Immobilized cellulase of magnetic mesoporous core-shell material, preparation method and application thereof

Abstract

The application belongs to the technical field of enzyme engineering and new material cross, and particularly relates to a kind of immobilized cellulase of magnetic mesoporous core-shell material and preparation method and application. The immobilized cellulase is obtained by physical adsorption of cellulase with magnetic mesoporous core-shell material F5HPU-46 as carrier. F5HPU-46 is a unique core-shell structure with paramagnetic Fe3O4 nanoparticles as core layer and UIO-66 mesoporous shell layer with stable structure, which has the dual advantages of magnetic response and high specific surface area mesoporous structure. The relative enzyme activity of the immobilized cellulase in the application is increased to 119% under acidic conditions at pH 4, while the relative enzyme activity of free enzyme is decreased to 75%.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of enzyme engineering and new materials, specifically relating to an immobilized cellulase made of magnetic mesoporous core-shell material, its preparation method, and its application. Background Technology

[0002] The green refining of lignocellulose into biomass fuels has become an important way to replace fossil resources. Cellulase is the core biocatalyst that drives the degradation of lignocellulose into fermentable sugars and then into biomass fuels. However, the molecular structure of free cellulase is unstable and easily affected by external conditions such as temperature and pH during the reaction, leading to conformational changes, decreased enzyme activity, or even inactivation. Furthermore, free cellulase is difficult to separate and recover from the reaction system and cannot be reused, which not only increases the production cost of biomass fuels but may also cause product pollution. In addition, in the refining process of biomass using lignocellulose as raw material, the pretreatment stage, which aims to efficiently remove lignin and hemicellulose to expose cellulose, usually uses acidic conditions such as dilute acids. Under this acidic pretreatment environment, the enzyme activity of free cellulase is easily inhibited by hydrogen ions, resulting in a significant decrease in stability. The acidic environment may also exacerbate the loss of free enzymes, further restricting its application in the green refining of lignocellulose into biomass fuels, affecting the degradation efficiency of lignocellulose and the yield of biomass fuels.

[0003] To address the aforementioned technical challenges, immobilized enzyme methods have emerged. The core of these methods involves immobilizing cellulase on the surface or within a specific carrier material using physical or chemical methods, thereby achieving enzyme recovery, reuse, and improved stability. The performance of the carrier material is a key factor determining the effectiveness of the immobilized enzyme. Currently, carrier materials used for immobilizing cellulase are mainly classified into three categories: inorganic carriers, organic carriers, and composite carriers. Inorganic carriers, such as silica, alumina, and hydroxyapatite, offer advantages such as high stability and good mechanical strength, but suffer from limited specific surface area, low enzyme loading capacity, and insufficient protection of enzyme activity. Organic carriers, such as chitosan, sodium alginate, and polyethylene glycol, exhibit excellent biocompatibility and are easily modified, but they have poor thermal stability and mechanical strength, and are prone to degradation in acidic or high-temperature environments, affecting the long-term effectiveness of the immobilized enzyme. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides an immobilized cellulase in a magnetic mesoporous core-shell material, along with its preparation method and applications.

[0005] The first aspect of this invention provides an immobilized cellulase on a magnetic mesoporous core-shell material, which is obtained by physically adsorbing cellulase using magnetic mesoporous core-shell material F5HPU-46 as a carrier. Using paramagnetic Fe3O4 nanoparticles as the core and a metal-organic framework material with a mesoporous structure as the shell, a core-shell structure was formed, resulting in magnetic mesoporous core-shell material F5HPU-46. The paramagnetic Fe3O4 nanoparticles were obtained by modifying Fe3O4 nanoparticles with poly-4-styrene sulfonate. The surface of Fe3O4 nanoparticles was modified with poly-4-styrene sulfonate, and the electrostatic effect and steric hindrance of the polyelectrolyte were used to promote the uniform nucleation and growth of MOF on the magnetic core surface. The process is simpler, has fewer components, and does not use organic pore-forming agents. The mass ratio of F5HPU-46 to the cellulase is 1:0.625~0.75.

[0006] In another preferred embodiment, the Fe3O4 nanoparticles have a particle size of 250 nm to 400 nm; the metal-organic framework material is UIO-66.

[0007] The pore size of the mesopore is between 3.4 nm and 4 nm. This pore size is adapted to the specific structural domain of cellulase, thereby achieving enhanced enzyme activity under acidic conditions (acid activation phenomenon).

[0008] A second aspect of this invention provides a method for preparing immobilized cellulase from the aforementioned magnetic mesoporous core-shell material, comprising the following steps: Soluble iron salt, citrate, and sodium acetate were mixed in an alcohol reagent and subjected to a solvothermal reaction to obtain Fe3O4 nanoparticles. The mass ratio of iron salt, citrate, and sodium acetate was 1.95–1.956: 0.6–0.625: 3.6–3.66. At this ratio, citrate, as a surface ligand, effectively controlled the nucleation and growth rate of Fe3O4 crystal nuclei, avoiding excessive agglomeration and obtaining paramagnetic nuclei with uniform particle size (250–400 nm) and good dispersion. Sodium acetate provided an alkaline environment and acted as an electrostatic stabilizer, promoting the uniform formation of Fe3O4 nanoparticles. The optimized ratio of the three components ensured high crystallinity and superparamagnetism of the magnetic nuclei, providing a stable core for subsequent coating.

[0009] The Fe3O4 nanoparticles were surface-modified with poly(4-styrene sulfonate) to obtain paramagnetic Fe3O4 nanoparticles. The mass ratio of paramagnetic Fe3O4 nanoparticles to poly(4-styrene sulfonate) was 0.6~0.62:5.4~5.46. High amounts of poly(4-styrene sulfonate) could form a dense polyelectrolyte modification layer on the Fe3O4 surface through electrostatic adsorption and steric hindrance. This modification layer not only enhanced the anti-agglomeration ability of the magnetic nuclei under acidic conditions, but more importantly, provided abundant negatively charged binding sites, effectively chelating zirconium ions (Zr). 4+This induces heterogeneous nucleation and uniform growth of the UIO-66 shell on the surface of the magnetic core, thus avoiding the isolated formation of free MOF crystals.

[0010] The paramagnetic Fe3O4 nanoparticles were mixed with a zirconium source, organic ligands, and a template agent in a mass ratio of 6~6.12:1.2~1.22:1.2~1.22:1~1.02 under an acidic environment, followed by a hydrothermal reaction to coat the surface of the paramagnetic Fe3O4 nanoparticles with a metal-organic framework shell. The template agent was then removed to obtain the magnetic mesoporous core-shell material. The mass ratio of the paramagnetic Fe3O4 nanoparticles with the zirconium source, organic ligands, and template agent of 6~6.12:1.2~1.22:1.2~1.22:1~1.02 balances the integrity of the shell coating with the construction of the mesoporous structure. Appropriate amounts of zirconium source and fluorinated organic ligand (tetrafluoroterephthalic acid) ensure good crystallinity and chemical stability of the UIO-66 shell; block polyether F127 serves as a template agent, which can self-assemble into regular micelles at this ratio, guiding the mesopores (pore size 3.4~4 nm) to be uniformly distributed in the shell; the relatively high proportion of Fe3O4 magnetic cores ensures that the final material has strong magnetic responsiveness, which facilitates rapid magnetic separation.

[0011] Specifically, the hydrothermal reaction temperature is 60℃~61℃, which differs from the high-temperature (>120℃) organic solvothermal synthesis of traditional UIO-66. This invention uses a medium-temperature aqueous phase crystallization at 60℃~61℃. This mild condition avoids the oxidation or agglomeration of Fe3O4 magnetic nuclei caused by high temperatures, and controls the slow and uniform growth of the UIO-66 shell, forming a continuous, dense, and moderately thick coating layer. The reaction time of 11.5h~12h is sufficient for the MOF crystals to fully develop to structural stability without excessive growth that blocks the mesopores, thus obtaining F5HPU-46 material with high specific surface area, regular mesopore channels, and a complete core-shell morphology.

[0012] F5HPU-46 was mixed with cellulase in buffer solution, and then subjected to physical adsorption, magnetic separation, and washing to obtain immobilized cellulase.

[0013] In another preferred embodiment, the soluble iron salt is FeCl3, the citrate is anhydrous trisodium citrate, and the alcohol reagent is ethylene glycol.

[0014] In another preferred embodiment, the temperature of the solvothermal reaction is 200°C to 201°C, and the time is 9.5h to 10h.

[0015] In another preferred embodiment, the zirconium source is zirconium tetrachloride, the organic ligand is tetrafluoroterephthalic acid, and the template agent is block polyether F127.

[0016] In another preferred embodiment, the template removal agent refers to extraction with ethanol to remove the block polyether F127 from the mesopores.

[0017] In another preferred embodiment, the acidic environment refers to the mixture of hydrochloric acid and glacial acetic acid in a volume ratio of 2 to 2.1:3.

[0018] In another preferred embodiment, the buffer solution is a citrate buffer solution; The physical adsorption refers to physical adsorption at 300~500 r / min for 2h~2.5h.

[0019] Specifically, cellulase was dissolved in 50 mM citrate buffer at pH 4.8. Magnetic mesoporous core-shell material F5HPU-46 was mixed with the cellulase solution at pH 4.8. The mixture was physically adsorbed for 2 to 2.5 hours at a speed of 300-500 r / min and 25°C. After magnetic separation, the mixture was washed twice with citrate buffer at pH 4.8 to obtain immobilized cellulase.

[0020] The third aspect of the present invention provides the application of the immobilized cellulase in the preparation of biomass fuel, characterized in that the biomass fuel is made from lignocellulose.

[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention employs a structurally stable metal-organic framework material with a mesoporous structure as the shell layer and paramagnetic Fe3O4 nanoparticles as the core layer, forming a unique core-shell structure. The resulting F5HPU-46 possesses the dual advantages of magnetic responsiveness and a high specific surface area mesoporous structure. After the immobilized enzyme reaction is complete, separation and recovery can be achieved within 10 seconds using an external magnetic field, eliminating the need for complex centrifugation and filtration operations, significantly simplifying the operation process and improving recovery efficiency. The immobilized cellulase in this invention exhibits a relative enzyme activity of 119% under acidic conditions at pH 4, while the relative enzyme activity of the free enzyme decreases to 75%. Furthermore, after 10 repeated uses, the enzyme activity retention rate under acidic conditions remains above 71%, improving the degradation efficiency of lignocellulose in acidic systems and reducing the cost of enzymatic hydrolysis. This invention is particularly suitable for systems following acidic pretreatment of lignocellulose.

[0022] The metal-organic framework material UIO-66 shell in this invention enables enzyme loading to reach over 180 mg / g, which significantly improves enzyme loading compared to ordinary magnetic carriers, effectively enhancing the catalytic efficiency of immobilized enzymes and meeting the needs of large-scale industrial production.

[0023] The UIO-66 shell in the carrier synthesis of this invention is produced using a 60°C aqueous phase medium-temperature crystallization method. Compared with the traditional solvothermal method for UIO-66, which typically involves reaction temperatures >120°C and the use of N,N-dimethylformamide organic solvent, this method avoids the use of toxic solvents, provides milder conditions, consumes less energy, and is more in line with the industrial development concept of green refining of lignocellulose. At the same time, it reduces the industrial preparation cost of the carrier, facilitates large-scale mass production, and further enhances the industrial application value of this invention. Attached Figure Description

[0024] Figure 1 These are structural characterization diagrams of the paramagnetic Fe3O4 nanoparticles and the magnetic mesoporous core-shell material F5HPU-46 of this invention; a is a SEM image of the Fe3O4 nanoparticles; b is a SEM image of the magnetic mesoporous core-shell material F5HPU-46.

[0025] Figure 2 These are the infrared spectra of the magnetic mesoporous core-shell material F5HPU-46 and the non-magnetic mesoporous material HPU-46 prepared in this invention.

[0026] Figure 3 The figures show the N2 adsorption isotherm and pore size distribution of the magnetic mesoporous core-shell material F5HPU-46 prepared by this invention. The inset in the figures is the pore size distribution.

[0027] Figure 4 This is a standard curve of protein concentration according to the present invention, in which the inset shows the values ​​of each parameter.

[0028] Figure 5 This is a graph showing the changes in the catalytic performance of the immobilized cellulase prepared in this invention on sodium carboxymethyl cellulose at different temperatures.

[0029] Figure 6 This is a graph showing the changes in the catalytic performance of the immobilized cellulase prepared in this invention on sodium carboxymethyl cellulose at different pH values.

[0030] Figure 7 This is a graph showing the relative enzyme activity of the immobilized cellulase prepared in this invention after 10 repeated operations at different pH values. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0032] 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.

[0033] Metal-organic frameworks (MOFs), as a novel class of porous crystalline materials, possess characteristics such as large specific surface area, tunable pore size, regular structure, and easy surface functionalization, and have attracted widespread attention in the field of immobilized enzymes in recent years. Among them, UIO-66, as a typical MOF material, exhibits excellent chemical and thermal stability, and its mesoporous structure can be obtained by controlling the preparation conditions, providing sufficient immobilization sites and diffusion channels for cellulase, which is beneficial for improving enzyme loading and enzymatic reaction efficiency. However, it is difficult to quickly separate UIO-66-immobilized enzymes from the system after the reaction, requiring complex operations such as centrifugation and filtration for recovery, which increases operating costs and process complexity, limiting its industrial application.

[0034] Paramagnetic iron oxide (Fe3O4) nanoparticles possess excellent magnetic responsiveness, biocompatibility, and chemical stability. Using them as core materials to prepare core-shell structured carriers can impart magnetic separation properties, enabling rapid and efficient recovery of immobilized enzymes. However, the surface of individual Fe3O4 nanoparticles lacks suitable immobilization sites, resulting in low enzyme loading. Furthermore, they are prone to aggregation and oxidation in acidic environments, leading to decreased magnetic properties and stability. This makes them unsuitable for providing a stable immobilization environment for cellulase and failing to meet the requirements of acidic operating conditions.

[0035] Existing technology discloses a method for synthesizing Fe3O4@HMUiO-66-NH2 magnetic core-shell hierarchical mesoporous metal-organic frameworks (MOFs) and applying them to the immobilization of porcine pancreatic lipase (PPL). This method achieves PPL immobilization by amylating UiO-66 and utilizing its enhanced adsorption with patulin. However, due to the fundamental differences between porcine pancreatic lipase and the cellulase of interest in this invention in terms of molecular size, catalytic mechanism, and optimal operating environment, this existing carrier cannot be directly applied to cellulase immobilization. Furthermore, this synthesis method relies on NaClO4 and tetramethylbenzidine (TMB) to construct a complex nanoemulsion system to induce mesopore formation and shell growth, resulting in a complex preparation process. More importantly, the enzyme activity of this existing system is significantly inhibited under acidic conditions: the relative enzyme activity of free porcine pancreatic lipase in an acidic environment drops sharply to 13.1%. Given that the present invention aims to achieve efficient immobilization of cellulase under acidic conditions and maintain high activity, the enzyme immobilization strategies disclosed in the prior art are obviously not applicable to the technical problems solved by the present invention.

[0036] This invention is based on UiO-66 core-shell material, introducing fluorinated ligands to regulate the material's hydrophobicity, chemical stability, and interfacial interaction with cellulase. Unlike existing amination modification strategies, this invention's system does not contain amino groups, thus avoiding the adverse effects of amino protonation on enzyme activity under acidic conditions. Regarding magnetic core modification, this invention uses poly-4-styrene sulfonate to modify the Fe3O4 surface, utilizing the electrostatic interaction and steric hindrance effect of the polyelectrolyte to promote uniform nucleation and growth of MOFs on the magnetic core surface. This synthetic route is simple, requires fewer components, and eliminates the need for organic porogens such as tetramethylbenzidine (TMB). Experimental results show that the cellulase immobilized using this method exhibits a unique acid activation phenomenon under acidic conditions, with enzyme activity increasing rather than decreasing; after 10 cycles, the immobilized enzyme still retains 71.8% of its residual activity, fully demonstrating the excellent stability of this immobilization system in acidic environments.

[0037] The following is a detailed description of an immobilized cellulase in a magnetic mesoporous core-shell material, its preparation method, and its application.

[0038] The block polyether F127 (Pluronic F127) used in the following examples was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and all room temperatures refer to 25°C.

[0039] Example 1: A method for preparing immobilized cellulase, comprising the following steps: S1. Preparation of magnetic mesoporous core-shell material F5HPU-46 Preparation of paramagnetic Fe3O4 nanoparticles: First, 1.95 g of FeCl3 and 0.6 g of anhydrous trisodium citrate were dissolved in 60 mL of ethylene glycol, and then 3.6 g of sodium acetate was added and stirred to obtain a mixture. The mixture was stirred at 800 r / min for 3 h, and then sealed in a stainless steel autoclave lined with polytetrafluoroethylene and reacted at 200 °C for 10 h. After cooling to room temperature, Fe3O4 nanoparticles were obtained. The Fe3O4 nanoparticles were washed three times with ethanol and deionized water, and then dried overnight at 60 °C in a vacuum drying oven and stored in a desiccator for later use.

[0040] Preparation of magnetic mesoporous core-shell material F5HPU-46: 5.4 g of poly-4-styrene sulfonate was dispersed in 360 mL of ultrapure water in an Erlenmeyer flask and sonicated for 0.5 h. Then, 0.6 g of Fe3O4 nanoparticles were added, and the mixture was sonicated for another 0.5 h. After magnetic separation, the nanoparticles were washed once with ultrapure water to obtain modified Fe3O4 nanoparticles, which are paramagnetic Fe3O4 nanoparticles that can be directly used for the next step of synthesis. 120 mg of ZrCl4 and paramagnetic Fe3O4 nanoparticles were placed in a test tube containing 6 mL of ultrapure water and sonicated for 1 h. Then, 100 mg of block polyether F127 was added to obtain a mixture. The mixture was stirred at 500 r / min for 10 min at room temperature until dissolved. Subsequently, 0.8 mL of concentrated hydrochloric acid (HCl) and 1.2 mL of glacial acetic acid were added. After standing for 30 min, the mixture was placed in a 60 °C water bath and 120 mg of tetrafluoroterephthalic acid was added. The reaction was carried out for 12 h. After the reaction was completed, the resulting reddish-brown nanoparticles were magnetically separated and washed at least three times each with water and ethanol. The block polyether F127 was removed from the mesopores by ethanol extraction for 3 days. The resulting reddish-brown nanoparticles were dried overnight at 60 °C in a vacuum drying oven to obtain the magnetic mesoporous core-shell material F5HPU-46.

[0041] S2, Preparation of cellulase solution Weigh 312.5 mg of cellulase, dissolve it in 50 mM citrate-sodium citrate buffer solution at pH 4.8 and bring the volume to 25 mL to obtain a cellulase solution with a concentration of 12.5 mg / mL. Store the solution at 4 °C for later use.

[0042] Take 10 mg of magnetic mesoporous core-shell material F5HPU-46 and place it in a 2 mL centrifuge tube. Then add 12.5 mg / mL cellulase solution with pH 4.8. Perform physical adsorption for 2.5 h in a constant temperature shaker at 25 °C and 500 r / min. After magnetic separation, wash twice with citrate buffer with pH 4.8 to obtain immobilized cellulase.

[0043] Figure 1 This is a SEM image of the magnetic properties of the paramagnetic Fe3O4 nanoparticles prepared in this invention and the mesoporous core-shell material F5HPU-46. Figure 1 It is evident that, compared to Fe3O4 nanoparticles, the surface of F5HPU-46 is rougher, and the UIO-66 shell covering the magnetic core can be clearly distinguished, indicating the formation of a core-shell structure.

[0044] Figure 2 This is the infrared spectrum of F5HPU-46, the magnetic mesoporous core-shell material prepared in this invention. Figure 2It is evident that F5HPU-46 exhibits characteristic peaks of both Fe3O4 and UIO-66, indicating the successful recombination of Fe3O4 and UIO-66.

[0045] Figure 3 This is an N2 adsorption isotherm and pore size distribution diagram of the magnetic mesoporous core-shell material F5HPU-46 prepared in this invention. Figure 3 It is evident that the main pores of F5HPU-46 are mesopores, with the pore size mainly concentrated at 3.4 nm, which is suitable for the size of cellulase.

[0046] Example 2 Measurement of the loading capacity of F5HPU-46 immobilized enzyme The supernatant and washing solution obtained after magnetic separation in Example 1 were used to determine the enzyme protein loading.

[0047] Preparation of bovine serum albumin standard solution: Accurately weigh 25 mg of bovine serum albumin powder, dissolve it in citrate buffer (pH 4.8), and bring the volume to 25 mL to obtain a 1 mg / mL stock solution. Then dilute the bovine serum albumin solution with citrate buffer (pH 4.8) to obtain bovine serum albumin solutions with concentrations ranging from 0.04 to 1 mg / mL. Specific concentrations of bovine serum albumin solutions are 0.04 mg / mL, 0.08 mg / mL, 0.16 mg / mL, 0.4 mg / mL, 0.64 mg / mL, 0.8 mg / mL, and 1.0 mg / mL.

[0048] Preparation of protein concentration standard curve: Take 24 5mL EP tubes and number them. Use three tubes as blanks and add 0.3mL of citrate buffer (pH 4.8) to each. Add 0.3mL of the different concentrations of bovine serum albumin (BSA) standard solution obtained above to the other tubes, then add 3mL of quinolinic acid (BCA) working solution to each and mix well. Establish three parallel samples for each concentration. Incubate at room temperature for 2 hours, and measure the absorbance at 595nm using a UV spectrophotometer. Zero the instrument using the absorbance value of the blanks. Plot the protein concentration standard curve with the concentration of the BSA standard solution as the x-axis and the absorbance as the y-axis. (See figure below.) Figure 4 As shown.

[0049] Protein loading determination: Take three 5mL EP tubes and add 0.3mL of the supernatant obtained from magnetic separation in step 3 to each tube. Take three 5mL EP tubes and add 0.3mL of the washing buffer obtained from magnetic separation to each tube. Then add 3mL of BCA working solution to each tube and mix well. Let stand at room temperature for 2 hours. Use a UV spectrophotometer to measure the absorbance of the cellulase supernatant and washing buffer at 595nm. Based on the absorbance values, find the protein concentration (mg / mL) of the supernatant and washing buffer on the standard curve.

[0050] The formula for calculating cellulase protein loading is as follows: ; Calculations show that the protein loading of the composite material F5HPU-46 after adsorbing cellulase is 186.08 mg / g.

[0051] Example 3 Enzymatic hydrolysis temperature stability of F5HPU-46 immobilized enzyme Using sodium carboxymethyl cellulose (CMC-Na) as a substrate, the activities of immobilized and free cellulase were determined by the dinitrosalicylic acid (DNS) method. The immobilized cellulase prepared in Example 1 was dispersed in 0.5 mL of citrate buffer solution, followed by the addition of 0.5 mL of 1% (w / w) CMC-Na (prepared in a citrate-sodium citrate buffer solution at pH 4.8). The mixtures were reacted at 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C for 1 h each. Three parallel samples were prepared at each temperature. For each reaction, 0.2 mL of the supernatant was mixed with 0.3 mL of deionized water, and 1 mL of DNS reagent was added. All samples were then boiled in a water bath for 5 min, removed, cooled to room temperature, and then 4.5 mL of deionized water was added and mixed thoroughly. The absorbance of the supernatant at 540 nm was measured using a UV spectrophotometer. Using the enzyme activity of immobilized cellulase and free cellulase at 50℃ as 100%, the relative activities of immobilized cellulase and free cellulase at different temperatures of 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ were calculated. Figure 5 It is evident that the activity of free enzymes changes more drastically with reaction temperature, while immobilized cellulases exhibit better thermal stability than free enzymes.

[0052] Example 4 pH stability of F5HPU-46 immobilized enzyme during enzymatic hydrolysis Using sodium carboxymethyl cellulose (CMC-Na) as a substrate, the activities of immobilized and free cellulase were determined using the dinitrosalicylic acid (DNS) method. The immobilized cellulase prepared in Example 1 was dispersed in 0.5 mL of citrate buffer solutions with pH values ​​of 2.2, 3, 4, 4.8, and 6, respectively. Then, 0.5 mL of 1% CMC-Na (prepared from citrate-sodium citrate buffer solutions with pH values ​​of 2.2, 3, 4, 4.8, and 6) was added, and the mixture was reacted at 50 °C for 1 h. Three parallel samples were prepared for each pH. 0.2 mL of the supernatant after the reaction was mixed with 0.3 mL of deionized water, and 1 mL of DNS reagent was added to each sample. The mixture was then boiled in a water bath for 5 min, removed, cooled to room temperature, and then 4.5 mL of deionized water was added to each sample and mixed thoroughly. The absorbance of the supernatant at 540 nm was measured using a UV spectrophotometer. With the enzyme activity of immobilized cellulase and free cellulase at pH 4.8 as 100%, the relative activities of immobilized cellulase and free cellulase at pH 2.2, 3, 4, 4.8 and 6 were calculated respectively.

[0053] Depend on Figure 6 It is evident that the activity of the free enzyme changes more drastically with environmental pH, while the immobilized cellulase exhibits better pH stability than the free enzyme. The immobilized cellulase showed acid activation; under acidic conditions of pH 4, its relative enzyme activity increased to 119%, while the relative enzyme activity of the free enzyme decreased to 75%. This indicates that the optimal enzymatic hydrolysis pH for the immobilized cellulase is 4, and also demonstrates that the immobilized cellulase has better acid resistance than the free enzyme.

[0054] Example 5 Reusability of F5HPU-46 immobilized enzyme Using sodium carboxymethyl cellulose (CMC-Na) as a substrate, the activity of immobilized cellulase at pH 4 and 4.8 after 10 reuses was determined using the dinitrosalicylic acid (DNS) method. The immobilized cellulase was dispersed in 0.5 mL of citrate buffer solution at pH 4 and 4.8, respectively, followed by the addition of 0.5 mL of 1% CMC-Na (prepared from citrate-sodium citrate buffer solution at pH 4 and 4.8, respectively). The reaction was carried out at 50 °C for 1 h. Three parallel samples were prepared for each pH. For each reaction, 0.2 mL of the supernatant was mixed with 0.3 mL of deionized water, and 1 mL of DNS reagent was added. The mixture was then boiled in a water bath for 5 min, removed, cooled to room temperature, and then 4.5 mL of deionized water was added and mixed thoroughly. The absorbance of the supernatant at 540 nm was measured using a UV spectrophotometer. After the reaction, the immobilized enzyme was magnetically separated and washed three times with buffer solutions at the corresponding pH values. The immobilized cellulase was then added back to the substrate solution at the corresponding pH, and the experiment was repeated. Using the initial enzyme activity of the immobilized cellulase as 100%, the relative activities of the immobilized cellulase after 10 repeated uses at pH 4 and 4.8 were calculated.

[0055] Depend on Figure 7 It can be seen that at pH=4.8, the immobilized enzyme still retains 68.3% of its activity after 10 reuses, while at pH=4, the immobilized enzyme retains 71.8% of its activity after 10 reuses, which indicates that the immobilized enzyme has good reusability.

[0056] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An immobilized cellulase in a magnetic mesoporous core-shell material, characterized in that, It was obtained by physically adsorbing cellulase using magnetic mesoporous core-shell material F5HPU-46 as a carrier; Using paramagnetic Fe3O4 nanoparticles as the core and a metal-organic framework material with a mesoporous structure as the shell, a core-shell structure was formed, resulting in magnetic mesoporous core-shell material F5HPU-46. The paramagnetic Fe3O4 nanoparticles were obtained by modifying Fe3O4 nanoparticles with poly-4-styrene sulfonate; The mass ratio of F5HPU-46 to the cellulase is 1:0.625~0.

75.

2. The immobilized cellulase in the magnetic mesoporous core-shell material according to claim 1, characterized in that, The Fe3O4 nanoparticles have a particle size of 250 nm to 400 nm. The pore size of the mesopores is between 3.4 nm and 4 nm; The metal-organic framework material is UIO-66.

3. A method for preparing immobilized cellulase from the magnetic mesoporous core-shell material of claim 2, characterized in that, Includes the following steps: Soluble iron salt, citrate, and sodium acetate were mixed in an alcohol reagent and subjected to a solvothermal reaction to obtain Fe3O4 nanoparticles; wherein the mass ratio of iron salt, citrate, and sodium acetate was 1.95~1.956:0.6~0.625:3.6~3.

66. The Fe3O4 nanoparticles were surface modified with poly-4-styrene sulfonate to obtain the paramagnetic Fe3O4 nanoparticles; the mass ratio of the paramagnetic Fe3O4 nanoparticles to poly-4-styrene sulfonate was 0.6~0.62:5.4~5.

46. The paramagnetic Fe3O4 nanoparticles were mixed with a zirconium source, an organic ligand, and a template agent in an acidic environment at a mass ratio of 6~6.12:1.2~1.22:1.2~1.22:1~1.

02. The mixture was then subjected to a hydrothermal reaction to coat the surface of the paramagnetic Fe3O4 nanoparticles with a metal-organic framework shell. The template agent was then removed to obtain the magnetic mesoporous core-shell material F5HPU-46. The magnetic mesoporous core-shell material F5HPU-46 was mixed with cellulase in a buffer solution, and after physical adsorption, magnetic separation and washing were performed to obtain immobilized cellulase.

4. The method for preparing immobilized cellulase from magnetic mesoporous core-shell material according to claim 3, characterized in that, The soluble iron salt is FeCl3, the citrate is anhydrous trisodium citrate, and the alcohol reagent is ethylene glycol.

5. The method for preparing immobilized cellulase using magnetic mesoporous core-shell material according to claim 3, characterized in that, The solvothermal reaction is carried out at a temperature of 200℃~201℃ for a time of 9.5h~10h.

6. The method for preparing immobilized cellulase from magnetic mesoporous core-shell material according to claim 3, characterized in that, The zirconium source is zirconium tetrachloride, the organic ligand is tetrafluoroterephthalic acid, and the template agent is block polyether F127.

7. The method for preparing immobilized cellulase from magnetic mesoporous core-shell material according to claim 3, characterized in that, The template removal agent refers to extraction with ethanol to remove block polyether F127 from the mesopores.

8. The method for preparing immobilized cellulase from magnetic mesoporous core-shell material according to claim 3, characterized in that, The acidic environment refers to the mixture of hydrochloric acid and glacial acetic acid in a volume ratio of 2 to 2.1:

3.

9. The method for preparing immobilized cellulase from magnetic mesoporous core-shell material according to claim 3, characterized in that, The buffer solution is a citrate buffer solution; The physical adsorption refers to physical adsorption at 300~500 r / min for 2h~2.5h.

10. The application of the immobilized cellulase according to claim 2 in the preparation of biomass fuel, characterized in that, The biomass fuel is made from lignocellulose.

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