Biological enzyme for catalyzing slow-release biological energy substance and fixing method thereof
By immobilizing dopamine and amylase on a ceramic matrix, the problem of poor enzyme stability was solved, enabling effective sustained release of glucose and nutritional support at bone defects, thus promoting cell proliferation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANGHAI INST OF CERAMIC CHEM & TECH CHINESE ACAD OF SCI
- Filing Date
- 2024-12-19
- Publication Date
- 2026-05-12
AI Technical Summary
Existing biological enzymes, such as amylase, have poor stability in application and are easily affected by changes in temperature, solution salt concentration, and pH, resulting in reduced catalytic activity and an inability to effectively release glucose to address the problem of energy deficiency at bone defects.
A method for immobilizing dopamine and amylase on a ceramic matrix was adopted. By mixing and treating the ceramic matrix, dopamine and amylase in a specific ratio, a stable bio-enzyme complex was formed. Coaxial scaffolds were then prepared using coaxial 3D printing for glucose slow release.
It significantly improved the stability of biological enzymes and the sustained release effect of glucose, promoted the proliferation of bone marrow mesenchymal stem cells, and enhanced nutritional support at bone defects.
Smart Images
Figure CN119736278B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of tissue engineering and bone repair technology, specifically relating to a bioenzyme for catalytically releasing bioenergy substances and its fixation method. Background Technology
[0002] Bone defects caused by infection, trauma, and various diseases (such as osteonecrosis, osteoporosis, and bone tumors) pose a serious threat to patients' health and quality of life. Traditional bone repair materials mainly work by releasing calcium... 2+ PO4 3- Mg 2+ and Zn 2+ Plasma can promote cell differentiation and activate related cellular pathways, thereby accelerating bone tissue repair. However, bone defects are often accompanied by severe blood supply impairment, which obstructs the transport channels for bioenergy substances (including glucose, fatty acids, and amino acids). This results in a lack of necessary nutritional support for the tissues and cells at the bone defect site, ultimately affecting the effectiveness of bone repair. Cells obtain adenosine triphosphate (ATP) by breaking down glucose, fatty acids, and amino acids obtained from food or stored in the body to meet normal cellular energy needs, and glucose is the preferred energy source for most cells in the body. Therefore, directly releasing glucose at the defect site is the best option to address the nutrient deficiency at the bone defect site, especially in the early stages.
[0003] Bioenzymes are organic compounds produced by living cells that possess catalytic functions. They can significantly lower the activation energy of chemical reactions, thereby accelerating the reaction rate. Based on the different substrates and catalyzed reaction types, bioenzymes can be further subdivided into various types, such as pectinases, lipases, amylases, and cellulases. Bioenzymes have advantages such as high catalytic activity, rapid catalytic efficiency, reaction specificity, low dosage, and mild reaction conditions, and have wide applications in pharmaceuticals, cosmetics, pulp and paper, biofuels, textiles, food, water treatment, petroleum, brewing, and biomedicine.
[0004] The glucose needed by the human body usually exists in food in the form of carbohydrates (such as starch). Foods rich in carbohydrates, such as rice, pasta, bread, fruits, and vegetables, are important sources of glucose. When the body ingests these foods, the digestive system uses various enzymes (salivary amylase, pancreatic amylase, and intestinal amylase) to break them down into glucose monosaccharides, which are then absorbed into body tissues to provide energy. Therefore, the in-situ catalytic slow release of glucose using enzymes can be used to address the problem of energy deficiency at bone defects.
[0005] Studies have found that amylase can catalyze the production of glucose monosaccharides from starch. However, its application currently faces the challenge of poor stability: the activity of biological enzymes is easily affected by various factors, such as temperature, changes in the concentration and pH of various salts in the solution, which may lead to structural changes in the enzyme and thus loss of catalytic activity. Furthermore, over time, it is also prone to inactivation and loss of catalytic activity due to its own structural instability. Therefore, improving the stability of biological enzymes is crucial. Summary of the Invention
[0006] To address the technical problem of poor stability of amylase, the present invention aims to provide a fixation method for improving the stability of amylase.
[0007] In a first aspect, the present invention provides a bio-enzyme for catalyzing the slow release of bioenergy substances, the bio-enzyme comprising: a ceramic matrix, dopamine loaded on the ceramic matrix, and amylase loaded on the dopamine.
[0008] Based on the total mass of the bio-enzymes used to catalyze the slow release of bioenergy substances as 100%, the ceramic matrix accounts for 80-90% of the mass, dopamine accounts for 5-10% of the mass, and amylase accounts for 1-10% of the mass.
[0009] Preferably, the ceramic matrix is at least one of hydroxyapatite, β-TCP, and dicalcium silicate, with β-TCP being the most preferred.
[0010] Secondly, the present invention provides a method for immobilizing the above-mentioned bioenzyme for catalytic slow-release of bioenergy substances, the immobilization method comprising the following steps:
[0011] (1) Mix ceramic powder with dopamine hydrochloride to obtain raw material mixed powder, then add deionized water and stir to obtain dopamine-loaded ceramic powder;
[0012] (2) The dopamine-loaded ceramic powder is mixed with amyloglucosidase, then deionized water is added and the mixture is shaken to ensure that the amyloglucosidase is fully loaded and bound to the dopamine, thereby obtaining the fixed bio-enzyme for catalyzing the slow release of bioenergy substances.
[0013] Preferably, in step (1), the ceramic powder has a particle size of 100 nm-10 μm, more preferably 300 nm-10 μm, and a specific surface area of 10-50 m². 2 / g.
[0014] Preferably, in step (1), the mass ratio of the ceramic powder to dopamine hydrochloride is 5-10:1.
[0015] Preferably, in step (1), the ratio of the raw material mixed powder to deionized water is 1g:5-10mL.
[0016] Preferably, in step (2), the mass ratio of the dopamine-loaded ceramic powder to the amylase is 1:0.1-0.4.
[0017] Preferably, in step (2), the ratio of the dopamine-loaded ceramic powder to deionized water is 1g:5-50mL, more preferably 1g:20-50mL.
[0018] Beneficial effects
[0019] This invention significantly improves the stability of biological enzymes by immobilizing amylase; at the same time, by preparing coaxial scaffolds using coaxial 3D printing of the immobilized biological enzymes, the coaxial scaffolds have a longer glucose release effect, thereby promoting the proliferation of bone marrow mesenchymal stem cells. Attached Figure Description
[0020] Figure 1 Characterization of amylase immobilization in Example 1: (a) and (b) are SEM morphologies of β-TCP powder after grinding, (c) and (d) are SEM morphologies of β-TCP powder loaded with dopamine, and (e) and (f) are SEM morphologies of β-TCP powder loaded with amylase.
[0021] Figure 2 Characterization of amyloglucosidase loading in Example 1: (a) and (b) are schematic diagrams of particle size distribution and specific surface area of β-TCP powder after grinding; (c) and (d) are XRD and Raman characterization of β-TCP powder under different loading conditions.
[0022] Figure 3 Performance characterization of amylase before and after fixation: (a) absorbance of glucose solutions of different concentrations reacted with DNS, (b) absorbance versus concentration curve, (c) activity of amylase and its loading after fixation, (d) activity curve of amylase and its fixed loading at different soaking times.
[0023] Figure 4 The coaxial 3D printed scaffolds with PCL / β-TCP as the outer layer are as follows: (A) solid structure scaffold (Control), (B) inner layer pure starch material (STA) scaffold, (C) inner layer starch material containing glucose (GLU) scaffold, (D) inner layer starch material containing 2% TDA (2% TDA) scaffold, (E) inner layer starch material containing 5% TDA (5% TDA) scaffold, and (F) inner layer starch material containing 10% TDA (10% TDA) scaffold.
[0024] Figure 5 The glucose release curve of the coaxial scaffold;
[0025] Figure 6 The diagram shows the proliferation and adhesion of rBMSCs cells on the scaffold: (A) cell proliferation is represented by the absorbance of the microplate reader, (B) cell morphology fluorescence imaging, and (C) cell SEM morphology. Detailed Implementation
[0026] The present invention will be further illustrated by the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.
[0027] First, the present invention provides a bioenzyme for catalyzing the slow release of bioenergy substances. The bioenzyme comprises: a ceramic matrix, dopamine loaded on the ceramic matrix, and an amylase loaded on the dopamine.
[0028] In some embodiments, the ceramic matrix may be at least one of hydroxyapatite, β-TCP, and dicalcium silicate, preferably β-TCP (β-tricalcium phosphate).
[0029] During the grinding process, β-TCP generates nanoscale lath-shaped hydroxyapatite on its surface, which further increases its specific surface area and is beneficial for dopamine loading.
[0030] In some embodiments, with the total mass of the bio-enzymes used to catalyze the slow release of bioenergy substances as 100%, the mass percentage of the ceramic matrix can be 80-90%, the mass percentage of dopamine can be 5-10%, and the mass percentage of amylase can be 1-10%.
[0031] If the proportion of ceramic mass is too large, it will result in low dopamine loading, thus affecting the loading of amylase; if the proportion of dopamine mass is too large, it will not be able to be fully loaded on ceramic particles, resulting in waste, and will also form a thicker dopamine layer, affecting the loading; if the proportion of biological enzyme mass is too large, it will be difficult for it to be fully loaded, resulting in substantial waste.
[0032] The following is an exemplary description of a method for immobilizing bioenzymes for catalytically releasing bioenergy substances, provided by the present invention. The immobilization method may include the following steps:
[0033] (1) The ceramic powder is mixed with dopamine hydrochloride to obtain raw material mixed powder, then deionized water is added to it and magnetic stirring is performed for 1-4 hours. After centrifugation, washing with deionized water and freeze drying, dopamine-loaded ceramic powder is obtained.
[0034] (2) The ceramic powder loaded with dopamine is mixed with amyloglucosidase, then deionized water is added and the mixture is shaken for 4-12 hours to allow the amyloglucosidase to be fully loaded and bound to dopamine. After washing and drying, the fixed bio-enzyme for catalyzing the slow release of bioenergy substances is obtained.
[0035] In some embodiments, in step (1), the particle size of the ceramic powder can be 100 nm-10 μm, preferably 300 nm-10 μm, and the specific surface area is 10-50 m². 2 / g.
[0036] If the particle size of ceramic powder is too large, the specific surface area of the particles will be small, resulting in a lower bio-enzyme loading; if the particle size of ceramic powder is too small, the specific surface area will be large, but it will be prone to agglomeration. By selecting a suitable medium-sized particle surface to generate nanostructures, the agglomeration problem can be avoided while ensuring the specific surface area of the powder.
[0037] In some embodiments, in step (1), the mass ratio of the ceramic powder to dopamine hydrochloride can be 5-10:1.
[0038] In some embodiments, in step (1), the ratio of the raw material mixed powder to deionized water can be 1g:5-10mL.
[0039] In some embodiments, in step (2), the mass ratio of the dopamine-loaded ceramic powder to amylase can be 1:0.1-0.4. Excessive use of amylase will result in some enzymes failing to be successfully loaded and thus failing to exert their catalytic effect, leading to waste; insufficient use of amylase will result in insufficient loading on the ceramic particles, affecting the final catalytic effect.
[0040] In some embodiments, in step (2), the ratio of the dopamine-loaded ceramic powder to deionized water can be 1g:5-50mL, preferably 1g:20-50mL.
[0041] Glucose concentration can be quantitatively determined using the 3,5-dinitrosalicylic acid colorimetric method and UV-Vis absorption spectroscopy. The specific method is as follows: Dissolve 1g of starch in 100ml of deionized water and heat to boiling; after cooling the starch solution, add 0.01-0.1g of fixed and unfixed enzymes and react for 30min; determine the glucose concentration of the starch solution using the DNS method, then calculate the true activities of the fixed and unfixed enzymes, and determine the fixed loading of amyloglucosidase; then determine the relative activities of the fixed and unfixed enzymes at 37℃ and pH=7.4 to assess the effect of fixation on activity.
[0042] Using the method disclosed in patent ZL202110277324.X, a coaxial scaffold with starch-immobilized amyloglucosidase as the inner layer and β-TCP / PCL as the outer layer can be printed to study the glucose release performance of the coaxial scaffold.
[0043] The coaxial scaffold was placed in a centrifuge tube and PBS solution was added. Samples were taken and the soaking solution was replaced periodically at preset times. The glucose release curves of the coaxial scaffold at 37°C for different times were measured to evaluate the glucose release performance of different coaxial scaffolds.
[0044] In addition, the aforementioned coaxial scaffolds can be used to culture bone marrow mesenchymal stem cells to study the effect of the coaxial scaffolds on their proliferation.
[0045] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the range based on the description herein, and are not intended to be limited to the specific values in the examples below. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
[0046] Example 1
[0047] The method for immobilizing bioenzymes for catalytic slow-release of bioenergy substances provided in this embodiment includes the following steps:
[0048] (1) β-TCP powder was ground in aqueous solution for 24 h using a planetary ball mill to obtain smaller particle size (MillingTCP, T), and its particle size and specific surface area were characterized using a laser particle size analyzer and a specific surface area pore size analyzer; the ground β-TCP powder was mixed with dopamine hydrochloride at a mass ratio of 10:1; then deionized water was added at a ratio of 10 ml / g powder, and the mixture was magnetically stirred at room temperature for 4 h; the dopamine-loaded β-TCP powder was centrifuged and washed 3 times with centrifuge and deionized water respectively, and then freeze-dried to obtain dopamine-loaded ceramic powder for later use (MillingTCP-Dopa, TD);
[0049] (2) Mix TD powder at a ratio of 1g TD powder / 0.2g amylase, and add deionized water at a ratio of 5ml deionized water / 1g powder. Shake in a shaker for 12h to fully load the amylase. Finally, centrifuge and wash 3 times, freeze dry the sample to obtain the fixed bio-enzyme (MillingTCP-Dopa-AGS, TDA) for catalyzing the slow release of bioenergy substances.
[0050] X-ray diffraction (XRD), scanning electron microscopy (SEM), and Raman spectroscopy were used to characterize the crystal structure, microstructure, and functional groups of the β-TCP powder (T), dopamine-loaded β-TCP powder (TD), and amylase-loaded β-TCP powder (TDA) prepared in Example 1, respectively, to determine the enzyme loading. (See Appendix) Figure 1 and 2 .from Figure 1 , 2 As can be seen, nanoscale lath-shaped hydroxyapatite was formed on the ground β-TCP, with a particle size distribution of 300 nm-10 μm and a specific surface area of 31.132 μm. 2 / g; XRD and Raman tests showed that dopamine and amylase were successfully loaded onto ceramic particles.
[0051] The following quantitative determination of glucose concentration was performed using the 3,5-dinitrosalicylic acid colorimetric method and a UV-Vis absorption spectrometer: First, a glucose solution with a concentration of 1 mg / ml was prepared. Then, based on this, glucose solutions with concentrations of 0.2, 0.4, 0.6, and 0.8 mg / ml were prepared. 0.5 ml of each of these glucose solutions was added to a 15 ml centrifuge tube, followed by 0.5 ml of DNS solution. The centrifuge tubes were incubated in a boiling water bath for 10 minutes, then cooled before measurement. 0.3 ml of the reaction solution was added to a cuvette and diluted to 3 ml with deionized water. The absorption spectra of glucose solutions of different concentrations in the 400-700 nm range were measured using a UV-Vis spectrophotometer, with each sample measured three times. The highest absorbance value of each curve was statistically analyzed, and an absorbance-concentration standard curve was plotted. In subsequent tests, if the measured glucose concentration was higher than 1 mg / ml, it needed to be diluted proportionally to the range of 0-1 mg / ml.
[0052] Take 1g of starch, dissolve it in 100ml of deionized water, and heat to boiling. After the starch solution cools, add 0.01g of fixed and unfixed enzymes and react for 30min. Use the DNS method to determine the glucose concentration of the starch solution, then calculate the true activities of the fixed and unfixed enzymes, and obtain the fixed loading of amyloglucosidase. Then, measure the relative activities of the fixed and unfixed enzymes at 37℃ and pH=7.4 for 1, 3, 7, 14, 21, and 28 days to evaluate the effect of fixation on activity.
[0053] Figure 3The performance characteristics of amylase before and after immobilization are shown in the figures: (a) absorbance of glucose solutions of different concentrations reacted with DNS; (b) absorbance versus concentration curves; (c) activity of amylase and its loading after immobilization; and (d) activity curves of amylase after immobilization at different soaking times. As can be seen from the figures, since absorbance has a highly linear relationship with glucose concentration, the DNS method can be used to infer the glucose concentration in the solution. The ceramic particles loaded with amylase exhibit an activity of 110 U / g, and the immobilized amylase shows higher stability and sustained catalytic activity compared to the unimmobilized form.
[0054] Starch slurry was prepared using a high-temperature boiling method, and different proportions of fixed amyloglucosidase (TDA) were uniformly added to the starch slurry at low temperature. The specific procedure was as follows: 1g, 1g, 0.98g, 0.95g, and 0.9g of starch were added to beakers respectively; 10g of water was added to the beakers, and the starch solution was boiled under magnetic stirring until it became a viscous, clear slurry; after the starch slurry cooled, starting from the second beaker, 1g of anhydrous glucose, 0.02g of TDA, 0.05g of TDA, and 0.1g of TDA were added to the above beakers respectively. Appropriate amounts of deionized water were added to all beakers to make the total mass of starch (+TDA) and deionized water 5g. The slurry in the beakers was stirred evenly and transferred to a container for later use. These were labeled STA, GLU, 2% TDA, 5% TDA, and 10% TDA, respectively. The glucose release curve in patent ZL202110277324.X is basically similar to that of the GLU group, so GLU is used instead of the data of this group.
[0055] Figure 4 The coaxial 3D printed scaffolds with PCL / β-TCP as the outer layer are shown in the figures: (A) solid structure scaffold (Control), (B) scaffold with inner pure starch material (STA), (C) scaffold with inner starch material containing glucose (GLU), (D) scaffold with inner starch material containing 2% TDA (2% TDA), (E) scaffold with inner starch material containing 5% TDA (5% TDA), and (F) scaffold with inner starch material containing 10% TDA (10% TDA). As can be seen from the figures, except for the Control group, all other groups have coaxial structures. The STA and GLU groups have no particles in their starch layers, while the 2%-10% TDA groups show a significant increase in starch particles.
[0056] Figure 5 The figure shows the glucose release curves of the coaxial scaffold. As can be seen from the figure, the GLU group had basically completed the release by the third day; other groups containing immobilized enzymes showed longer sustained release times, and the total amount of glucose released also increased with the increase of immobilized enzymes.
[0057] Cell proliferation assays were performed using the Cell Counting Kit-8 (CCK-8) method. The experiment included coaxial scaffolds with inner layers containing no starch (solid structure), pure starch, starch containing glucose, starch containing 2% TDA, starch containing 5% TDA, and starch containing 10% TDA, designated as Control, STA, GLU, 2% TDA, 5% TDA, and 10% TDA, respectively.
[0058] First, all scaffolds used in cell experiments were soaked in 75% ethanol for half an hour and washed 3-5 times with sterile PBS buffer, 5 minutes each time. Then, the treated scaffolds were added to 48-well plates and seeded with 1×10⁶ cells / well. 4 rBMSCs cells were cultured at 37°C, 5% CO2, and 95% humidity for 1, 3, and 7 days, with medium changes on days 3 and 5. Then, CCK-8 working solution diluted 10-fold with pure DMEM was added to each well containing the scaffold, and the cells were cultured for another 2 hours. The solution was then transferred to new 96-well plates. Finally, the OD value of the solution at 450 nm was measured using a microplate reader (six replicates per group) to assess the effect of the coaxial scaffold on rBMSC cell proliferation.
[0059] First, the cultured rBMSCs cells were digested with trypsin and centrifuged at 1000 rpm for 5 min. They were then diluted with the appropriate culture medium and counted. The sterilized Control, STA, GLU, 2% TDA, 5% TDA, and 10% TDA scaffolds were placed into 48-well plates, 1 × 10⁶ cells per well. 5 Cell implantation. Cells were cultured at 37°C, 5% CO2, and 95% humidity for 3 days, and the culture medium was aspirated. Cells were fixed with 2.5% glutaraldehyde for 2 hours, followed by aspiration of the glutaraldehyde solution and washing three times with PBS. The fixed samples were then gradually dehydrated by immersing them in 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and 100% ethanol solutions three times each for 10 minutes. The samples were then treated with 50% hexamethyldisilazane / ethanol solution for 10 minutes, and finally immersed in 100% hexamethyldisilazane solution for 10 minutes. The samples were then dried overnight in a fume hood for SEM observation of cell adhesion behavior.
[0060] Figure 6Schematic diagram of the proliferation and adhesion of rBMSCs cells on the scaffold: (A) cell proliferation represented by absorbance from the microplate reader, (B) cell morphology fluorescence imaging, (C) cell SEM morphology. As can be seen from the figures, there was no significant difference on day 1, and the GLU group showed an inhibitory effect; on day 3, the immobilized enzyme group showed higher cell proliferation, but excessive glucose release affected cell viability; the results on day 7 were basically consistent with those on day 3; the SEM images of cell adhesion and cell staining were basically consistent with the cell proliferation results on day 3.
[0061] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.
Claims
1. A bioenzyme for catalyzing the slow release of bioenergy substances, characterized in that, The bioenzyme for catalyzing the slow release of bioenergy substances comprises: a ceramic matrix, dopamine hydrochloride loaded on the ceramic matrix, and amylase loaded on the dopamine hydrochloride; the ceramic matrix has a particle size of 300 nm-10 μm and a specific surface area of 10-50 m². 2 / g; The ceramic matrix is β-TCP; Based on the total mass of the bio-enzymes used to catalyze the slow release of bioenergy substances as 100%, the ceramic matrix accounts for 80-90% of the mass, dopamine hydrochloride accounts for 5-10% of the mass, and amylase accounts for 1-10% of the mass.
2. A method for immobilizing a bioenzyme for catalytically releasing bioenergy substances as described in claim 1, the immobilization method comprising the following steps: (1) The ceramic powder is mixed with dopamine hydrochloride to obtain raw material mixed powder, and then deionized water is added to it and stirred to obtain ceramic powder loaded with dopamine hydrochloride; (2) The ceramic powder loaded with dopamine hydrochloride is mixed with amyloglucosidase, then deionized water is added and the mixture is shaken to ensure that the amyloglucosidase is fully loaded and bound to dopamine hydrochloride, so as to obtain the fixed bio-enzyme for catalyzing the slow release of bioenergy substances.
3. The fixing method according to claim 2, characterized in that, In step (1), the particle size of the ceramic powder is 100nm-10μm.
4. The fixing method according to claim 2, characterized in that, In step (1), the ratio of the raw material mixed powder to deionized water is 1g:5-10mL.
5. The fixing method according to claim 2, characterized in that, In step (2), the ratio of the ceramic powder loaded with dopamine hydrochloride to deionized water is 1g:5-50mL.
6. The fixing method according to claim 2, characterized in that, In step (2), the ratio of the ceramic powder loaded with dopamine hydrochloride to deionized water is 1g:20-50mL.