High-temperature-resistant immobilized cutinase degradation promoter, and preparation method and application thereof

Abstract

This invention provides a high-temperature resistant immobilized keratinase degradation accelerator, its preparation method, and its application, relating to the field of biodegradable materials. The method includes: grafting and modifying cellulose nanocrystals with polyester and / or polyester-polyether block copolymers at a mass ratio of 2:1-3:1, then immobilizing them with keratinase at a mass ratio of 10:1-15:1 through physical adsorption, followed by freeze-drying to obtain the accelerator. This invention utilizes grafted and modified cellulose nanocrystals as a carrier to immobilize keratinase, effectively improving the enzyme's heat resistance and compatibility with the polyester matrix. After heat treatment at 160 °C, the activity retention rate is not less than 70%. When this accelerator is melt-blended with PBS masterbatch, its degradation efficiency under composting conditions (55 °C, 50% humidity) reaches over 11% / d, far exceeding the 1.82% / d of pure PBS, achieving controllable and complete degradation of polyester materials and demonstrating promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biodegradable materials technology, specifically relating to a high-temperature resistant immobilized keratinase degradation promoter for degradable plastics and its preparation method, as well as the application of the promoter in the preparation of controllable degradable polybutylene succinate composite materials. Background Technology

[0002] In recent years, biodegradable materials, such as polylactic acid (PLA), polybutylene adipate / terephthalate (PBAT), and polybutylene succinate (PBS), have gradually increased their competitiveness and are widely used in packaging materials, agricultural and forestry films, and biomedicine, becoming ideal alternatives that balance environmental protection and practicality. However, with development, biodegradable materials still face challenges in effectively degrading in the natural environment. This is mainly due to the scarcity of specific degrading enzymes, their low enzyme activity, and their stringent environmental requirements, necessitating industrial composting. Consequently, they cannot completely replace traditional non-biodegradable materials in practical applications. Therefore, designing biodegradable materials that can adapt to different environments and degrade efficiently is one effective way to address this problem.

[0003] "Enzyme-embedded" polymer systems refer to polymers incorporating specific enzymes to effectively enhance the degradation rate and completeness of materials in natural environments such as soil and water, while ensuring controllable degradation cycles. Currently, enzyme-embedded methods are divided into melt blending and solvent casting. The former is suitable for commercial extrusion and large-scale production processes, but faces the problem of enzyme inactivation during high-temperature processing; the latter, while avoiding high-temperature heating, suffers from a lack of suitable solvents and poor enzyme dispersibility in the polymer. Therefore, there is an urgent need to develop a highly heat-resistant enzyme-embedded degradation promoter to achieve effective control over the degradation cycle of a wide range of degradable materials.

[0004] Currently, enzyme heat resistance modification technologies are mainly divided into two categories: protein engineering modification and immobilization modification. Protein engineering modification improves the intrinsic heat resistance of enzymes through gene mutation, but it suffers from problems such as poor expression stability, long experimental cycles, and high costs. Enzyme immobilization technology refers to using chemical or physical methods to immobilize enzymes on natural polymers or composite carriers, thereby improving the enzyme's thermal stability, acid-base stability, and facilitating enzyme recycling. However, existing immobilization technologies mainly target the modification of enzyme heat resistance in aqueous environments, with heat resistance temperatures generally below 100℃, which is insufficient to meet the requirements of plastic melt processing (typically 120-200℃).

[0005] In recent years, a few studies have attempted to use immobilized enzymes in plastic melt processing. For example, patent CN115895046B discloses a biodegradation promoter that uses cellulose nanocrystals to immobilize lipases. This lipase retains some degradation activity after melt extrusion at 80°C and hot pressing at 150°C. However, this technology did not evaluate the enzyme's activity retention rate, and its heat resistance is insufficient for processing at higher temperatures (e.g., above 160°C). Another example is patent CN118580659A, which uses a metal-organic framework (MAF-7) to in-situ encapsulate proteinase K or lipases. While this improves the enzyme's thermal stability, the overly tight encapsulation makes it difficult for the enzyme to contact the polymer substrate in the degradation environment, leading to activation difficulties and limited degradation efficiency. It is evident that existing technologies face a dilemma: on the one hand, if a mild immobilization method (such as physical adsorption) is used, the enzyme is easily deactivated during high-temperature processing, resulting in insufficient heat resistance; on the other hand, if a tight encapsulation protection method (such as metal-organic framework embedding) is used, although heat resistance can be improved, the excessively tight encapsulation makes it difficult for the enzyme to contact the substrate in the degradation environment, making activation difficult and limiting degradation efficiency.

[0006] Therefore, how to achieve a balance between protecting the enzyme during high-temperature processing and rapidly activating the enzyme in the degradation environment is a technical challenge that urgently needs to be solved in the field of embedded enzyme technology. Summary of the Invention

[0007] One of the objectives of this invention is to provide a method for preparing a high-temperature resistant immobilized keratinase degradation promoter. This method involves grafting cellulose nanocrystals with polyester and / or polyester-polyether block copolymers, immobilizing the keratinase on the surface of the modified carrier using physical adsorption, and then freeze-drying to obtain the promoter. The preparation process is mild and controllable, and suitable for industrial production.

[0008] The second objective of this invention is to provide a high-temperature resistant immobilized keratinase degradation promoter prepared by the above-mentioned method. In the promoter, the keratinase is firmly loaded on a grafted modified cellulose nanocrystal carrier and maintains a high activity retention rate after high-temperature melt blending processing, exhibiting good thermal stability and storage stability.

[0009] The third objective of this invention is to provide an application of the above-mentioned high-temperature resistant immobilized keratinase degradation promoter in the preparation of controllable degradation polybutylene succinate composite material. By melt-blending the promoter with polybutylene succinate masterbatch, the degradation efficiency of the composite material under composting conditions is significantly improved, thereby achieving controllable and complete degradation of the polyester material.

[0010] One of the technical solutions adopted by this invention to achieve its objective is: providing a method for preparing a high-temperature resistant immobilized keratinase degradation promoter, comprising the following steps: S1. Cellulose nanocrystals are mixed with polyester and / or polyester-polyether block copolymers at a mass ratio of 2:1-3:1, and grafting reaction is carried out under heating conditions. After separation and purification, grafted modified cellulose nanocrystals are obtained. S2. Disperse the grafted modified cellulose nanocrystals in water, adjust the pH to 3-9, add the keratinase according to the mass ratio of grafted modified cellulose nanocrystals to keratinase of 10:1-15:1, and obtain an immobilized keratinase mixture through physical adsorption and immobilization reaction. S3. Freeze-dry the immobilized keratinase mixture to obtain a high-temperature resistant immobilized keratinase degradation promoter.

[0011] The overall concept of this invention is as follows: This invention provides a high-temperature resistant immobilized keratinase degradation promoter, which utilizes cellulose nanocrystals grafted with polyester and / or polyester-polyether block copolymers to immobilize keratinase. This feature simultaneously brings three technical advantages: First, the grafting modification improves the hydrophobicity of the cellulose nanocrystals, enhancing the coating rate of the keratinase, and the immobilized keratinase maintains a high activity retention rate after high-temperature melt blending processing; Second, the polyester grafted segments effectively improve the interfacial compatibility between the immobilized enzyme and the polyester matrix resin, which is beneficial to the uniform dispersion of the enzyme in the matrix and avoids agglomeration; Third, this immobilized enzyme degradation promoter is easily activated under composting conditions and can effectively catalyze the controllable and complete degradation of the polyester matrix under appropriate temperature and humidity conditions. Based on the above three synergistic effects, this invention can simultaneously overcome the technical problems of significant reduction in enzyme activity after high-temperature processing and low degradation efficiency in natural environments in the prior art.

[0012] In this invention, the mass ratio of cellulose nanocrystals to polyester and / or polyester-polyether block copolymers is controlled within the range of 2:1 to 3:1. This is determined based on the matching relationship between the content of active groups (hydroxyl and carboxyl groups) on the surface of cellulose nanocrystals and the molar ratio of grafted chains. If the proportion of cellulose nanocrystals is too high (i.e., higher than 3:1), multiple active sites on the surface of a single cellulose nanocrystal are prone to cross-linking reactions with different grafted chains or grafted chains on different cellulose nanocrystals, forming a three-dimensional network structure of CNC-polyester-CNC. This structure reduces the dispersibility of the product and, due to steric hindrance, buries enzyme binding sites, which is detrimental to the effective loading and activity maintenance of the enzyme. If the proportion of cellulose nanocrystals is too low (i.e., lower than 2:1), a large number of grafted chains in the system fail to bond with the active groups on the surface of the cellulose nanocrystals and are removed during subsequent purification and elution, resulting in waste of raw materials. Therefore, limiting the ratio to the range of 2:1 to 3:1 ensures both the efficient progress of the grafting reaction and good product dispersibility, while avoiding the formation of a network structure and waste of raw materials.

[0013] Furthermore, the ratio of grafted modified cellulose nanocrystals to keratinase affects the enzyme's adsorption capacity and efficiency. When the amount of polyester-grafted modified cellulose nanocrystals is small (e.g., a mass ratio of 5:1), the enzyme loading per unit mass of carrier increases. However, since keratinase is sensitive to pH, excessively high local enzyme concentrations can easily lead to enzyme molecule aggregation and conformational changes, which is detrimental to maintaining enzyme activity. This invention controls the mass ratio of grafted modified cellulose nanocrystals to keratinase to be 10:1-15:1. Within this range, the carrier can provide sufficient adsorption sites to achieve effective loading of keratinase. Experiments have verified that the adsorption efficiency is highest when the mass ratio is 10:1, and the polyester grafting layer on the carrier surface can provide a better protective microenvironment for keratinase, improving its heat resistance and catalytic activity under high-temperature processing conditions.

[0014] In this invention, keratinase is used as the degradation active component, which is immobilized on a polyester-grafted modified cellulose nanocrystal carrier to form an embedded enzyme system. Keratinase belongs to the carboxylesterase family of α / β hydrolases, and its catalytic unit is closer to the enzyme molecule surface, exhibiting stronger catalytic degradation efficiency for polyesters with high crystallinity and high melting points. Compared to ordinary lipases, keratinase has a higher optimal degradation temperature and better heat resistance, making it more suitable for the preparation of polyester degradation accelerators that require high-temperature melt blending.

[0015] Further, in step S1, before mixing the cellulose nanocrystals with the polyester, the cellulose nanocrystals are first dispersed in water, and then the water is replaced with one of acetone, toluene, or methanol through solvent replacement. The polyesters and / or polyester-polyether block copolymers used in this invention are mostly hydrophobic, making them difficult to dissolve and disperse uniformly in aqueous solutions. Directly carrying out the grafting reaction in an aqueous system leads to low reaction efficiency. Replacing the dispersion medium of the cellulose nanocrystals with one of acetone, toluene, or methanol through solvent replacement allows the cellulose nanocrystals to be better dispersed in the organic medium. Simultaneously, it facilitates sufficient contact between the hydrophobic polyester segments and the active groups on the surface of the cellulose nanocrystals, improving the efficiency of the grafting reaction and the uniformity of the grafted segments.

[0016] Furthermore, the grafting reaction is carried out at a heating temperature of 50-80 °C for 18-36 h. The surface of the cellulose nanocrystals is rich in active groups such as hydroxyl and carboxyl groups. During the grafting reaction, the hydroxyl or carboxyl groups at the ends of the polyester segments can undergo esterification or condensation reactions with the active groups on the surface of the cellulose nanocrystals, forming stable chemical bonds between them.

[0017] Further, the separation and purification process involves first separating and centrifuging to obtain an aqueous solution of grafted cellulose nanocrystals (CNC-g), then freeze-drying to obtain CNC-g lyophilized powder, and finally adding deionized water to obtain a CNC-g solution. Preferably, after centrifugation, purification is performed using solvents such as chloroform. This utilizes the characteristic that CNC-g is insoluble in chloroform but some polyesters and / or polyester-polyether block copolymers are soluble in chloroform to remove unreacted polyesters and / or polyester-polyether block copolymers.

[0018] Furthermore, the polyester and / or polyester-polyether block copolymer includes one or more of polybutylene succinate (PBS), polycaprolactone (PCL), and polybutylene succinate-polyethylene glycol block copolymer (PBS-b-PEG). In addition, the core of graft modification lies in improving the hydrophobicity of cellulose nanocrystals. On the one hand, this enhances the affinity between the carrier and the hydrophobic regions on the surface of the keratinase molecules, increasing the enzyme coating rate; on the other hand, it improves the interfacial compatibility between the cellulose nanocrystals and the polyester matrix resin, reducing interfacial energy and enhancing the dispersibility and stability of the immobilized enzyme during the blending stage. Based on the above principles, in addition to the polyesters and polyethers mentioned above, the grafted segments of the present invention can also be extended to linear and nonlinear polyester and polyether segments of single or multi-component blocks prepared by polycondensation of diacids and diols or ring-opening of lactones, such as polybutylene adipate terephthalate, polylactic acid and other biodegradable segments. All of these can improve the compatibility of blends and the stability of immobilized enzymes by regulating the hydrophilicity and hydrophobicity of the surface of cellulose nanocrystals.

[0019] Furthermore, the molecular weight of the polyester and / or polyester-polyether block copolymer is 1000-10000 g / mol. In this invention, the molecular weight of the polyester and / or polyester-polyether block copolymer determines the length of the graft chain, which in turn affects the immobilization efficiency, the protection efficiency of enzyme activity, and the compatibility during the blending stage. If the molecular weight is below 1000 g / mol, the graft chain is too short and cannot form an effective protective layer around the enzyme molecule, resulting in insufficient enzyme activity protection efficiency and limited improvement on the hydrophobicity of the cellulose nanocrystal surface, with little improvement in compatibility with the polyester matrix. If the molecular weight is above 10000 g / mol, the graft chain is too long, increasing the degree of freedom of molecular chain movement, which can easily generate steric hindrance during immobilization, reducing the immobilization efficiency. At the same time, excessively long graft chains may completely encapsulate the enzyme molecule, hindering the contact between the enzyme active site and the substrate, making it difficult to exert catalytic activity.

[0020] Furthermore, the grafting rate of the polyester and / or polyester-polyether block copolymer onto the cellulose nanocrystals is 60%-70%, where the grafting rate refers to the ratio of the mass of the polyester and / or polyester-polyether block copolymer grafted onto the cellulose nanocrystals to the mass of the cellulose nanocrystals before grafting. In this invention, the grafting rate is determined by thermogravimetric analysis, and the resulting grafted cellulose nanocrystals have a grafting rate as high as 60%-70%, resulting in better enzyme protection and correspondingly superior heat resistance and degradation performance under composting conditions.

[0021] Furthermore, the cellulose nanocrystals used in this invention have a length of 500 nm.

[0022] Further, in step S2, the pH is adjusted using NaOH or HCl solution. Preferably, the pH is 3-5. In this invention, the pH value affects the immobilization process. When pH = 3-5, the pH of the solution is lower than the isoelectric point of the keratinase, causing the keratinase to carry a positive charge, while CNC-g carries a negative charge under these conditions. The electrostatic effect effectively improves its immobilization efficiency, giving it higher heat resistance and enhanced catalytic degradation activity compared to other pH values ​​(such as pH = 6-9).

[0023] Furthermore, the temperature of the physical adsorption reaction is 0-10 °C, and the time is 4-12 h. Preferably, the physical adsorption reaction is carried out in an ice-water bath environment of 0-4 °C for 6-8 h.

[0024] The second objective of this invention is to provide a high-temperature resistant immobilized keratinase degradation promoter, prepared by the method described in the first objective of this invention. After heat treatment at 160 °C for 5 min, the keratinase activity retention rate of the high-temperature resistant immobilized keratinase degradation promoter is not less than 70%. Here, the keratinase activity retention rate refers to the ratio of the activity of the enzymatically hydrolyzed PBS after heat treatment to the activity before heat treatment.

[0025] In this invention, the keratinase activity retention rate is not less than 70%, indicating that the promoter has excellent heat resistance. This is due to the polyester-grafted modified cellulose nanocrystal carrier firmly loading the keratinase through hydrophobic interactions, while the polyester grafted segments on the carrier surface form a hydrophobic protective layer around the enzyme molecules, effectively inhibiting conformational changes of the enzyme molecules at high temperatures, thereby endowing it with excellent thermal stability.

[0026] The third objective of this invention is to provide the application of the high-temperature resistant immobilized keratinase degradation promoter described in the second objective of this invention in the preparation of controllably degradable polybutylene succinate composite materials, comprising: mixing the high-temperature resistant immobilized keratinase degradation promoter with polybutylene succinate masterbatch to obtain a premix; and melting and hot-pressing the premix at 120-140 °C to obtain an enzyme-PBS embedded composite material.

[0027] Preferably, the grafted segments used in preparing the degradation promoter have the same or similar molecular structure as the polyester matrix resin to improve the dispersibility of cellulose nanocrystals in the matrix and further enhance composting degradation efficiency. For example, when using polybutylene succinate (PBS) as the matrix, the PBS grafted segments have the same molecular structure as the substrate, and their compatibility is better than that of PCL grafted segments. This is more conducive to the uniform dispersion of cellulose nanocrystals in the matrix and promotes the efficient degradation of the substrate by enzymes under composting conditions.

[0028] Furthermore, the amount of the high-temperature resistant immobilized keratinase degradation promoter added accounts for 0.1 wt.%-3 wt.% of the polybutylene succinate masterbatch. Preferably, the amount added is 1-3 wt.%.

[0029] Furthermore, the hot pressing process conditions are as follows: preheating for 2-4 minutes, hot pressing for 0.5-2 minutes, and cold pressing for 3-6 minutes.

[0030] Furthermore, the embedded enzyme-PBS composite material is degraded under composting conditions of 55 °C and 50% humidity, with a degradation efficiency of not less than 10% / d.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The method for preparing the high-temperature resistant immobilized keratinase degradation promoter provided by the present invention uses cellulose nanocrystals and keratinase as the main raw materials. After grafting modification of CNC with polyester and / or polyester-polyether block copolymer, the keratinase is immobilized as a carrier. By utilizing the physical constraint of the carrier on the enzyme molecules and the multiple forces such as hydrogen bonds and hydrophobic interactions between the carrier and the enzyme, the thermal motion of the enzyme molecules at high temperature is effectively restricted, the tertiary structure of the enzyme is stabilized, and the heat resistance of the keratinase is significantly improved. The hydrophobic segments introduced by grafting form an amphiphilic protective microenvironment around the enzyme molecules, which further enhances the conformational stability of the immobilized enzyme under high temperature processing conditions. After heat treatment at 160℃ for 5 min, the activity retention rate of the keratinase can still reach more than 77%, which is much higher than that of the ungrafted modified immobilized enzyme (23.86%) and the free enzyme.

[0032] (2) The high-temperature resistant immobilized keratinase degradation promoter provided by this invention effectively improves the interfacial compatibility between the immobilized enzyme and the polyester matrix resin through polyester grafting segments, improves the uniformity of enzyme dispersion in the matrix, and avoids the problems of insufficient exposure of enzyme active sites and decreased degradation efficiency caused by agglomeration. Experimental results show that the degradation efficiency of the PBS-b-PEG grafted immobilized enzyme after melt blending (0.25% / h) is better than that of the ungrafted immobilized enzyme (0.21% / h), verifying the positive effect of grafting modification on blending dispersion and enzyme activity retention.

[0033] (3) The high-temperature resistant immobilized keratinase degradation promoter provided by this invention is applied to the preparation of controllably degradable polybutylene succinate composite materials. The resulting enzyme-PBS embedded composite material still exhibits high catalytic activity under composting conditions. Composting degradation experiments show that after grafting modified CNC-PCL-HiC into PBS, the degradation efficiency reaches 11.06% / d under composting conditions of 55℃ and 50% humidity, which is much higher than the 1.82% / d of the pure PBS control group. This proves that the grafted modified immobilized enzyme can still effectively catalyze the degradation of the polyester matrix under natural composting conditions. The application of this promoter enables the controllable and complete degradation of polyester materials in the natural environment, providing an effective solution to the technical problem of low degradation efficiency of existing degradable materials in the natural environment. Attached Figure Description

[0034] Figure 1 For the performance testing of this invention, the weight loss curves of the biodegradation promoter and pure enzyme (Comparative Example 2) prepared by grafting PBS with 500nm CNC (Comparative Example 1) and 500nm CNC (Example 1) were applied to the degradation of pure polyester sheets under non-heat-treated conditions. Figure 2 In the performance test of this invention, the biodegradation promoter and pure enzyme (Comparative Example 2) prepared by grafting PBS with 500nm CNC (Comparative Example 1) and 500nm CNC (Example 1) respectively were subjected to high temperature heat treatment and applied to the degradation of pure polyester sheet, and the weight loss curves were obtained. Figure 3 In the performance test of this invention, the immobilized keratinase prepared in Example 2 and Comparative Example 1 were melt-blended with PBS and then used to prepare sample pieces for degradation weight loss curves. Figure 4 In the performance test of this invention, the PCL grafted modified CNC immobilized enzyme (CNC-PCL-Hic) prepared in Example 3 was melt-blended with PBS to form a sample, and then the weight loss curves were obtained under composting and high solids conditions. The control group was pure PBS. Detailed Implementation

[0035] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] The present invention will be further described below with reference to specific embodiments, but these are not intended to limit the scope of the invention.

[0037] The main parameters and variables of the various embodiments and comparative examples of the present invention are shown in Table 1 below.

[0038] Table 1

[0039] In various embodiments of the present invention, the keratinase used is a keratinase derived from Humicola insolens (its protein structure database number is PDB code: 4OYY).

[0040] Example 1 This embodiment provides a method for preparing a high-temperature resistant immobilized keratinase degradation promoter, including the following steps: (1) Preparation of polybutylene succinate grafted modified CNC: A quantitative amount of CNC (500 nm) solution was added to deionized water to prepare a CNC solution. The CNC solution was prepared by ultrasonic dispersion and centrifugation to replace the deionized water solvent with acetone solvent. Dry polybutylene succinate was added to fix the CNC mass ratio at 2:1 with the polybutylene succinate mass. The mixture was then transferred to an oil bath at 60 °C and reacted for 24 h. After the reaction was completed, the precipitate was collected by centrifugation. The precipitate was dissolved in chloroform and then centrifuged again to purify the product. The precipitate was washed with deionized water and centrifuged three times. After ultrasonic dispersion with deionized water, a grafted cellulose nanoparticle (CNC-PBS) solution was obtained. The grafted cellulose nanocrystal (CNC-PBS) lyophilized powder was obtained by freeze-drying. (2) Physical adsorption and immobilization of keratinase: A clean beaker was filled with a quantitative amount of CNC-PBS lyophilized powder, which was dissolved in deionized water and then dispersed by ultrasonic vibration using an ultrasonic cleaner. The pH of the solution was adjusted to 4 with HCl solution and magnetically stirred. The quantitative amount of keratinase solution was then added to the CNC-PBS solution. The mass ratio of CNC-PBS to keratinase was 10:1. The mixed solution was kept at a constant temperature and magnetically stirred in an ice-water bath at 4°C for 6 hours to obtain a CNC-PBS-HiC solution. After freeze-drying, immobilized keratinase lyophilized powder was obtained.

[0041] Example 2 This embodiment provides a method for preparing a high-temperature resistant immobilized keratinase degradation promoter, including the following steps: (1) Preparation of polybutylene succinate and polyethylene glycol block copolymer: A certain amount of succinic acid, butanediol and polyethylene glycol were added to a three-necked round bottom flask in sequence. After heating to 60°C, isopropyl titanate was added, nitrogen gas was introduced, the temperature was raised to 110°C and held for 30 min, then the temperature was raised to 180°C and reacted at this temperature for 2 h. Then, the same mass of succinic acid was added, the end was sealed at 180°C, and the reaction was continued for 2 h. The product was dissolved in chloroform and stirred for 4 h. Methanol was added dropwise for extraction. After filtration, the mixture was ventilated for 1 day and dried at 40°C to obtain the product polybutylene succinate and polyethylene glycol block copolymer (PBS-b-PEG).

[0042] (2) Preparation of CNC grafted with polybutylene succinate and polyethylene glycol block copolymer: A quantitative amount of CNC (500 nm) solution was added to deionized water to prepare the CNC solution. The acetone solvent was replaced with deionized water solvent by ultrasonic dispersion and centrifugation. Dry PBS-b-PEG was added to fix the mass ratio of CNC to PBS-b-PEG at 2:1. The mixture was then transferred to an oil bath at 60 °C and reacted for 24 h. After the reaction was completed, the precipitate was collected by centrifugation, dissolved in chloroform, and then centrifuged again to purify the product. The precipitate was washed with deionized water and centrifuged three times. After ultrasonic dispersion with deionized water, the grafted cellulose nanoparticles (CNC-PBS-b-PEG) solution was obtained. The grafted cellulose nanocrystals (CNC-PBS-PEG) lyophilized powder was obtained by freeze-drying.

[0043] (3) Physical adsorption and immobilization of keratinase: A clean beaker was filled with a quantitative amount of CNC-PBS-b-PEG lyophilized powder, which was dissolved in deionized water and then dispersed by ultrasonic vibration using an ultrasonic cleaner. The pH of the solution was adjusted to 4 with HCl solution and magnetically stirred. The quantitative keratinase solution was then added to the CNC-PBS-b-PEG solution. The mass ratio of CNC-PBS-b-PEG to keratinase was 10:1. The mixed solution was subjected to constant temperature magnetic stirring in an ice-water bath at 4°C for 6 hours to obtain CNC-PBS-b-PEG-HiC solution. After freeze-drying, immobilized keratinase lyophilized powder was obtained.

[0044] Example 3 This embodiment provides a method for preparing a high-temperature resistant immobilized keratinase degradation promoter, including the following steps: (1) Preparation of polycaprolactone diol grafted modified CNC: 78.1 mL of 3.10% CNC (500 nm) solution was added to deionized water to prepare 140 mL of CNC solution. The acetone solvent was replaced by deionized water solvent by ultrasonic dispersion and centrifugation. 1.215 g of dried polycaprolactone diol was added, and the mixture was transferred to an oil bath at 60 °C and reacted for 24 h. After the reaction was completed, the lower layer of liquid was separated, centrifuged, and the precipitate was washed three times with deionized water. After ultrasonic dispersion with 150 mL of deionized water, polycaprolactone diol grafted modified cellulose nanocrystals (CNC-PCL) solution was obtained. (2) Physical adsorption and immobilization of keratinase: Take 81.1 mL of CNC-PCL solution and ultrasonically disperse it for 10 min using an ultrasonic cleaner. Prepare NaOH solution to adjust the pH of the mixed solution to 9, perform magnetic stirring, add 640 μL of keratinase solution, the mass ratio of CNC-PCL to keratinase is 15:1, and standardize the system to 100 mL with deionized water. The mixed solution is subjected to constant temperature magnetic stirring in an ice-water bath at 4 ℃ for 6 h, and finally obtains the keratinase immobilized CNC-PCL-Hic solution. Dilute with deionized water and then freeze-dry to obtain polycaprolactone-grafted modified cellulose nanocrystal immobilized keratinase (CNC-PCL-Hic) lyophilized powder.

[0045] Comparative Example 1 This comparative example provides a method for preparing a physically adsorbed immobilized keratinase degradation promoter, comprising the following steps: A clean beaker is filled with a quantitative CNC (500nm) solution, which is then dissolved in deionized water and dispersed by ultrasonic oscillation using an ultrasonic cleaner. The pH is adjusted to 4 and 6 with HCl solution, and to 9 with NaOH solution, followed by magnetic stirring. A quantitative keratinase solution is then added to the CNC solution, with a CNC to keratinase mass ratio of 10:1. The mixed solution is then subjected to constant temperature magnetic stirring in an ice-water bath at 4°C for 6 hours to obtain a CNC-HiC solution. After freeze-drying, immobilized keratinase lyophilized powders prepared under different pH conditions are obtained.

[0046] Comparative Example 2 This comparative example uses freeze-dried solid keratinase, without immobilization treatment.

[0047] Application performance testing (a) Routine activity testing The products prepared in Example 1, Comparative Example 1 and Comparative Example 2 were subjected to routine activity tests, all of which were performed in a Tris-HCl buffer environment.

[0048] The test method is as follows: A quantitative amount of lyophilized powder was placed in 5 mL of Tris-HCl buffer to make the enzyme activity concentration of the solution 0.2409 mg / mL. Then, a PBS sample was placed in the buffer, and the activity was tested under water bath conditions of 60℃ and 80 rpm. Among them, Example 1 was an enzyme immobilized by grafting PBS onto 500 nm CNC at pH 4; Comparative Example 1 was an enzyme immobilized by 500 nm CNC at pH 4, 6, and 9; Comparative Example 2 was a pure enzyme at the same concentration.

[0049] The test results are shown in Table 2 below.

[0050] Table 2

[0051] Conventional activity tests show that pH 4 is the optimal condition for immobilized enzymes, resulting in the highest enzyme adsorption efficiency.

[0052] (ii) Activity test after high temperature treatment The products prepared in Example 1, Comparative Example 1, and Comparative Example 2 were subjected to high-temperature treatment followed by activity testing. The testing method was as follows: A quantitative amount of lyophilized powder was placed in 5 mL of Tris-HCl buffer to make the enzyme activity concentration of the solution 0.2409 mg / mL (Comparative Example 2 used the same concentration of pure enzyme). The product was heat-treated at 150 °C or 160 °C for 5 min (Comparative Example 2 had completely lost its activity at 160 °C, so it was only heat-treated at 150 °C). Then, PBS samples were placed in the buffer and the activity was tested in a water bath at 60 °C and 80 rpm. The results are shown in Table 3.

[0053] Table 3

[0054] As shown in the table above, in Example 1, after CNC grafting and modifying the PBS chain, the hydrophobic PBS chain provides more hydrophobic interactions, which restricts its swinging and unfolding at high temperatures, greatly improving the heat resistance of the immobilized keratinase degradation promoter, so that its activity retention rate can still reach 77.19% after high temperature treatment.

[0055] (III) Activity test after blending and dispersion The immobilized keratinase lyophilized powders prepared in Examples 1, 2, and Comparative Example 1 (pH=4) were respectively blended with polybutylene succinate: a quantitative amount of lyophilized powder and a quantitative amount of PBS masterbatch were weighed and melt-blended at 130°C (the amount of lyophilized powder added was 2 wt% of the PBS masterbatch), and hot-pressed into shape under a specific mold. The process conditions were: preheating at 130°C for 3 min, hot pressing for 1 min, and cold pressing for 5 min. The thickness of the hot-pressed film was controlled between 0.2-0.3 mm. Then, the film was made into an enzyme-embedded sample with a diameter of 1 cm by a punch. The sample was then placed in 5 mL of Tris-HCl buffer and the activity was tested under the conditions of 60°C and 50 rpm water bath. The results are shown in Table 4.

[0056] Table 4

[0057] As shown in the table above, the grafted enzyme exhibits significantly higher activity. This is because the grafted PBS / PBS-b-PEG chains encapsulate and immobilize the enzyme, preventing conformational damage caused by high temperatures. In addition, the grafted segments have good compatibility with the PBS matrix, which improves the dispersibility of the immobilized enzyme in PBS, thereby enhancing its heat resistance and catalytic efficiency.

[0058] (iv) Degradation performance test under composting and high solids conditions The CNC-PCL-Hic lyophilized powder of immobilized keratinase prepared in Example 3 was melt-blended with polybutylene succinate (PBS) at 130°C (the amount of lyophilized powder added was 2 wt% of the PBS masterbatch). Then, it was preheated at 130°C for 3 min, hot-pressed for 1 min, and cold-pressed for 5 min, controlling the thickness of the hot-pressed film to be between 1-2 mm, to obtain the embedded enzyme system. The film was then cut into square samples with a side length of 1 cm and tested for composting degradation according to the national standard GB / T19277.1-2011 at a temperature of 55°C and a humidity of 50%. The composting degradation efficiency and catalytic efficiency were calculated. At the same time, pure PBS samples of the same size were prepared as a control group. The test results are shown in Table 5 below.

[0059] Table 5

[0060] In the table above, the grafted modified CNC-g-Hic prepared in Example 3 was embedded in PBS and subjected to composting and high-solids treatments, respectively; the control group was treated with pure PBS and subjected to both composting and high-solids treatments. The test results showed that the degradation efficiency of the grafted modified immobilized enzyme (CNC-PCL-Hic) was significantly higher than that of pure PBS. This is because the polyester-grafted modified CNC carrier provides stable conformational protection for the enzyme molecules, reducing the denaturation and inactivation of the enzyme in the composting environment due to factors such as temperature and humidity; at the same time, the grafted PCL segments have good compatibility with the PBS matrix, promoting the uniform dispersion of the immobilized enzyme in the matrix, making full use of the catalytic active sites, thereby significantly improving the degradation efficiency and enhancing the degradation adaptability of the biodegradable material under natural environmental conditions.

[0061] It is worth noting that although PCL grafted segments of a different type than the PBS matrix were used in Example 3, a composting degradation efficiency of 11.06% / d was still achieved, which is much higher than the 1.82% / d of pure PBS. This indicates that the degradation promoter prepared in this invention can achieve efficient degradation without using grafted segments that are exactly the same as the matrix resin, and has good versatility. It can be expected that when the grafted segments are made of polyesters with the same or similar molecular structure as the matrix resin (such as PBS grafted segments used in the PBS matrix), the degradation efficiency is expected to be further improved due to the further enhancement of compatibility and better dispersibility.

[0062] In summary, this invention significantly improves the thermal stability and processing adaptability of keratinase by immobilizing it on a polyester-grafted modified cellulose nanocrystal carrier. Simultaneously, the introduction of grafted segments effectively improves the enzyme's dispersibility and interfacial compatibility within the polyester matrix. High-temperature melt blending and composting degradation verification demonstrated that this promoter maintains high catalytic activity in the natural environment, achieving controllable and efficient degradation of polyester materials. This provides a feasible technical solution for improving the degradation performance of biodegradable polyester materials under natural conditions.

[0063] The above are merely preferred embodiments of the present invention and are not intended to limit the implementation methods and protection scope of the present invention. Those skilled in the art should recognize that any equivalent substitutions and obvious changes made based on the content of this specification should be included within the protection scope of the present invention.

Claims

1. A method for preparing a high-temperature resistant immobilized keratinase degradation promoter, characterized in that, Includes the following steps: S1. Cellulose nanocrystals are mixed with polyester and / or polyester-polyether block copolymers at a mass ratio of 2:1-3:1, and grafting reaction is carried out under heating conditions. After separation and purification, grafted modified cellulose nanocrystals are obtained. S2. Disperse the grafted modified cellulose nanocrystals in water, adjust the pH to 3-9, add the keratinase according to the mass ratio of grafted modified cellulose nanocrystals to keratinase of 10:1-15:1, and obtain an immobilized keratinase mixture through physical adsorption and immobilization reaction. S3. Freeze-dry the immobilized keratinase mixture to obtain a high-temperature resistant immobilized keratinase degradation promoter.

2. The preparation method according to claim 1, characterized in that, In step S1, before mixing the cellulose nanocrystals with the polyester and / or polyester-polyether block copolymer, the cellulose nanocrystals are first dispersed in water, and then the water is replaced with one of acetone, toluene or methanol by solvent displacement; the heating temperature of the grafting reaction is 50-80 ℃, and the heating time of the grafting reaction is 18-36 h.

3. The preparation method according to claim 1, characterized in that, The polyester and / or polyester-polyether block copolymer includes one or more of polybutylene succinate, polycaprolactone, and polybutylene succinate-polyethylene glycol block copolymer; the molecular weight of the polyester and / or polyester-polyether block copolymer is 1000-10000 g / mol.

4. The preparation method according to claim 1, characterized in that, The grafting rate of the polyester and / or polyester-polyether block copolymer onto cellulose nanocrystals is 60%-70%.

5. The preparation method according to claim 1, characterized in that, In step S2, the pH is 3-5; the temperature of the physical adsorption reaction is 0-10 °C, and the time is 4-12 h.

6. A high-temperature resistant immobilized keratinase degradation promoter, prepared by the method according to any one of claims 1-5, characterized in that, The heat-resistant immobilized keratinase degradation promoter retains no less than 70% of the keratinase activity after heat treatment at 160℃ for 5 minutes.

7. The application of the high-temperature resistant immobilized keratinase degradation promoter according to claim 6 in the preparation of controllably degradable polybutylene succinate composite materials, characterized in that, The high-temperature resistant immobilized keratinase degradation promoter is mixed with polybutylene succinate masterbatch to obtain a premix; the premix is ​​melt-blended at 120-140℃ and hot-pressed to obtain an enzyme-PBS embedded composite material.

8. The application according to claim 7, characterized in that, The amount of the high-temperature resistant immobilized keratinase degradation promoter added accounts for 0.1 wt.%-3 wt.% of the polybutylene succinate masterbatch.

9. The application according to claim 7, characterized in that, The hot pressing process conditions are: preheating for 2-4 minutes, hot pressing for 0.5-2 minutes, and cold pressing for 3-6 minutes.

10. The application according to claim 7, characterized in that, The embedded enzyme-PBS composite material was degraded under composting conditions of 55 ℃ and 50% humidity, with a degradation efficiency of not less than 10% / d.

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