Ternary composite sponge and application thereof

Abstract

The invention discloses a ternary composite sponge and application thereof, and belongs to the field of biotechnology and enzyme engineering. The ternary composite sponge is prepared from chitosan, pollen wall microgel and diatomaceous earth through ionic crosslinking and freeze drying processes, and the mass ratio of the chitosan to the pollen wall microgel to the diatomaceous earth is (1.25): (0.52): (0.20.6). The carrier has high porosity, large specific surface area and excellent biocompatibility, is suitable for immobilized protease, especially protein glutaminase, shows high enzyme loading capacity, high carrier enzyme activity and good reuse stability, and has wide application prospects in the fields of food processing and enzyme catalysis.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and enzyme engineering, specifically to a composite sponge carrier for immobilizing enzymes, its preparation method, and its application. Background Technology

[0002] Plant proteins, containing high levels of glutamine, are suitable substrates for deamidation. Enzymatic deamidation exhibits strong substrate specificity and can selectively modify protein structure. Protein glutaminase (PGase) is the most commonly used enzyme for deamidation modification, significantly improving protein functional properties. It specifically catalyzes the conversion of glutamine in proteins to glutamate without affecting asparagine or causing protein hydrolysis. However, free PGase suffers from poor stability, low activity, difficulty in recovery, and limitations in reaction conditions. Immobilizing PGase can improve its stability and reaction efficiency, facilitating separation and enabling reuse and controlled reactions.

[0003] Carriers suitable for immobilizing PGases should possess high biocompatibility, high mechanical stability, low mass transfer resistance, non-toxicity, and high protein affinity. Simultaneously, since both PGases and substrates are macromolecules, the carrier also needs a large porous structure to ensure unimpeded enzyme diffusion and substrate contact. The porosity and high surface area of ​​sponges make them a potential enzyme carrier. Sponges provide abundant immobilization sites, increasing the contact area between the enzyme and the carrier. Their pores provide reaction space for the enzyme, facilitating substrate access and thus improving immobilization and catalytic efficiency. For example, CN107254460A discloses a composite sponge prepared using carboxymethyl chitosan and sodium alginate for immobilizing papain, showing good recovery rate and stability. Chitosan (CS) is a commonly used sponge carrier material, but its relatively dense structure and limited internal space restrict the enzyme's mass transfer rate.

[0004] In contrast, sunflower pollen wall microgels (SPMGs) possess a large number of micron-sized pores and are rich in functional groups such as hydroxyl and carboxyl groups, providing natural pore sizes and attachment sites for biomolecules. For example, CN118345067A discloses that pollen wall microgels can act as a stabilizer for PGases, significantly improving their activity. Furthermore, due to their porous structure, pollen wall microgels also show potential in the preparation of sponge carriers. However, no sponge carriers made using pollen wall microgels have been reported to date, mainly because their poor formability makes it difficult to form a stable sponge structure. Based on this, this invention successfully developed a ternary composite sponge carrier with uniform structure and excellent performance by compounding pollen wall microgels with chitosan and introducing diatomaceous earth, and applied it to immobilized enzymes. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a ternary composite sponge with uniform structure and excellent performance, as well as its preparation method and application.

[0006] In a first aspect, the present invention provides a ternary composite sponge, which is prepared from raw materials comprising the following components through an ion crosslinking and freeze-drying process: chitosan, pollen wall microgel and diatomaceous earth, wherein the mass ratio of chitosan, pollen wall microgel and diatomaceous earth is (1.2-5):(0.5-2):(0.2-0.6).

[0007] Preferably, the mass ratio of chitosan, pollen wall microgel and diatomaceous earth is 1.2 : 1 : 0.3.

[0008] Secondly, the present invention provides a method for preparing the ternary composite sponge, comprising the following steps: (1) Dissolve chitosan in acetic acid solution to prepare chitosan solution; (2) Add diatomaceous earth to the chitosan solution and disperse it evenly to obtain solution A; (3) Add pollen wall microgel to solution A and disperse it evenly to obtain composite solution B; (4) The composite solution B is injected into the sodium tripolyphosphate solution for ionic cross-linking to form the composite gel C; (5) The composite gel C is freeze-dried to obtain the ternary composite sponge.

[0009] Preferably, the chitosan solution has a mass concentration of 1.5-3%, and / or the sodium tripolyphosphate solution has a mass concentration of 5-15%.

[0010] Preferably, the average particle size of the diatomaceous earth is 7-22 μm.

[0011] Preferably, the freeze-drying process parameters are: freezing temperature of -60℃ to -20℃; and / or, sublimation temperature of 10 to 40℃, and vacuum pressure of 1 to 5 Pa.

[0012] More preferably, the freeze-drying process parameters are: freezing temperature of -40°C; and / or, sublimation temperature of 20°C, and vacuum pressure of 2.5 Pa.

[0013] Thirdly, the present invention provides the application of the composite sponge in immobilized proteases.

[0014] Fourthly, the present invention provides a method for immobilizing protein glutaminase, comprising the following steps: (1) The composite sponge was activated using glutaraldehyde solution; (2) The activated composite sponge was reacted with a protein glutaminase solution to obtain immobilized protein glutaminase.

[0015] Preferably, in step (1), the glutaraldehyde solution has a mass concentration of 0.4-0.8%, an activation temperature of 20-40℃, and an activation time of 3-8 hours. Preferably, in step (2), the mass concentration of the protein glutaminase solution is 1-3%, the reaction temperature is 20-30℃, and the reaction time is 2-4 hours.

[0016] The beneficial effects of this invention are as follows: In this invention, pollen wall microgels provide abundant functional groups and natural micropores, playing a role in pore formation and enzyme stabilization. Chitosan constitutes the basic three-dimensional framework of the sponge, while diatomaceous earth not only acts as a dispersant, but its rigid inorganic framework and high porosity enhance the sponge's mechanical strength and pore connectivity. These three components work synergistically to form an ideal carrier with high porosity and a hierarchical pore structure. Immobilizing proteases using this carrier exhibits high carrier enzyme activity and high loading capacity. Simultaneously, the immobilized enzyme demonstrates excellent stability, reusability, and catalytic activity, expanding the application range of PGase.

[0017] This invention effectively inhibits electrostatic aggregation between chitosan and pollen wall microgels, significantly improves the formability and pore structure uniformity of the composite sponge, uses biocompatible and biodegradable raw materials, and has mild preparation conditions, making it suitable for large-scale production and showing great promise for industrial applications. Attached Figure Description

[0018] Figure 1 Photographs and SEM microstructure diagrams of the sponges used in the examples and comparative examples.

[0019] Figure 2 Thermogravimetric analysis (TG) curves of different sponge carriers.

[0020] Figure 3 Example 1 and Comparative Example 1 3. FTIR spectrum comparison diagram.

[0021] Figure 4 : Comparison of the infrared (FTIR) spectra of immobilized PGase and the raw material. Detailed Implementation

[0022] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that these embodiments are only for illustrating the present invention and not for limiting the scope of the present invention. Experiments in the following embodiments that do not specify specific methods are generally conventional operations in the art and can be carried out in accordance with the methods listed in reference books or other prior patents filed by the applicant.

[0023] In previous studies, we demonstrated that pollen wall microgels (SPMGs), due to their unique spatial physical microenvironment and surface functional groups, can significantly improve the catalytic activity and stability of Pgase enzymes, and based on this, we have developed several industrial carriers with promising applications. In this study, we developed a high-performance composite sponge carrier to immobilize PGase enzymes, solving the problems of poor stability and difficulty in recovery of free enzymes. The key innovation of this study lies in the synergistic effect of chitosan (CS), pollen wall microgels (SPMGs), and diatomaceous earth to successfully prepare a high-performance ternary composite sponge (CS / SPMG / diatomaceous earth), and to solve the technical challenges of poor sponge moldability and low enzyme immobilization efficiency.

[0024] Materials: SPMG, prepared by the applicant, method referenced CN 118345067A; Chitosan, purchased from Aladdin Reagent (Shanghai) Co., Ltd., catalog number C105802; Diatomaceous earth, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., catalog number C828579; Sodium tripolyphosphate, purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S30235; PGase enzyme, purchased from Tano Corporation, Japan, enzyme activity 660 U / g; Alkaline protease, purchased from Shanghai Yuanye Biotechnology Co., Ltd., enzyme activity 200 U / mg; Papain, purchased from Shanghai Yuanye Biotechnology Co., Ltd., enzyme activity 800 U / mg; Bromelain, purchased from Shanghai Yuanye Biotechnology Co., Ltd., enzyme activity 600 U / mg; Glutaraldehyde, purchased from Sinopharm Chemical Reagent Co., Ltd., chemically pure; Substrate: Z-glutamylglycine (Z-Gln-Gly), purchased from Aladdin Reagent (Shanghai) Co., Ltd., catalog number G121389.

[0025] Instruments: Scanning electron microscope (SEM), Carl Zeiss AG, Germany, model ZEISS Gemini 30; Automated surface area and porosity analyzer (BET), Micromeritics, USA, model ASAP 2460; Thermogravimetric analyzer (TG), TA Instruments, USA, model Q500; Fourier transform infrared spectrometer (FTIR), Bruker, Germany, model Tensor 27; Microplate reader, VICTOR Nivo, USA.

[0026] Part 1: Preparation and Performance Evaluation of Composite Sponges SPMG contains abundant functional groups and natural micropores, which can protect PGase enzymes and improve their catalytic activity. However, SPMG has poor formability and cannot form a stable sponge structure on its own. Combining SPMG with other sponge carrier materials such as CS is one of the effective solutions to improve formability. However, there is an electrostatic aggregation effect between positively charged CS and negatively charged SPMG, resulting in uneven pore distribution in the sponge and affecting the immobilization effect of the enzyme. Therefore, we introduced diatomaceous earth to improve the aggregation behavior of the two materials. In the preparation of the composite sponge, in addition to the ratio of the above three materials, solution concentration, diatomaceous earth particle size, and freeze-drying parameters will affect the sponge performance. We finally determined the range of these parameters through a large number of experiments. Some examples are shown below.

[0027] Example 1: Preparation of ternary composite sponge Chitosan was dissolved in a 2% (w / w) acetic acid solution to prepare a 2% (w / w) chitosan solution. 13 μm diatomaceous earth was added to the chitosan solution and dispersed evenly to obtain solution A. SPMG was slowly added to A while stirring, maintaining a chitosan:SPMG:diatomaceous earth weight ratio of 1.2:1:0.3. The mixture was then ultrasonically dispersed to obtain composite solution B. Using a 2 ml syringe, B was dispersed in a 7.15% (w / w) STPP solution and allowed to stand for 6 hours to obtain composite gel C. C was frozen at -40℃ for 12 hours to obtain cryogel D. D was placed in a vacuum freeze dryer and freeze-dried at 2.5 Pa and a sublimation temperature of 20℃ for 12 hours to remove moisture, yielding a dried composite sponge.

[0028] Example 2: Preparation of ternary composite sponge Chitosan was dissolved in a 2% acetic acid solution to prepare a 2% chitosan solution. 13 μm diatomaceous earth was added to the chitosan solution and dispersed evenly to obtain solution A. SPMG was slowly added to A while stirring, maintaining a chitosan:SPMG:diatomaceous earth weight ratio of 5:2:0.4. The mixture was then ultrasonically dispersed to obtain composite solution B. B was dispersed in a 7.15% STPP solution using a 2 ml syringe and allowed to stand for 6 hours to obtain composite gel C. C was frozen at -40℃ for 12 hours to obtain cryogel D. D was placed in a vacuum freeze dryer and freeze-dried at 2.5 Pa and a sublimation temperature of 20℃ for 12 hours to remove moisture, resulting in a dried composite sponge.

[0029] Example 3: Preparation of ternary composite sponge Chitosan was dissolved in a 2% acetic acid solution to prepare a 2% chitosan solution. 13 μm diatomaceous earth was added to the chitosan solution and dispersed evenly to obtain solution A. SPMG was slowly added to A while stirring, maintaining a chitosan:SPMG:diatomaceous earth weight ratio of 2:0.5:0.6. The mixture was then ultrasonically dispersed to obtain composite solution B. B was dispersed in a 7.15% STPP solution using a 2 ml syringe and allowed to stand for 6 hours to obtain composite gel C. C was then frozen at -40℃ for 12 hours to obtain cryogel D. D was placed in a vacuum freeze dryer and freeze-dried at 2.5 Pa and a sublimation temperature of 20℃ for 12 hours to remove moisture, resulting in a dried composite sponge.

[0030] Example 4: Preparation of ternary composite sponge Chitosan was dissolved in a 2% acetic acid solution to prepare a 2% chitosan solution. 13 μm diatomaceous earth was added to the chitosan solution and dispersed evenly to obtain solution A. SPMG was slowly added to A while stirring, maintaining a chitosan:SPMG:diatomaceous earth weight ratio of 1.5:2:0.2. The mixture was then ultrasonically dispersed to obtain composite solution B. B was dispersed in a 7.15% STPP solution using a 2 ml syringe and allowed to stand for 6 hours to obtain composite gel C. C was then frozen at -40℃ for 12 hours to obtain cryogel D. D was placed in a vacuum freeze dryer and freeze-dried at 2.5 Pa and a sublimation temperature of 20℃ for 12 hours to remove moisture, resulting in a dried composite sponge.

[0031] Example 5: Preparation of ternary composite sponge Chitosan was dissolved in a 2% acetic acid solution to prepare a 1.5% chitosan solution. 7μm diatomaceous earth was added to the chitosan solution and dispersed evenly to obtain solution A. SPMG was slowly added to A while stirring, maintaining a chitosan:SPMG:diatomaceous earth weight ratio of 1.2:1:0.3. The mixture was then ultrasonically dispersed to obtain composite solution B. B was dispersed in a 15% STPP solution using a 2ml syringe and allowed to stand for 6 hours to obtain composite gel C. C was frozen at -20℃ for 12 hours to obtain cryogel D. D was placed in a vacuum freeze dryer and freeze-dried at 1 Pa and a sublimation temperature of 40℃ for 12 hours to remove moisture, yielding a dried composite sponge.

[0032] Example 6: Preparation of ternary composite sponge Chitosan was dissolved in a 2% acetic acid solution to prepare a 3% chitosan solution. 22 μm diatomaceous earth was added to the chitosan solution and dispersed evenly to obtain solution A. SPMG was slowly added to A while stirring, maintaining a chitosan:SPMG:diatomaceous earth weight ratio of 1.2:1:0.3. The mixture was then ultrasonically dispersed to obtain composite solution B. B was dispersed in a 5% STPP solution using a 2 ml syringe and allowed to stand for 6 hours to obtain composite gel C. C was then frozen at -60℃ for 12 hours to obtain cryogel D. D was placed in a vacuum freeze dryer and freeze-dried at 5 Pa and a sublimation temperature of 10℃ for 12 hours to remove moisture, resulting in a dried composite sponge.

[0033] Comparative Example 1 (CS+SPMG) Chitosan was dissolved in a 2% acetic acid solution to prepare a 2% chitosan solution. SPMG was slowly added to the chitosan solution while stirring, until the weight ratio of chitosan to SPMG was 1.2:1. The mixture was then sonicated to obtain composite solution A. Using a 2ml syringe, A was dispersed in a 7.15% STPP solution and allowed to stand for 6 hours to obtain composite gel B. B was then frozen at -40℃ for 12 hours to obtain cryogel C. C was then placed in a vacuum freeze dryer and freeze-dried at 2.5 Pa and a sublimation temperature of 20℃ for 12 hours to remove moisture, yielding the dried product.

[0034] Comparative Example 2 (SPMG) Using a 2ml syringe, SPMG was dispersed in a 7.15% STPP solution to prepare a 1.5% SPMG solution. After standing for 6 hours, gel A was obtained. Gel A was then frozen at -40℃ for 12 hours to obtain cryogel B. Gel B was then placed in a vacuum freeze dryer and freeze-dried at 2.5 Pa and a sublimation temperature of 20℃ for 12 hours. After the water in the sample was removed, the dried product was obtained.

[0035] Comparative Example 3 (CS) Chitosan was dissolved in a 2% acetic acid solution to prepare a 2% chitosan solution. Using a 2ml syringe, the chitosan solution was dispersed in a 7.15% STPP solution and allowed to stand for 6 hours to obtain gel A. Gel A was then frozen at -40℃ for 12 hours to obtain cryogel B. Gel B was placed in a vacuum freeze dryer and freeze-dried at 2.5 Pa and a sublimation temperature of 20℃ for 12 hours to remove moisture, yielding the dried product.

[0036] Experiment 1: Appearance and Morphology of Composite Sponge Figure 1The images show photographs of the physical appearance of the examples and comparative samples, as well as SEM microstructure images of their cross-sections. The product prepared from SPMG (Comparative Example 2) exhibits structural collapse, failing to form a stable sponge structure. Scanning electron microscopy reveals a loose structure containing numerous pores. The product prepared from CS (Comparative Example 3) demonstrates good formability, with a smooth and flat surface. SEM results show a dense external structure lacking obvious pores, while the internal structure presents a flat, sheet-like structure with few pores. When SPMG and CS are combined (Comparative Example 1), the sponge surface becomes rough, forming a relatively loose network structure with increased internal pores. However, due to the electrostatic interaction between SPMG and CS, SPMG aggregates extensively, resulting in uneven pore distribution. Introducing diatomaceous earth (Example 1) suppresses SPMG aggregation, significantly improves the sponge's uniformity, and achieves a highly uniform pore distribution within the microstructure, forming a stable network structure.

[0037] Experiment 2: Porosity of Composite Sponge Macroscopic porosity can be used to characterize the looseness of a sponge carrier. The porosity is calculated by measuring the mass difference of the sample before and after immersion in ethanol, and the ratio of this mass to the sample volume. The specific measurement method is as follows: The sponge sample is weighed and recorded as M1. It is then immersed in ethanol to fully absorb the liquid, removed, and excess liquid is absorbed with filter paper. The sample is weighed again and recorded as M2. Simultaneously, the diameter of the sponge is measured using vernier calipers, and its volume is calculated. The porosity measurement results for each sample are shown in Table 1.

[0038] Table 1: Porosity of different sponge carriers

[0039] Note: Due to the irregular shape, the volume could not be measured, so no data is given for Comparative Example 2; all the above data are the average of three repetitions.

[0040] The results showed that Comparative Example 3 had the lowest porosity, Comparative Example 1 had a significantly higher porosity, and the porosity of each embodiment was further increased, reaching up to nearly 90%. This is mainly due to the synergistic pore-forming effect of SPMG and diatomaceous earth. SPMG contains a large number of natural micropores, while diatomaceous earth, as a rigid inorganic particle, can reduce the excessive aggregation between CS and SPMG, improve the connectivity of the channels, and make the pores more uniform in spatial distribution.

[0041] Experiment 3: Specific surface area and average pore size of composite sponge The specific surface area and average pore size of the sponge carrier were determined using the BET method, and the results are shown in Table 2.

[0042] Table 2: Specific surface area and average pore size of different sponge carriers

[0043] The results showed that all sponge carriers exhibited typical porous adsorption characteristics, with samples composed of mesopores (2-50 nm) of different sizes, along with a small number of micropores (< 2 nm) and macropores (> 50 nm). Comparative Example 3 had a specific surface area of ​​5.99 m² / g, with a relatively dense pore structure. The addition of SPMG increased the average pore size but slightly decreased the specific surface area. In contrast, the introduction of diatomaceous earth significantly increased the specific surface area, and the uniform pore structure and high specific surface area provided a favorable microenvironment for enzyme immobilization.

[0044] Experiment 4: Stability of Composite Sponge (1) Mechanical stability The ability of a sponge carrier to resist breakage or swelling under external force or fluid impact, maintaining its structural integrity, is a prerequisite for its effective application. The mechanical stability of a sponge carrier can be determined by measuring the percentage of its dry mass remaining after treatment under specific mechanical disturbance conditions (vibration, stirring, etc.) relative to its initial dry mass. The method is as follows: Approximately 1 g of sample was weighed and immersed in 20 mL of deionized water, then shaken at 250 rpm for 2 h. The collected sponge and 1 g of unshaken sponge were then dried to equilibrium and weighed. The mechanical stability of the sample was calculated from the percentage of dry weight. The results are shown in Table 3.

[0045] Table 3 Mechanical stability of different sponge carriers

[0046] Note: The above data are the average of three repetitions.

[0047] The results show that Comparative Example 2 has very low mechanical stability due to its loose structure. Comparative Example 3 has a dense structure and high mechanical stability. The mechanical stability of Comparative Example 1 is lower than that of Comparative Example 3. This is because the electrostatic aggregation of CS and SPMG disrupts the uniform molecular chain network of CS to some extent, hindering the effective crosslinking of STPP and CS, resulting in a decrease in the crosslinking density of the sponge. Introducing diatomaceous earth into the composite system reduces the aggregates formed by electrostatic interactions, increases the crosslinking density, and makes the three-dimensional network structure of the composite sponge more continuous. Furthermore, the inorganic inert framework of diatomaceous earth itself provides additional support for the material, thereby improving the mechanical stability of the sponge.

[0048] 4.2 Thermal stability The thermal stability of the sample was determined by thermogravimetric analysis (TG). The results are as follows: Figure 2 As shown.

[0049] TG results showed that all samples exhibited slight weight loss below 150℃, mainly due to the evaporation of residual moisture. Within the 240–360℃ range, the CS-containing sample showed a significant weight loss peak, corresponding to the thermal decomposition of the chitosan backbone. Comparative Example 2 showed poor thermal stability; after being combined with CS, the weight loss curve of Comparative Example 1 was flatter than that of Comparative Example 3, but its final mass loss was still higher than that of Comparative Example 3. Further introduction of diatomaceous earth resulted in a smoother weight loss process in the composite sponge, with a significant reduction in total weight loss. This is mainly due to the high thermal inertia of diatomaceous earth hindering heat conduction and decomposition diffusion, giving the composite sponge a higher thermal decomposition temperature and structural stability. The final mass loss rates were: 50.7% for Comparative Example 3, 75.17% for Comparative Example 2, 51.28% for Comparative Example 1, and 43.7% for Example 1.

[0050] Experiment 5: Interactions of Composite Sponges FTIR infrared spectroscopy can be used to analyze the interactions between different materials and samples. The infrared spectra of Example 1 and Comparative Examples 1, 2, and 3 are as follows: Figure 3 As shown.

[0051] FTIR results showed that CS was at 2930.31 cm⁻¹. -1 The methylene (-CH2) stretching vibration peak redshifted to approximately 2927 cm⁻¹ after interaction with STPP. -1 (Comparative Example 3, Comparative Example 1, Example 1) demonstrates that STPP, as a polyvalent anion, can undergo ionic crosslinking with CS. In the spectrum of CS, 3443.66 cm⁻¹ -1 The amino (NH) stretching vibration peak in Comparative Example 3 was significantly shifted to 3413.58 cm⁻¹. -1 This further explains the phosphate (PO4) in STPP. 3- It can form hydrogen bonds with the amino group of CS, thereby altering its vibrational environment. Simultaneously, the phosphorus oxyphosphorus (POP) stretching vibration peak of STPP changes from 896.16 cm⁻¹. -1 Moved to 850.97 cm -1 This also indicates a strong interaction between CS and STPP. In contrast, the changes in the POP peak of STPP in Comparative Example 2, Comparative Example 1, and Example 1 are not significant. Even with the presence of CS (Comparative Example 1 and Example 1), there is no obvious shift as seen in Comparative Example 3. This suggests that the introduction of SPMG has suppressed the changes in the STPP structure to some extent, and the main interactions in the system are still concentrated between CS and STPP.

[0052] In the spectrum of SPMG, 3411.81 cm⁻¹ -1 The hydroxyl (-OH) stretching vibration peaks and the peaks at 1661.13 and 1434.05 cm⁻¹ -1The carboxylate peak remained almost unchanged in Comparative Example 2, indicating a weak interaction with STPP. However, in Comparative Example 1, the carboxylate peaks red-shifted to 1656.62 cm⁻¹. -1 and 1430.53 cm -1 This indicates a significant enhancement in the electrostatic adsorption between CS (cations) and SPMG (anions). However, after the introduction of diatomaceous earth (Example 1), the carboxylate peak returned to a position similar to that of SPMG (1662.48, 1433.45 cm⁻¹). -1 This indicates that diatomaceous earth effectively suppressed the strong electrostatic bonding between CS / SPMG, resulting in a more uniform network structure. Furthermore, the asymmetric stretching peak of the silicon-oxygen bond (Si-O-Si) in diatomaceous earth changed from 1090.32 cm⁻¹. -1 The blue shifted to 1126.30 cm. -1 This indicates that its silicon-oxygen network participated in the construction of the composite structure. The presence of diatomite not only improved the dispersion of CS and SPMG, but also weakened the excessive electrostatic interaction, thereby avoiding the formation of large aggregates and helping to construct a more stable three-dimensional network.

[0053] Overall, the ionic crosslinking and hydrogen bonding between CS and STPP are the main sources of composite structure enhancement, while the electrostatic interaction between SPMG and CS has a significant impact on network uniformity. Diatomaceous earth, by regulating interfacial interactions and inhibiting excessive composite formation, enables the ternary composite sponge to form a more stable and uniform structure. These multiple interactions achieve a synergistic effect, giving the composite carrier both high structural stability and good mass transfer performance, which is beneficial for improving the application effect of immobilized enzymes.

[0054] Experiment 6: Immobilization effect of composite sponge on PGase enzyme The composite sponge carrier was added to a glutaraldehyde solution (0.65%, w / w) and activated at 25°C and 150 rpm for 6.5 h, then washed and ready for use. A 2% (w / w) PGase solution was prepared, and the activated composite sponge was added to the PGase solution. The reaction was carried out at 20°C and 150 rpm for 4 h, followed by washing to obtain composite sponge immobilized with PGase enzyme.

[0055] The enzyme activity and loading capacity of each vector for immobilizing PGase were investigated, and the results are shown in Table 4 below.

[0056] Table 4. Effects of different vectors on enzyme activity and loading capacity of immobilized PGase vectors

[0057] Note: The above data are the average of three repetitions.

[0058] The results showed that Comparative Example 3, due to its dense structure and low porosity, had high mass transfer resistance, resulting in low enzyme activity and enzyme loading. Introducing SPMG into the carrier (Comparative Example 1) allowed the -COOH and -OH functional groups in the SPMG molecule to provide more reaction sites for immobilized PGase, while simultaneously promoting the formation of a porous structure in chitosan, significantly improving pore connectivity and reducing mass transfer resistance, thereby providing more attachment sites for PGase molecules and enhancing immobilization efficiency. Furthermore, the incorporation of diatomaceous earth (Examples 1-6) further increased the enzyme activity and enzyme loading of the carrier. This was mainly because the addition of diatomaceous earth effectively inhibited excessive aggregation between CS and SPMG, resulting in a more uniform distribution of reaction sites and significantly improving overall enzyme activity.

[0059] Experiment 7: Immobilization effect of composite sponges on other enzymes The composite sponge carrier was added to a glutaraldehyde solution (0.65%, w / w) and activated at 25℃ and 150 rpm for 6.5 h, then washed and ready for use. Alkaline protease, papain, and bromelain solutions with concentrations of 2% w / w were prepared. The activated composite sponge was added to the enzyme solutions, reacted at 20℃ and 150 rpm for 4 h, and then washed to obtain composite sponge immobilized protease. The enzyme activity and loading of different immobilized proteases are shown in Table 5 below.

[0060] Table 5. Immobilization effect of composite sponge carrier on different proteases

[0061] Note: The above data are the average of three repetitions.

[0062] The composite sponge carrier prepared by this invention can not only successfully immobilize PGase, but also exhibits good immobilization ability for a variety of other proteases, proving that the carrier has good versatility and providing possibilities for its expanded application in food processing and biocatalysis.

[0063] Part Two: Applications of Composite Sponges The composite sponge carrier prepared in this invention is composed of CS, SPMG, and diatomaceous earth. CS is rich in amino (–NH2) and hydroxyl (–OH) groups, SPMG is rich in carboxyl (–COOH) and hydroxyl (–OH) groups, and PGase molecules have amino and carboxyl groups on their surface. Glutaraldehyde, as a bifunctional cross-linking agent, can chemically react with these groups. First, the aldehyde group preferentially undergoes a Schiff base reaction with the amino group to form a C=N bond, achieving covalent bonding. Second, the aldehyde group can also undergo an aldol condensation reaction with the hydroxyl groups in SPMG or CS, further enhancing the activity and structural stability of the carrier. By first activating the composite sponge carrier with glutaraldehyde to introduce aldehyde groups onto the carrier surface, and then cross-linking with the amino groups on the PGase surface, covalent bonding between the enzyme molecule and the carrier can be achieved. The concentration of glutaraldehyde, activation temperature, concentration of the PGase solution, and reaction temperature have a significant impact on the immobilization effect. The following are some experimental examples.

[0064] Example 7 The composite sponge carrier was added to a glutaraldehyde solution (0.65%, w / w) and activated at 25°C and 150 rpm for 6.5 h, then washed and ready for use. A 2% (w / w) PGase solution was prepared, and the activated composite sponge was added to the PGase solution. The reaction was carried out at 20°C and 150 rpm for 4 h, followed by washing to obtain composite sponge immobilized with PGase enzyme.

[0065] Example 8 The composite sponge carrier was added to a glutaraldehyde solution (0.8%, w / w), activated at 30°C and 150 rpm for 5 h, and then washed for later use. A 1.5% (w / w) PGase solution was prepared, and the activated composite sponge was added to the PGase solution. The reaction was carried out at 30°C and 150 rpm for 3 h, and then washed to obtain composite sponge immobilized with PGase enzyme.

[0066] Example 9 The composite sponge carrier was added to a glutaraldehyde solution (0.4%, w / w) and activated at 35°C and 150 rpm for 3.5 h, then washed and ready for use. A 2.5% (w / w) PGase solution was prepared, and the activated composite sponge was added to the PGase solution. The reaction was carried out at 25°C and 150 rpm for 2 h, then washed to obtain composite sponge immobilized with PGase enzyme.

[0067] Experiment 1: Determination of enzyme immobilization effect Immobilized PGase was prepared using the methods described above, and the enzyme activity, loading capacity, and reusability of the immobilized PGase were investigated for each method. The results are shown in Table 6 below.

[0068] Table 6. Enzyme immobilization effects of different immobilization methods

[0069] Note: The above data are the average of three repetitions.

[0070] Experiment 2: Interactions of immobilized PGase Infrared spectroscopy of the free enzyme, the composite sponge carrier, and the immobilized PGase can determine whether PGase has been successfully immobilized. The measurement results are as follows: Figure 4 As shown.

[0071] The results showed that the hydroxyl stretching vibration peaks of Example 1 and PGase were located at 3419.23 cm⁻¹. -1 and 3423.15cm -1 In Examples 7, 8, and 9, the hydroxyl stretching vibration peak shifted to 3414.31 cm⁻¹, respectively. -1 3417.54 cm -1 3414.33 cm -1 The amide I band is one of the main characteristic absorption peaks of protein molecules, caused by the stretching vibration of the C=O bond, and is located at 1600–1700 cm⁻¹. -1 The asymmetric and symmetric stretching vibration peaks of the carboxylate group in PGase are located at 1659.55 cm⁻¹. -1 and 1430.26 cm -1 In Example 1, the asymmetric and symmetric stretching vibration peaks of the carboxylate group were located at 1662.48 cm⁻¹. - ¹ and 1433.45 cm - ¹. In Example 7 (1662.63 cm) - ¹ and 1427.63 cm - ¹), Example 8 (1661.93 cm) - ¹ and 1417.11 cm - ¹), Example 9 (1661.38 cm) - ¹ and 1418.25 cm - In the FTIR spectrum of ¹), the characteristic peaks of the carboxylate groups showed a significant shift. Based on the changes in the characteristic peaks of amide I and the hydroxyl group, it is indicated that an interaction occurred between PGase and the sponge support of Example 1. Simultaneously, the asymmetric stretching vibration peak of the silicon-oxygen bond in Example 1 was located at 1126.3 cm⁻¹. -1 In Examples 7, 8, and 9, the peak shifted to 1078.06 cm⁻¹, respectively. -1 1070.59 cm -1 1080.01 cm -1This indicates that the incorporation of PGase had a significant impact on the diatomaceous earth in the composite sponge, confirming the interaction between the sponge carrier and PGase, and further demonstrating that PGase was successfully immobilized on the sponge carrier prepared in Example 1.

[0072] Infrared spectroscopy results showed that there was a significant interaction between PGase and the composite sponge carrier, and the diatomaceous earth component in the carrier also participated in the binding process, indicating that PGase was successfully fixed on the composite sponge.

[0073] Experiment 3: Determination of kinetic parameters of immobilized enzymes Enzyme kinetic parameters Km (Michaelis constant) and Vmax (maximum reaction rate) can be used to characterize the affinity between the enzyme and its substrate and its maximum catalytic activity. These parameters can be obtained by measuring the reaction rates at different substrate concentrations and performing kinetic fitting on the rate data. The specific measurement method is as follows: PGase is placed in the reaction system, and substrate solutions of different concentrations are added at a constant temperature, recording the initial reaction rate v. Subsequently, the substrate concentrations and corresponding initial rates are substituted into the Michaelis equation, and the Km and Vmax values ​​are calculated using the Lineweaver–Burk double reciprocal method. The kinetic parameter measurement results for free PGase and Example 8 are shown in Table 7.

[0074] Table 7 Kinetic parameters of free PGase and immobilized PGase

[0075] Note: The above data are the average of three repetitions.

[0076] The results showed that both Km and Vmax in Example 8 were significantly higher than those in free PGase. The increase in Km indicates a decrease in the enzyme's affinity for the substrate after immobilization, mainly due to the internal and external diffusion resistance introduced by immobilization, further confirming that the enzyme was successfully immobilized on the support. Although Km increased, Vmax increased simultaneously, indicating that the immobilization process effectively stabilized the conformation of PGase, increased the number of accessible active sites, and enabled it to maintain higher catalytic efficiency at higher substrate concentrations. In summary, immobilization not only endows the enzyme with better structural stability but also improves catalytic performance while maintaining its immobilized state, demonstrating the significant advantages of immobilized enzymes in high substrate loading environments.

Claims

1. A ternary composite sponge, characterized by, The ternary composite sponge is prepared from raw materials comprising the following components through ion crosslinking and freeze-drying processes: chitosan, pollen wall microgel and diatomaceous earth, wherein the mass ratio of chitosan, pollen wall microgel and diatomaceous earth is (1.2-5):(0.5-2):(0.2-0.6).

2. The ternary composite sponge according to claim 1, wherein: The mass ratio of chitosan, pollen wall microgel, and diatomaceous earth is 1.2:1:0.

3.

3. A method of preparing the ternary composite sponge of claim 1 or 2, characterized in that, Includes the following steps: (1) Dissolve chitosan in acetic acid solution to prepare chitosan solution; (2) Add diatomaceous earth to the chitosan solution and disperse it evenly to obtain solution A; (3) Add pollen wall microgel to solution A and disperse it evenly to obtain composite solution B; (4) The composite solution B is injected into the sodium tripolyphosphate solution for ionic cross-linking to form the composite gel C; (5) The composite gel C is freeze-dried to obtain the ternary composite sponge.

4. The method of claim 3, wherein: The chitosan solution has a mass concentration of 1.5-3%, and / or the sodium tripolyphosphate solution has a mass concentration of 5-15%.

5. The method of claim 3, wherein: The average particle size of the diatomaceous earth is 7-22 μm.

6. The method of claim 3, wherein, The freeze-drying process parameters are: freezing temperature of -60℃ to -20℃; and / or, sublimation temperature of 10 to 40℃, and vacuum pressure of 1 to 5 Pa.

7. The method of claim 6, wherein, The freeze-drying process parameters are: freezing temperature of -40℃; and / or sublimation temperature of 20℃ and vacuum pressure of 2.5Pa.

8. The application of the ternary composite sponge according to claim 1 or 2 in immobilized protease.

9. A method of immobilizing a protein glutaminase, characterized by, Includes the following steps: (1) Activate the composite sponge as described in claim 1 or 2 using glutaraldehyde solution; (2) The activated composite sponge was added to the protein glutaminase solution to react and obtain immobilized protein glutaminase.

10. The method of claim 9, wherein: In step (1), the mass concentration of the glutaraldehyde solution is 0.4-0.8%, the activation temperature is 20-40℃, and the activation time is 3-8 hours; and / or, in step (2), the mass concentration of the protein glutaminase solution is 1-3%, the reaction temperature is 20-30℃, and the reaction time is 2-4 hours.

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