temperatures (~10°C), the absorbance of all samples is close to 1.0, indicating that the polymer is in an insoluble state; as the temperature rises to 30-35°C, the absorbance decreases rapidly, indicating that the polymer gradually dissolves. UCST can be determined by the absorbance mutation point in the heating curve, which is about 30-35°C. At this time, the UCST of the optimal synthesis ratio LN-1 is 30°C. In addition, by comparing the UCST of lignin synthesized with different lignin addition amounts, it can be seen that with the increase in lignin addition, the UCST of the synthesized thermosensitive lignin-based copolymer increases slightly. This may be due to the increase in the content of lignin, which makes the formed thermosensitive lignin-based copolymer require a higher temperature to be completely dissolved. During the cooling process, the absorbance of the solution rises, indicating that the polymer re-precipitates, but it precipitates at around 10°C, which is lower than the UCST, showing a certain hysteresis effect.

[0057] 3. Conclusion

[0058] The lignin-based copolymer with UCST thermosensitive properties was synthesized by this preparation method, and its temperature response performance was adjustable. The copolymer with the best ratio (lignin:NAGA=60:540) showed reversible solubility at 30℃.

[0059] Comparative Example 1

[0060] The preparation method of LN-1 in this comparative example is roughly the same as that in Example 1, except that the amount of DMSO added is 10 mL and 30 mL respectively. Under this ratio condition, thermosensitive lignin cannot be synthesized.

[0061] Comparative Example 2

[0062] The preparation method of LN-1 in this comparative example is substantially the same as that in Example 1, except that the reaction temperatures are 60° C. and 80° C. respectively. Under this reaction temperature condition, thermosensitive lignin cannot be synthesized.

[0063] Example 3: Optimization of conditions for immobilizing β-glucosidase on thermosensitive lignin-based copolymers

[0064] This example introduces the synthesis method of immobilized enzyme, and measures the effects of five factors, namely, the amount of ECH added, activation pH, immobilization time, immobilization temperature, and initial enzyme concentration, on the immobilized enzyme. While measuring the immobilized loading, the residual enzyme specific activity after immobilization was also measured to further optimize the immobilization conditions. The optimal immobilization conditions were selected by comprehensively considering the immobilized loading and the residual enzyme specific activity.

[0065] 1. Experimental Methods

[0066] (1) Preparation of thermosensitive lignin-based copolymer-immobilized β-glucosidase

[0067] Weigh 10 mg of the thermosensitive lignin-based copolymer LN-1 prepared in Example 1, dissolve it in 1 mL of sodium phosphate buffer, add a certain amount of ECH and mix well, and place it in a metal bath to react for a period of time. After the reaction is completed, centrifuge at 4°C and 6000rpm for 10 minutes, remove the supernatant, obtain the activated carrier, and wash the precipitate three times with deionized water to remove the unreacted ECH. Then, add a certain amount of β-glucosidase and citrate buffer. After the mixed solution is placed in a metal bath for a period of time, centrifuge at 4°C and 6000rpm for 10 minutes. Remove the supernatant, wash the precipitate three times with deionized water, and then resuspend it in sodium citrate buffer to obtain an immobilized enzyme. The immobilized enzyme is called LN-βG (lignin-β-glucosidase).

[0068] (2) Optimization of immobilization conditions of β-glucosidase immobilized on thermosensitive lignin-based copolymers

[0069] In order to optimize the immobilization conditions and improve the immobilized enzyme loading and residual enzyme specific activity, the effects of ECH addition amount, activation pH, immobilization time, immobilization temperature and initial enzyme concentration were systematically investigated.

[0070] First, the effect of ECH addition on the immobilization effect was studied. The amount of ECH added was adjusted to 5, 10, 15, 20, and 25 mg, and the immobilization amount and residual enzyme specific activity under different ECH additions were measured to determine the optimal ECH addition amount. Subsequently, the effect of pH on the immobilization process was explored. The pH of the sodium phosphate buffer was adjusted to 4, 5, 6, 7, and 8, and the immobilization amount and residual enzyme specific activity under different pH conditions were measured to determine the optimal activation pH.

[0071] In the immobilization time optimization experiment, the polymer solution and enzyme solution were mixed and fixed for 1h, 3h, 6h, 9h, and 12h, respectively, and the immobilization amount and residual enzyme specific activity at each time point were measured to screen the optimal immobilization time. At the same time, the effect of immobilization temperature on enzyme immobilization was investigated. The mixed solution was placed at 4°C, 15 ℃ The immobilization was carried out in a constant temperature oscillator at 25℃, 30℃ and 50℃, and the immobilization amount and residual enzyme specific activity under different temperature conditions were measured to determine the optimal immobilization temperature.

[0072] Finally, by optimizing the initial enzyme concentration, the amount of β-glucosidase added in the immobilized system was adjusted to make the final enzyme concentrations of 2, 4, 6, 8, and 10 mg / mL, and the immobilized capacity and residual enzyme specific activity at different enzyme concentrations were measured to determine the optimal initial enzyme concentration.

[0073] 2. Results Analysis

[0074] Figure 4(a) The effect of ECH addition on immobilized LN-βG. Figure 4 (a) Analysis shows that when the added ECH is 5 mg (i.e., the mass ratio of lignin and ECH is 2:1), the solid loading is 26.7 mg / g. When the amount of ECH added is increased to 20 mg (i.e., the mass ratio of lignin and ECH is 1:2), the solid loading reaches the best, which is 31.4 mg / g. As the amount of ECH added continues to increase, the solid loading increases accordingly, but the enzyme specific activity at this time decreases from 282.4 U / mg to 210.2 U / mg. This is because ECH has a certain toxicity to proteins, which will cause the enzyme to lose some activity, thereby reducing the enzyme specific activity. After considering factors such as the solid loading amount and the enzyme specific activity, the condition of 20 mg of ECH added (i.e., the mass ratio of lignin and ECH is 1:2) was selected for the immobilized enzyme implementation.

[0075] Figure 4 (b) The effect of activation pH on immobilized LN-βG. Figure 4 (b) Analysis shows that as the pH increases, the solid loading increases, and reaches a maximum value of 34.1 mg / g at pH = 8. At this time, the enzyme specific activity decreases from 292.9 U / mg to 240.0 U / mg. This is because under alkaline conditions, the epoxy group is easily attacked, and the etherification reaction between the alkyl halide and the alcohol hydroxyl group makes it easier for the immobilization reaction to proceed, thereby increasing the solid loading. At this time, there is no obvious trend in the enzyme specific activity. After considering factors such as the solid loading amount and the enzyme specific activity, the pH = 8 condition was selected for the immobilized enzyme implementation.

[0076] Figure 4 (c) The effect of immobilization time on immobilized LN-βG. Figure 4 (c) Analysis shows that under the five fixed time conditions of 1h, 3h, 6h, 9h, and 12h, the corresponding solid loadings are 31.4mg / g, 34.3mg / g, 32.9mg / g, 32.5mg / g, and 32.9mg / g, respectively. The solid loading reaches the highest level of 34.3mg / g when the fixed time is 3h. After the fixed time increases from 1h to 12h, the enzyme specific activity decreases from 292.9U / mg to 203.0U / mg. It is speculated that the enzyme loses some activity due to the long fixed time. After considering factors such as solid loading and enzyme specific activity, the fixed time of 3h was selected for the implementation of immobilized enzyme.

[0077] Figure 4 (d) The effect of immobilization temperature on immobilized LN-βG. Figure 4(d) Analysis shows that under the five fixed temperature conditions of 4°C, 15°C, 25°C, 35°C, and 50°C, the corresponding immobilized capacities are 28.5 mg / g, 25.5 mg / g, 28.4 mg / g, 28.4 mg / g, and 26.5 mg / g, respectively, and there is no obvious trend in the overall immobilized capacities. Under the five fixed temperature conditions, the corresponding enzyme specific activities are 275.3 U / mg, 273.3 U / mg, 234.5 U / mg, 238.2 U / mg, and 216.7 U / mg, respectively. When the fixed temperature is 4°C, the enzyme specific activity of the immobilized enzyme is the highest, and the enzyme is more able to maintain activity at low temperatures. After considering factors such as the immobilized capacities and the enzyme specific activity, the fixed temperature of 4°C is selected for the implementation of the immobilized enzyme.

[0078] Figure 4 (e) The effect of the added β-glucosidase concentration on the immobilized LN-βG. Figure 4 (e) Analysis shows that within the range of β-glucosidase concentration of 2 to 10 mg / mL, as the β-glucosidase concentration increases, the solid loading reaches 34.2 mg / g at a β-glucosidase concentration of 4 mg / mL, and reaches 34.4 mg / g at a β-glucosidase concentration of 6 mg / mL. However, as time continues to increase, the solid loading remains almost unchanged. This is because the reaction of the thermosensitive lignin-based copolymer with β-glucosidase reaches saturation. At this time, there is no obvious trend in the enzyme specific activity. After considering factors such as the solid loading amount and the enzyme specific activity, the condition of a β-glucosidase concentration of 4 mg / mL was selected for the immobilized enzyme implementation.

[0079] 3. Conclusion

[0080] The optimal conditions for activating the thermosensitive lignin-based copolymers were 20 mg of ECH addition (i.e., the mass ratio of lignin to ECH was 1:2) and pH 8. The optimal conditions for immobilizing the enzyme were 4 °C temperature, 3 h immobilization time, and 4 mg / mL β-glucosidase concentration.

[0081] Example 4: pH, thermal stability and recyclability of thermosensitive lignin-based copolymer immobilized βG

[0082] This example compares the differences between the immobilized enzyme and the free enzyme in terms of pH stability, thermal stability and recyclability to evaluate the stability and application potential of the thermosensitive lignin-based copolymer immobilized β-glucosidase (LN-βG).

[0083] 1. Experimental Methods

[0084] (1) Preparation of Thermosensitive Lignin-Based Copolymer Immobilized βG Refer to Example 3.

[0085] (2) Determine the pH stability of the immobilized enzyme and the free enzyme. Specifically, prepare 50 mM citrate buffers of pH 4, pH 5, pH 6, pH 7, and pH 8 at 30°C, and measure the initial enzyme activities of the immobilized enzyme and the free enzyme at 30°C. After the sample is left to stand for 3 h, the residual enzyme activity is measured, and the relative enzyme activity is calculated using the initial enzyme activity as the benchmark (100%).

[0086] (3) Determine the thermal stability of the immobilized enzyme and the free enzyme, specifically: perform enzyme activity determination at five different temperature conditions: 30°C, 40°C, 50°C, 60°C, and 70°C. After the enzyme sample is preheated to the target temperature, the initial enzyme activity is measured, and then the residual enzyme activity is measured again after standing for 3 hours, and the relative enzyme activity is calculated to evaluate the heat resistance of the immobilized enzyme and the free enzyme.

[0087] (4) Determine the recyclability of the immobilized enzyme and the free enzyme. Specifically, the immobilized enzyme was centrifuged and washed after each round of hydrolysis. It was then redissolved in 1 mL of citrate buffer and supplemented with the same substrate for the next cycle. The hydrolysis reaction was continued for 8 times, and the initial enzyme activity was defined as 100%. The residual enzyme activity after each round of reaction was determined to evaluate the recyclability of the immobilized enzyme.

[0088] 2. Results Analysis

[0089] Figure 5 (a) pH stability analysis of immobilized enzyme and free enzyme. Figure 5 (a) Analysis shows that the immobilized enzyme exhibits high activity in the pH range of 4 to 8, especially in weakly alkaline conditions (pH 7 to 8), where it can still maintain more than 50% activity. The stability of the free enzyme is poor, and it only maintains a certain activity under pH 4 to 6 conditions. In a slightly alkaline environment (pH 7 to 8), the activity drops significantly, and only retains less than 30% activity. This shows that the immobilization process effectively improves the stability of the enzyme in a wider pH range, allowing it to maintain a higher catalytic efficiency in a wider range of reaction environments.

[0090] Figure 5 (b) Thermal stability analysis of immobilized enzyme and free enzyme. Figure 5 (b) Analysis shows that the activity of the immobilized enzyme remains above 80% in the temperature range of 30°C to 50°C, showing good thermal stability. Even at high temperature (70°C), it still retains 28% of its activity, while the activity of the free enzyme decreases significantly after exceeding 50°C, leaving only 6% of its activity at 70°C. The results show that the immobilization process significantly improves the heat resistance of the enzyme, allowing it to still have good catalytic performance at higher temperatures, which is conducive to its application in a wider temperature range.

[0091] Figure 6To verify the recyclability of the immobilized enzyme and the free enzyme, the residual enzyme activity of the immobilized LN-βG after eight cycles was determined. Figure 6 Analysis shows that immobilized LN-βG still maintains 60% of its enzyme activity after 5 cycles. The decrease in enzyme activity with increased reuse may be caused by multiple factors, including partial loss of immobilized enzyme material, decreased activity due to structural changes in the enzyme during multiple uses, and possible influence of substrate diffusion. However, compared with the characteristic that free enzymes cannot be recycled and reused, immobilized enzymes can be recycled many times, which significantly improves reusability and reduces the cost of enzyme use.

[0092] 4. Conclusion

[0093] The pH stability, thermal stability and recycling performance of immobilized LN-βG were significantly better than those of free enzyme, and the immobilized LN-βG still maintained 60% of the enzyme activity after 5 cycles.

[0094] Example 5: Optimal pH and temperature for immobilization of βG by thermosensitive lignin-based copolymers

[0095] The purpose of this example is to compare the optimal pH and temperature of the immobilized enzyme and the free enzyme, so as to evaluate the pH adaptability and temperature adaptability of the thermosensitive lignin-based copolymer immobilized β-glucosidase (LN-βG).

[0096] 1. Experimental Methods

[0097] (1) Preparation of Thermosensitive Lignin-Based Copolymer Immobilized βG Refer to Example 3.

[0098] (2) For the optimal pH of the immobilized enzyme, five 50 mM citrate buffer solutions with different pH values, pH 4, pH 5, pH 6, pH 7, and pH 8, were prepared at 30°C. The immobilized enzyme was dissolved in solutions of different pH values ​​and the enzyme activity was measured. The highest enzyme activity obtained during the implementation was taken as the benchmark of 100%, and the relative enzyme activity was calculated.

[0099] (3) For the optimal temperature of the immobilized enzyme, the activity of the immobilized enzyme was measured at five different temperatures: 30°C, 40°C, 50°C, 60°C, and 70°C. The highest enzyme activity obtained during the implementation was taken as the benchmark 100%, and the relative enzyme activity was calculated.

[0100] 2. Results Analysis

[0101] Figure 7 (a) is the optimal pH study result of immobilized enzyme and free enzyme. Figure 7(a) Analysis showed that the optimum pH for free β-glucosidase and LN-βG was 5. However, at lower and higher pH, such as pH 4 and pH 8, the enzyme activity of LN-βG was higher than that of free β-glucosidase, which may be due to the fact that the immobilization process may form a stable enzyme-carrier interaction, reducing the direct impact of extreme pH conditions on the enzyme active center.

[0102] Figure 7 (b) is the optimal temperature study result of immobilized enzyme and free enzyme. Figure 7 (b) Analysis shows that the optimum temperature of free β-glucosidase and LN-βG is 50°C. However, when the temperature is low or high, the enzyme activity of LN-βG is higher than that of free β-glucosidase. This phenomenon indicates that the immobilization process may improve the thermal stability and low temperature adaptability of the enzyme, enabling it to maintain high activity in a wider temperature range. The possible mechanisms include the conformational changes of the immobilized restriction enzyme, the reduction of thermal denaturation, and the protective microenvironment provided by the carrier, which reduces the effect of temperature on enzyme activity.

[0103] 3. Conclusion

[0104] The experimental results showed that the optimal pH of the immobilized enzyme was 5 and the optimal temperature was 50°C. In addition, compared with the free enzyme, the immobilized LN-βG showed higher catalytic activity in a wider pH and temperature range, proving the enhancement effect of the immobilization process on the enzyme stability.

[0105] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention as long as they do not depart from the technical solution of the present invention.

Claims

1. A method for preparing a thermosensitive lignin-based copolymer, characterized in that: The following steps are involved: (1) Dissolving lignin and NAGA in DMSO to form a mixed solution, and then subjecting the solution to ultrasonic-vacuum treatment to remove gas; (2) Dissolve the initiator AIBN in DMSO and perform ultrasonic-vacuum treatment to remove gas; (3) The AIBN solution was added to the mixed solution in a ratio of lignin, NAGA, AIBN and DMSO of 60-120 mg: 480-540 mg: 6.22 mg: 20 mL, and the polymerization reaction was carried out in a water bath at 70 °C for 1-12 h. After the reaction, the mixture was precipitated with cold methanol, centrifuged, washed and dried to obtain a thermosensitive lignin-based copolymer with UCST properties.

2. The preparation method according to claim 1, characterized in that In step (3), the water bath temperature is 70°C and the reaction time is 3 h.

3. The preparation method according to claim 1, characterized in that In step (3), the drying temperature is 40-50°C and the drying time is 12-24 h.

4. The thermosensitive lignin-based copolymer prepared according to the preparation method according to any one of claims 1 to 3.

5. Use of the hunger temperature-sensitive lignin-based copolymer according to claim 4 in enzyme immobilization.

6. The use according to claim 5, characterized in that: The enzyme is β-glucosidase, cellulase or xylanase.

7. The use according to claim 5, characterized in that: The application method is as follows: dissolving the thermosensitive lignin-based copolymer in a buffer solution, adding ECH for activation, removing the supernatant by centrifugation after complete activation, washing the precipitate with water, adding enzyme and buffer solution, carrying out an immobilization reaction, and obtaining an immobilized enzyme.

8. The use according to claim 7, characterized in that: The buffer is citrate buffer or phosphate buffer.

9. The use according to claim 7, characterized in that: The activation reaction conditions were as follows: the mass ratio of the thermosensitive lignin-based copolymer to ECH was 1:2, and the buffer solution pH was 8.

10. The use according to claim 7, characterized in that The enzyme was β-glucosidase, and the immobilization reaction conditions were: buffer pH = 5, immobilization reaction temperature of 4°C, immobilization time of 3 h, the mass ratio of activated thermosensitive lignin-based copolymer, buffer, and enzyme was 10:1000:2~10, and the β-glucosidase concentration was 4 mg / mL.

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