A biological enzyme immobilization medium that can be prepared in various forms and its preparation method

Abstract

This invention belongs to the field of immobilized enzyme preparation technology. More specifically, it relates to a biological enzyme immobilization medium that can be prepared in various forms and its preparation method. This invention provides a medium for immobilizing enzymes, comprising silk fibroin and protocatechuic aldehyde; the mass ratio of silk fibroin to protocatechuic aldehyde is (10-30):(0.06906-172.65). The biological enzyme immobilization medium of this invention has simple components, a simple preparation method, and low cost. It also effectively solves the problem of reduced enzyme activity caused by existing immobilization media. It can immobilize different types of enzymes, improving the enzyme's cycling stability and temperature tolerance. Furthermore, this immobilization medium can be prepared in various forms, such as coatings, films, hydrogels, and microspheres, and can stably bind to different material surfaces to meet the needs of different application scenarios. In addition, the biological enzyme immobilization medium of this invention has good biocompatibility and has excellent application prospects and value.

Description

Technical Field

[0001] This invention belongs to the field of immobilized enzyme preparation technology. More specifically, it relates to a biological enzyme immobilization medium that can be prepared in various forms and its preparation method. Background Technology

[0002] Enzymes are substances produced by living cells that possess high specificity and catalytic efficiency towards their substrates. Also known as biological enzymes, most are proteins, with a very small percentage being RNA. Biological enzymes exhibit high substrate specificity and high catalytic efficiency, enabling them to catalyze reactions under mild conditions, and possess the advantages of high efficiency and high specificity. Compared to chemical catalysts, biological enzymes are environmentally friendly catalysts, being non-toxic and producing no harmful byproducts during the catalytic process. Currently, enzyme-catalyzed reactions are widely used in biomedicine, environmental protection, food engineering, and many other fields. However, biological enzymes have poor environmental tolerance and are easily affected by environmental factors such as temperature, pH, solvents, and heavy metal ions, leading to enzyme inactivation and loss of catalytic ability. Furthermore, biological enzymes are easily diffused in solution, and there is a lack of efficient recycling and reuse methods, making their use costly. Solving the problems of poor environmental tolerance and difficult recycling of biological enzymes has become crucial for reducing their usage costs.

[0003] To address the problems of poor environmental tolerance and difficulty in enzyme recovery, enzyme immobilization technology has been developed. This involves treating enzymes using physical or chemical methods to prevent diffusion in solution and immobilizing them on various carrier materials. Immobilized enzymes generally exhibit increased stability, are easily separated from reaction systems, are easily controlled, and can be reused repeatedly. They are also easier to transport and store, facilitating automated production. Immobilized enzymes are an enzyme application technology that has developed over the past decade and shows promising application prospects in industrial production, chemical analysis, and pharmaceuticals.

[0004] However, the immobilization media used in current immobilized enzyme technology are expensive, and the preparation methods for immobilized enzyme materials are complex, which is not conducive to the promotion of the technology; moreover, it will lead to a certain degree of reduction in enzyme activity. Summary of the Invention

[0005] The present invention aims to overcome the problems of high cost, complex preparation process, and reduced enzyme activity of the immobilization media used in the existing immobilized enzyme technology, and to develop an immobilization medium for preparing immobilized enzymes that is simple in composition, simple in preparation method, low in cost, and can better solve the problem of reduced enzyme activity.

[0006] The first objective of this invention is to provide a medium for immobilizing enzymes.

[0007] A second objective of this invention is to provide a solution for immobilizing biological enzymes.

[0008] A third objective of this invention is to provide microspheres for immobilizing enzymes.

[0009] A fourth object of the present invention is to provide a hydrogel solution for immobilizing enzymes.

[0010] A fifth object of the present invention is to provide applications of the above-mentioned media, solutions, microspheres or hydrogel solutions.

[0011] The sixth objective of this invention is to provide a method for enzyme immobilization.

[0012] The seventh object of the present invention is to provide an immobilized enzyme.

[0013] The above-mentioned objective of this invention is achieved through the following technical solution:

[0014] Silk fibroin is a natural biomolecule derived from silkworm cocoons, containing abundant glycine, alanine, and serine, as well as numerous hydroxyl, amino, carbonyl, and carboxyl groups. These groups interact with each other or with enzyme molecules. Protocatechuic aldehyde is a water-soluble polyphenol antioxidant. This invention has found that protocatechuic aldehyde can enhance the stability of the silk fibroin network and the enzyme immobilization efficiency. Adding protocatechuic aldehyde to silk fibroin can effectively improve the adhesion between silk fibroin and the substrate. Therefore, this invention claims protection for the following:

[0015] The present invention provides a medium for immobilizing enzymes, comprising silk fibroin and protocatechuic aldehyde; wherein the mass ratio of silk fibroin to protocatechuic aldehyde is (10-30):(0.06906-172.65).

[0016] This medium can be used to immobilize biological enzymes, effectively solving the problem of reduced enzyme activity caused by existing immobilization media. It can immobilize different types of enzymes, improving their cycling stability and temperature tolerance. Furthermore, this immobilization medium can be made into various forms, such as coatings, films, hydrogels, and microspheres, and can stably bind to different material surfaces to meet the needs of different application scenarios.

[0017] As one of the preferred embodiments, the present invention provides a solution for immobilizing biological enzymes, which can be used to prepare immobilized enzymes in the form of coatings. The solution comprises silk fibroin, protocatechuic aldehyde, and a solvent; the mass ratio of silk fibroin to protocatechuic aldehyde is (10-30):(0.06906-34.53); preferably, the final concentration of silk fibroin is 10-30 mg / mL, and the final concentration of protocatechuic aldehyde is 0.5-250 mmol / L.

[0018] More preferably, the mass ratio of silk fibroin to protocatechuic aldehyde is 20:(0.17265~34.53), the final concentration of silk fibroin is 20mg / mL, and the final concentration of protocatechuic aldehyde is 1.25~250mmol / L.

[0019] More preferably, the mass ratio of silk fibroin to protocatechuic aldehyde is 20:(0.6906~34.53), the final concentration of silk fibroin is 20mg / mL, and the final concentration of protocatechuic aldehyde is 5~250mmol / L.

[0020] More preferably, the mass ratio of silk fibroin to protocatechuic aldehyde is 20:(0.6906~17.265), the final concentration of silk fibroin is 20mg / mL, and the final concentration of protocatechuic aldehyde is 5~125mmol / L.

[0021] As an alternative implementation, the final concentration of silk fibroin is 20 mg / mL, and the final concentration of protocatechuic aldehyde is 5 mmol / L.

[0022] As an alternative implementation, the final concentration of silk fibroin is 20 mg / mL, and the final concentration of protocatechuic aldehyde is 125 mmol / L.

[0023] As a second preferred embodiment, the present invention provides microspheres for immobilizing enzymes, comprising silk fibroin, protocatechuic aldehyde and solvent; the mass ratio of silk fibroin to protocatechuic aldehyde is (10-20):(69.06-172.65); preferably, the final concentration of silk fibroin is 10-20 mg / mL and the final concentration of protocatechuic aldehyde is 0.5-1.25 mol / L.

[0024] As an alternative implementation, the microspheres are prepared by first preparing a silk fibroin solution and a protocatechuic aldehyde solution, and then adding the silk fibroin solution to the protocatechuic aldehyde solution and mixing them to obtain a microsphere solution; preferably, based on the total volume of the silk fibroin solution and the protocatechuic aldehyde solution, the final concentration of silk fibroin is 10-20 mg / mL and the final concentration of protocatechuic aldehyde is 0.5-1.25 mol / L.

[0025] As an alternative implementation, a silk fibroin solution of 30–40 mg / mL is added to a protocatechuic aldehyde solution of 1.5–2.5 mol / L and mixed to obtain a microsphere solution; the volume ratio of the silk fibroin solution to the protocatechuic aldehyde solution is (1–2):(1–2).

[0026] As an alternative implementation, a 35 mg / mL silk fibroin solution is added to a 2 mol / L protocatechuic aldehyde solution and mixed to obtain a microsphere solution; the volume ratio of the silk fibroin solution to the protocatechuic aldehyde solution is 1:1.

[0027] As a third preferred embodiment, the present invention provides a hydrogel solution for immobilizing enzymes, comprising silk fibroin, protocatechuic aldehyde, glycerol and a solvent; the mass ratio of silk fibroin to protocatechuic aldehyde is (10-30):(0.469-6.906); preferably, the final concentration of silk fibroin in the hydrogel solution is 10-30 mg / mL and the final concentration of protocatechuic aldehyde is 3.4-50 mmol / L.

[0028] As an alternative embodiment, the hydrogel solution includes silk fibroin, protocatechuic aldehyde, glycerol, and a solvent; the mass ratio of silk fibroin to protocatechuic aldehyde is (20-22):(0.469-6.906); preferably, the final concentration of silk fibroin in the hydrogel solution is 20-22 mg / mL, and the final concentration of protocatechuic aldehyde is 3.4-50 mmol / L.

[0029] As an alternative implementation, the hydrogel solution is prepared by first preparing a silk fibroin solution and a protocatechuic aldehyde solution, and then mixing the silk fibroin solution, protocatechuic aldehyde solution, glycerol and solvent evenly to obtain a hydrogel solution; the hydrogel solution is then solidified to obtain a hydrogel.

[0030] As an alternative implementation, the glycerol accounts for 29-30% of the volume fraction of the mixed solution.

[0031] As an alternative implementation, the mass ratio of silk fibroin to protocatechuic aldehyde is 21:6.906, the final concentration of silk fibroin in the hydrogel solution is 21 mg / mL, and the final concentration of protocatechuic aldehyde is 50 mmol / L.

[0032] As an alternative implementation, the mass ratio of silk fibroin to protocatechuic aldehyde is 21:0.469, the final concentration of silk fibroin in the hydrogel solution is 21 mg / mL, and the final concentration of protocatechuic aldehyde is 3.4 mmol / L.

[0033] As an alternative implementation, the solvent in the above solution, the above microspheres, or the above hydrogel solution is water.

[0034] The use of the above-mentioned medium, solution, microspheres or hydrogel solution as an enzyme immobilization medium or in the preparation of immobilized enzymes should also be within the scope of protection of this invention.

[0035] This invention provides a method for enzyme immobilization, wherein an enzyme solution is mixed with an immobilization medium; the immobilization medium is the aforementioned medium, the aforementioned solution, the aforementioned microspheres, or the aforementioned hydrogel solution.

[0036] This invention provides an immobilized enzyme, prepared by the enzyme immobilization method described above.

[0037] In addition, the present invention provides an immobilized enzyme, wherein the enzyme solution is mixed with the immobilization medium and then dried according to the above-described enzyme immobilization method to obtain the immobilized enzyme.

[0038] As an alternative implementation, the immobilized enzyme may also contain a substrate, the material of which includes metals, inorganic non-metals, and polymers.

[0039] The present invention has the following beneficial effects:

[0040] This invention provides a fixation medium for preparing immobilized enzymes, comprising silk fibroin and protocatechuic aldehyde. The bio-enzyme fixation medium of this invention has simple components, a simple preparation method, and low cost. It effectively solves the problem of reduced enzyme activity caused by existing fixation media, can fix different types of enzymes, improves enzyme cycling stability and temperature tolerance, and can be fabricated in various forms, such as coatings, films, hydrogels, and microspheres, to stably bind to different material surfaces and meet the needs of various application scenarios. Furthermore, the bio-enzyme fixation medium of this invention has good biocompatibility and excellent application prospects and value. Attached Figure Description

[0041] Figure 1 This is a schematic diagram illustrating the principle of the present invention.

[0042] Figure 2 The images show the state of the coatings on the Ti-ALP@SF / PA samples before drying.

[0043] Figure 3 The results show the contact angle test results of the alkaline phosphatase immobilized coating on the Ti surface.

[0044] Figure 4 The results of temperature tolerance cycling experiments on the activity of alkaline phosphatase immobilized coatings on Ti surfaces are shown in Figure a (a) schematic diagram of the temperature tolerance cycling experiment process; Figure b) results of the enzyme activity changes of different samples under 25℃ temperature treatment; Figure c) results of the enzyme activity changes of different samples under 37℃ temperature treatment; Figure d) results of the enzyme activity changes of different samples under 50℃ temperature treatment.

[0045] Figure 5 The results show the long-term stability test results of the alkaline phosphatase immobilized coating on the Ti surface.

[0046] Figure 6 Examples of immobilized coatings for glucose oxidase and horseradish peroxidase on Ti surface (Figure a shows the results of inhibition zone diameter measurement for different samples; Figure b shows the results of glucose oxidase activity evaluation for different samples; Figure c shows the results of horseradish peroxidase activity measurement for different samples).

[0047] Figure 7The results of cyclic assays of the enzyme activity of PEEK, MCE, and Si films with different coatings are shown in Figure a (a) for the change in ALP activity of the PEEK surface coating; b) for the change in ALP activity of the Si surface coating; and c) for the change in ALP activity of the mixed cellulose filter membrane coating.

[0048] Figure 8 The results of ALP enzyme activity determination for titanium sheet samples coated with different protocatechuic aldehyde concentrations.

[0049] Figure 9 The images show the state of PTFE-ALP@SF / PA and PTFE-ALP@SF samples (the top image in Figure A shows the surface coating state of the PTFE-ALP@SF sample, and the bottom image shows the film state after the coating of the PTFE-ALP@SF sample has been peeled off; the top image in Figure B shows the surface coating state of the PTFE-ALP@SF / PA sample, and the bottom image shows the film state after the coating of the PTFE-ALP@SF / PA sample has been peeled off).

[0050] Figure 10 The results of observation of the silk fibroin / protocatechuic aldehyde microsphere solution under a microscope (magnification 400x).

[0051] Figure 11 Images of the appearance of glycerol + SF hydrogel and glycerol + SF / PA hydrogel.

[0052] Figure 12 Figure 1 shows the ALP enzyme activity assay results of the hydrogel washing solution at different time points (Figure 2 shows the state diagram of ALP@SF hydrogel and ALP@SF / PA hydrogel; Figure 3 shows the ALP enzyme activity assay results of the hydrogel washing solution from 0 to 12 hours; Figure 4 shows the ALP enzyme activity assay results of the hydrogel washing solution from 12 to 24 hours; Figure 5 shows the ALP enzyme activity assay results of the hydrogel washing solution from 24 to 36 hours; Figure 6 shows the ALP enzyme activity assay results of the hydrogel washing solution from 36 to 48 hours). Detailed Implementation

[0053] The schematic diagram of the present invention is as follows: Figure 1 As shown.

[0054] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0055] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0056] LB agar plates are prepared by autoclaving LB agar powder mixed with pure water at a ratio of 36 g / L. Approximately 15 mL of the solution is poured into a 9 cm diameter petri dish and allowed to cool to obtain LB agar plates. The LB agar powder was purchased from Huankai Microbiology. The LB agar powder formula is as follows: each 36 g of dry powder culture medium contains 10 g of peptone, 5 g of yeast extract, 5 g of sodium chloride, 1 g of glucose, and 15 g of agar.

[0057] Hydrogen peroxide content detection kit, brand name BOXBIO, catalog number AKAO009M.

[0058] Protocatechuic aldehyde, CAS number 139-85-5, structural formula is

[0059] Silk fibroin, also known as silk protein, is commercially available or can be prepared using the following method: cut silkworm cocoons to 1cm lengths. 2 Silk fragments of various sizes were boiled in a 0.02M sodium carbonate solution for 30 minutes to remove sericin. After washing several times in pure water, the silk was air-dried at room temperature to obtain silk. 0.25g of silk and 1mL of 9.3M lithium bromide solution were placed in a glass bottle and heated at 60℃ for 4 hours to dissolve the silk fibroin. The solution was poured into a dialysis bag (MWCO 3500) and dialyzed for 2 days, changing the water every 12 hours, to obtain a crude silk fibroin extract. The extract was centrifuged at 10000rpm at 4℃, and the precipitate was discarded; the supernatant was the silk fibroin solution.

[0060] Example 1

[0061] I. Solution Preparation

[0062] (1) Preparation of silk fibroin solution

[0063] Dilute the silk fibroin solution with pure water to 25 mg / mL for later use.

[0064] (2) Preparation of protocatechuic aldehyde solution

[0065] Take 27.624g of protocatechuic aldehyde and dissolve it by heating and sonication at 60℃ in 1L of pure water. Then cool it to room temperature to prepare a 27.624g / L (0.2mol / L) protocatechuic aldehyde solution.

[0066] (3) Alkaline phosphatase storage solution

[0067] Dissolve 1 mg of alkaline phosphatase in 1 mL of pure water to prepare a 1 mg / mL alkaline phosphatase stock solution (4 KU / mL, Yuanye Bio).

[0068] II. Preparation of Coating Solution

[0069] Different coating solutions were prepared according to schemes (i), (ii), and (iii), as detailed below:

[0070] (i) The silk fibroin solution, protocatechuic aldehyde solution, and pure water were mixed thoroughly to obtain a mixed solution with a final concentration of 20 mg / mL for silk fibroin and a final concentration of 0.6906 mg / mL (5 μmol / mL) for protocatechuic aldehyde. 10 μL of alkaline phosphatase stock solution was added to each milliliter of the mixed solution to obtain the coating solution (denoted as ALP@SF / PA).

[0071] (ii) The silk fibroin solution and pure water were mixed thoroughly to obtain a mixed solution; the final concentration of silk fibroin in the mixed solution was 20 mg / mL. 10 μL of alkaline phosphatase stock solution was added to each milliliter of the mixed solution to obtain the coating solution (denoted as ALP@SF).

[0072] (iii) Take a certain amount of pure water and add 10 μL of alkaline phosphatase storage solution to each milliliter of pure water to obtain the coating solution (denoted as ALP).

[0073] III. Sample Preparation

[0074] The titanium sheet (1cm in diameter) was polished smooth with 400, 800 and 2000 grit sandpaper in sequence, then ultrasonically cleaned with acetone, ethanol and water for 10 minutes each, and then dried for later use.

[0075] (1) Take 50 μL of the coating solution prepared by scheme (i) onto the surface of the titanium sheet and air dry at room temperature to obtain the Ti-ALP@SF / PA sample.

[0076] (2) Take 50 μL of the coating solution prepared by scheme (ii) onto the surface of the titanium sheet and air dry at room temperature to obtain the Ti-ALP@SF sample.

[0077] (3) Take 50 μL of the coating solution prepared by scheme (iii) onto the surface of the titanium sheet and air dry at room temperature to obtain the Ti-ALP sample.

[0078] IV. Experimental Methods and Results

[0079] (1) Coating stability

[0080] The Ti-ALP@SF / PA and Ti-ALP@SF samples were placed in a 24-well plate, each immersed in 2 mL of pure water, and shaken at 120 rpm for 1 h at 37 °C. The pure water was then removed, and the plates were rinsed once with pure water. 2 mL of pure water was added to each well, and the plates were shaken at 120 rpm for 1 h at 37 °C. The pure water was then removed, and the plates were rinsed twice with pure water. The morphology of the coating was photographed and recorded.

[0081] The coatings of Ti-ALP@SF and Ti-ALP@SF / PA samples before drying are shown in the following figures. Figure 2As shown, the results indicate that obvious wrinkles appeared at the edge of the coating on the Ti-ALP@SF sample surface, while no wrinkles appeared on the Ti-ALP@SF / PA sample surface. The wrinkles on the Ti-ALP@SF sample disappeared after the coating dried as moisture evaporated.

[0082] (2) Contact angle test

[0083] 2.5 μL of pure water was dropped onto the surface of different samples, and the contact angle of the samples was measured using a contact angle meter (JC2000CS). The contact angle was calculated using the instrument's software. The test results are as follows: Figure 3 As shown, the results indicate that protocatechuic aldehyde and alkaline phosphatase were successfully modified or encapsulated in the silk fibroin coating.

[0084] (3) Test the coating enzyme activity of different samples

[0085] ALP enzyme activity cyclic assay method:

[0086] 121.14 mg of tris(hydroxymethyl)aminomethane, 58.44 mg of sodium chloride, and 4.7605 mg of magnesium chloride were dissolved in 10 mL of pure water, and the pH was adjusted to 9.5 to obtain a buffer solution. 4-Nitrophenyl phosphate was dissolved in the buffer solution at a concentration of 0.93 mg / mL to obtain the ALP enzyme activity assay solution.

[0087] Three temperature treatments were set up (25℃, 37℃, and 50℃). Different samples (Ti-ALP@SF / PA, Ti-ALP@SF, and Ti-ALP) were treated at different temperatures (25℃, 37℃, and 50℃) for 12 hours. Then, each sample was immersed in 0.5 mL of ALP enzyme activity assay solution and incubated at 37℃ with shaking at 120 rpm for 30 min. 100 μL of the reacted ALP enzyme activity assay solution was then measured at 405 nm in a 96-well plate to evaluate the ALP enzyme activity of different samples. After the enzyme activity test, the samples were washed twice with 2 mL of pure water, air-dried overnight at room temperature, and stored. This constituted one cycle of the ALP enzyme activity assay.

[0088] The ALP enzyme activity of different samples was measured cyclically every 24 hours.

[0089] ALP enzyme activity assay results of different samples are as follows Figure 4 As shown, the results indicate that the ALP activity of the Ti-ALP@SF / PA sample was significantly higher than that of the Ti-ALP@SF and Ti-ALP samples before temperature treatment (at 0 cycles). After cyclic heating treatment at different temperatures, the ALP activity of the Ti-ALP@SF / PA sample remained significantly higher than that of the Ti-ALP@SF and Ti-ALP samples.

[0090] In summary, the experimental results demonstrate that increasing the content of protocatechuic aldehyde can improve the adhesion between the coating and the substrate, as well as enhance the enzyme activity and temperature resistance cycling stability of the coating.

[0091] Example 2

[0092] I. Experimental Methods

[0093] Following the example in Example 1, Ti-ALP, Ti-ALP@SF, and Ti-ALP@SF / PA samples were prepared, and ALP enzyme activity was tested immediately after sample preparation.

[0094] The method for measuring ALP enzyme activity is as follows:

[0095] 121.14 mg of tris(hydroxymethyl)aminomethane, 58.44 mg of sodium chloride, and 4.7605 mg of magnesium chloride were dissolved in 10 mL of pure water, and the pH was adjusted to 9.5 to obtain a buffer solution. 4-Nitrophenyl phosphate was dissolved in the buffer solution at a concentration of 0.93 mg / mL to obtain the ALP enzyme activity assay solution.

[0096] Different samples (Ti-ALP@SF / PA, Ti-ALP@SF, Ti-ALP) were immersed in 0.5 mL of ALP enzyme activity detection solution and placed in a shaker at 37℃ with shaking at 120 rpm for 30 min. 100 μL of the ALP enzyme activity detection solution after the reaction was taken and the absorbance at 405 nm was measured in a 96-well plate.

[0097] After the sample activity test, the samples were washed twice with 2 mL of pure water, air-dried overnight at room temperature, and stored at room temperature. ALP enzyme activity was tested after 1 month and 2 months of storage at room temperature.

[0098] II. Experimental Results

[0099] ALP enzyme activity assay, such as Figure 5 As shown, the results indicate that, before storage, the ALP enzyme activity of the Ti-ALP@SF / PA sample was significantly higher than that of the Ti-ALP@SF sample.

[0100] After two months of storage, the ALP enzyme activity of the Ti-ALP@SF / PA sample remained significantly higher than that of the Ti-ALP@SF sample. This indicates that the addition of protocatechuic aldehyde to the coating solution can significantly enhance the enzyme activity of the coated samples and maintain this advantage.

[0101] Example 3

[0102] I. Material Preparation

[0103] (1) Dissolve glucose oxidase (SIGMA-ALDRICH) in pure water to obtain a glucose oxidase stock solution of 0.16 KU / mL (1 mg / mL).

[0104] (2) Following the method in Example 1, a protocatechuic aldehyde solution (27.624 mg / mL) and a silk fibroin solution (25 mg / mL) were prepared to obtain a titanium sheet.

[0105] II. Preparation of Coating Solution

[0106] Different coating solutions were prepared according to schemes (i) and (ii), as follows:

[0107] (i) The silk fibroin solution, protocatechuic aldehyde solution, and pure water were mixed thoroughly to obtain a mixed solution with a final concentration of 20 mg / mL for silk fibroin and a final concentration of 0.6906 mg / mL (5 μmol / mL) for protocatechuic aldehyde. 10 μL of glucose oxidase stock solution was added to each milliliter of the mixed solution to obtain the coating solution (denoted as GOx@SF / PA).

[0108] (ii) The silk fibroin solution, protocatechuic aldehyde solution, and pure water were mixed thoroughly to obtain a mixed solution, which is the control coating solution (denoted as SF / PA). The final concentration of silk fibroin in the mixed solution was 20 mg / mL, and the final concentration of protocatechuic aldehyde was 0.6906 mg / mL (5 μmol / mL).

[0109] III. Sample Preparation

[0110] (1) Take 50 μL of the coating solution prepared by scheme (i) onto the surface of the titanium sheet and air dry at room temperature to obtain the Ti-GOx@SF / PA sample.

[0111] (2) Take 50 μL of the coating solution prepared by scheme (ii) onto the surface of the titanium sheet and air dry at room temperature to obtain the Ti-SF / PA sample.

[0112] The Ti-GOx@SF / PA and Ti-SF / PA samples were placed in 24-well plates, each immersed in 2 mL of pure water, and shaken at 120 rpm for 1 h at 37 °C. The pure water was then aspirated and rinsed once with clean pure water. 2 mL of pure water was added to each well and shaken at 120 rpm for 1 h at 37 °C. The pure water was then aspirated and rinsed twice with clean pure water. The samples were then air-dried at room temperature.

[0113] IV. Experimental Methods and Results

[0114] (1) Antibacterial test

[0115] Spread 50 μL of Staphylococcus aureus bacterial suspension (10 μL) onto the surface of an LB agar plate. 5 The samples (CFU / mL) were soaked in PBS (pH 5.5, 10 mM) for 30 min, then inverted onto LB agar plates. After 12 h, photographs were taken, and the diameter of the inhibition zone was recorded. The results of the inhibition zone diameter determination are as follows: Figure 6As shown in Figure a, the results indicate that the glucose oxidase coated with the coating has an antibacterial effect against Staphylococcus aureus.

[0116] (2) Testing the coating enzyme activity of different samples

[0117] Place the sample in a 24-well plate, add 500 μL of PBS (pH = 5.5, 10 mM) containing 0.1 g / mL glucose to each well, and react at 37°C and 200 rpm in the dark for 24 h. Then, use a hydrogen peroxide content detection kit (BOXBIO) to measure the hydrogen peroxide content in the solution. Wash the sample with pure water, replace the reaction solution (PBS solution containing 0.1 g / mL glucose), react again for 24 h, and measure the hydrogen peroxide content in the solution. Wash the sample with pure water, replace the reaction solution, react again for 24 h, and measure the hydrogen peroxide content in the solution.

[0118] The glucose oxidase activity of different samples was evaluated by detecting the hydrogen peroxide content in the reaction solution. The results are as follows: Figure 6 As shown in Figure b, the results indicate that the sample coated with glucose oxidase can continuously decompose glucose to produce hydrogen peroxide.

[0119] Example 4

[0120] I. Material Preparation

[0121] (1) Horseradish peroxidase (Maclean) was dissolved in pure water to obtain a horseradish peroxidase stock solution of 3KU / mL (10mg / mL).

[0122] (2) Following the method in Example 1, a protocatechuic aldehyde solution (27.624 mg / mL) and a silk fibroin solution (25 mg / mL) were prepared to obtain a titanium sheet.

[0123] II. Preparation of Coating Solution

[0124] Different coating solutions were prepared according to schemes (i), (ii), and (iii), as detailed below:

[0125] (i) The silk fibroin solution, protocatechuic aldehyde solution and pure water are mixed evenly to obtain a mixed solution; the final concentration of silk fibroin in the mixed solution is 20 mg / mL and the final concentration of protocatechuic aldehyde is 0.6906 mg / mL (5 μmol / mL); 10 μL of horseradish peroxidase stock solution is added to each milliliter of the mixed solution to obtain the coating solution (denoted as HRP@SF / PA).

[0126] (ii) Mix the silk fibroin solution and pure water evenly to obtain a mixed solution with a final concentration of 20 mg / mL of silk fibroin. Add 10 μL of horseradish peroxidase stock solution to each milliliter of the mixed solution to obtain a coating solution (denoted as HRP@SF).

[0127] (iii) Take a certain amount of pure water and add 10 μL of horseradish peroxidase storage solution to each milliliter of pure water to obtain the coating solution (denoted as HRP).

[0128] III. Sample Preparation

[0129] (1) Take 50 μL of the coating solution prepared by scheme (i) onto the surface of the titanium sheet and air dry at room temperature to obtain the Ti-HRP@SF / PA sample.

[0130] (2) Take 50 μL of the coating solution prepared by scheme (ii) onto the surface of the titanium sheet and air dry at room temperature to obtain the Ti-HRP@SF sample.

[0131] (3) Take 50 μL of the coating solution prepared by scheme (iii) onto the surface of the titanium sheet and air dry at room temperature to obtain the Ti-HRP sample.

[0132] The Ti-HRP@SF / PA, Ti-HRP@SF, and Ti-HRP samples were placed in 24-well plates, each immersed in 2 mL of pure water. The plates were shaken at 120 rpm for 1 hour at 37°C. The pure water was then removed and the plates were rinsed once with clean pure water. 2 mL of pure water was added to each well and the plates were shaken at 120 rpm for 1 hour at 37°C. The pure water was then removed and the plates were rinsed twice with clean pure water. The samples were then air-dried at room temperature.

[0133] IV. Experimental Methods and Results

[0134] Different samples (Ti-HRP@SF / PA, Ti-HRP@SF, and Ti-HRP) were placed in 24-well plates. Each well was supplemented with 800 μL of sodium acetate buffer (pH 4.5), 100 μL of 0.2 mM 3,3,5,5-tetramethylbenzidine solution, and 100 μL of 1 mM hydrogen peroxide solution. The plates were incubated at 37°C with a shaker at 60 rpm for 60 min in the dark. 100 μL of the resulting solution was then plotted in a 96-well plate at 652 nm to measure the absorbance and evaluate the horseradish peroxidase activity of the different samples.

[0135] The results of enzyme activity assays for horseradish peroxidase in different samples are as follows: Figure 6 As shown in Figure c, the results indicate that the horseradish peroxidase activity of the Ti-HRP@SF / PA sample is significantly higher than that of the Ti-HRP@SF sample, suggesting that the addition of protocatechuic aldehyde (PA) can significantly enhance the enzyme activity of the horseradish peroxidase coating.

[0136] Example 5

[0137] I. Material Preparation

[0138] (1) Polyetheretherketone (PEEK, 1cm in diameter) was polished smooth with 400, 800 and 2000 grit sandpaper in sequence. PEEK wafers and silicon wafers (4mm×4mm) were ultrasonically cleaned with acetone, ethanol and water for 10 minutes each, and then dried for later use.

[0139] (2) Protocatechuic aldehyde solution (27.624 mg / mL), silk fibroin solution (25 mg / mL) and alkaline phosphatase storage solution were prepared according to the method of Example 1.

[0140] II. Preparation of Coating Solution

[0141] Different coating solutions were prepared according to schemes (i), (ii), and (iii), as detailed below:

[0142] (i) The silk fibroin solution, protocatechuic aldehyde solution and pure water are mixed evenly to obtain a mixed solution. The final concentration of silk fibroin in the mixed solution is 20 mg / mL and the final concentration of protocatechuic aldehyde is 0.6906 mg / mL (5 μmol / mL). 10 μL of alkaline phosphatase stock solution is added to each milliliter of the mixed solution to obtain the coating solution (denoted as ALP@SF / PA).

[0143] (ii) Mix the silk fibroin solution and pure water evenly to obtain a mixed solution with a final concentration of 20 mg / mL of silk fibroin. Add 10 μL of alkaline phosphatase storage solution to each milliliter of the mixed solution to obtain a coating solution (denoted as ALP@SF).

[0144] (iii) Take a certain amount of pure water and add 10 μL of alkaline phosphatase storage solution to each milliliter of pure water to obtain the coating solution (denoted as ALP).

[0145] III. Sample Preparation

[0146] (1) Take 50 μL of the coating solution prepared by scheme (i) and apply it to the surface of PEEK sheet and mixed cellulose filter membrane (MCE, pore size 0.8 μm, diameter 1 cm), respectively. Take 10 μL of the coating solution prepared by scheme (i) and apply it to the surface of silicon wafer (Si). Let it air dry at room temperature to obtain the sample.

[0147] (2) Take 50 μL of the coating solution prepared by scheme (ii) and apply it to the PEEK sheet and the mixed cellulose filter membrane respectively. Take 10 μL of the coating solution prepared by scheme (ii) and apply it to the surface of the silicon wafer (Si). Let it air dry at room temperature to obtain the sample.

[0148] (3) Take 50 μL of the coating solution prepared by scheme (iii) and apply it to the PEEK sheet and the mixed cellulose filter membrane respectively. Take 10 μL of the coating solution prepared by scheme (iii) and apply it to the surface of the Si sheet. Let it air dry at room temperature to obtain the sample.

[0149] Different samples were placed in a 24-well plate, each sample was immersed in 2 mL of pure water, and shaken at 120 rpm for 1 h at 37 °C. The pure water was aspirated and rinsed once with clean pure water. 2 mL of pure water was added to each well and shaken at 120 rpm for 1 h at 37 °C. The pure water was aspirated and rinsed twice with clean pure water. The samples were then air-dried at room temperature to obtain samples with different coatings.

[0150] IV. Experimental Methods and Results

[0151] ALP enzyme activity cyclic assay method:

[0152] 121.14 mg of tris(hydroxymethyl)aminomethane, 58.44 mg of sodium chloride, and 4.7605 mg of magnesium chloride were dissolved in 10 mL of pure water, and the pH was adjusted to 9.5 to obtain a buffer solution. 4-Nitrophenyl phosphate was dissolved in the buffer solution at a concentration of 0.93 mg / mL to obtain the ALP enzyme activity assay solution.

[0153] PEEK and MCE wafers with different coatings were each immersed in 1 mL of ALP enzyme activity assay solution, while silicon wafers with different coatings were each immersed in 0.2 mL of ALP enzyme activity assay solution. The solutions were incubated at 37°C and shaken at 120 rpm for 60 min. 100 μL of the reacted ALP enzyme activity assay solution was then plotted in a 96-well plate at 405 nm absorbance to evaluate the ALP enzyme activity of different samples. After the enzyme activity test, the samples were washed twice with 2 mL of pure water, air-dried overnight at room temperature, and stored. This constituted one cycle of the ALP enzyme activity assay. The ALP enzyme activity of different samples was measured cyclically every 24 h.

[0154] Results of cyclic assays of enzyme activity in PEEK, MCE, and Si wafers with different coatings are shown below. Figure 7 As shown, the results indicate that, compared with silk fibroin coatings, silk fibroin coatings with added protocatechuic aldehyde can enhance the enzyme activity of coatings on organic polymer (PEEK), inorganic non-metallic (Si), and porous membrane (MCE) materials.

[0155] Example 6

[0156] I. Preparation of Coating Solution

[0157] Following the method of Example 1, a protocatechuic aldehyde solution and a silk fibroin solution were prepared to obtain a titanium sheet.

[0158] Silk fibroin solution, protocatechuic aldehyde solution, and pure water were mixed in different proportions to obtain mixed solutions with different protocatechuic aldehyde concentrations. The following mixed solutions were prepared respectively:

[0159] Mixed solution 1: silk fibroin final concentration 20 mg / mL, protocatechuic aldehyde final concentration 0 mM;

[0160] Mixed solution 2: silk fibroin final concentration 20 mg / mL, protocatechuic aldehyde final concentration 0.5 mM;

[0161] Mixed solution 3: silk fibroin final concentration 20 mg / mL, protocatechuic aldehyde final concentration 1.25 mM;

[0162] Mixed solution 4: silk fibroin final concentration 20 mg / mL, protocatechuic aldehyde final concentration 12.5 mM;

[0163] Mixed solution 5: silk fibroin final concentration 20 mg / mL, protocatechuic aldehyde final concentration 125 mM;

[0164] Mixed solution 6: The final concentration of silk fibroin is 20 mg / mL, and the final concentration of protocatechuic aldehyde is 250 mM.

[0165] 1 μL of alkaline phosphatase stock solution (1 mg / mL, 4 KU / mL, Yuanye Biotechnology) was added to 100 μL of different mixed solutions (mixed solutions 1-6) to obtain different coating solutions.

[0166] In addition, a control group was set up, in which 1 μL of alkaline phosphatase stock solution (1 mg / mL, 4 KU / mL) was added to 100 μL of pure water.

[0167] II. Sample Preparation

[0168] Take 50 μL of different coating solutions and control solutions and add them to the surface of different titanium sheets respectively. Air dry overnight, wash twice with pure water, and air dry again to obtain titanium sheet samples with different coatings.

[0169] III. Experimental Methods and Results

[0170] After air drying, the ALP enzyme activity of different samples was tested. The method for ALP enzyme activity determination is as follows:

[0171] 121.14 mg of tris(hydroxymethyl)aminomethane, 58.44 mg of sodium chloride, and 4.7605 mg of magnesium chloride were dissolved in 10 mL of pure water, and the pH was adjusted to 9.5 to obtain a buffer solution. 4-Nitrophenyl phosphate was dissolved in the buffer solution at a concentration of 0.93 mg / mL to obtain the ALP enzyme activity assay solution.

[0172] Different samples were immersed in 0.5 mL of ALP enzyme activity detection solution and placed in a shaker at 37℃ with a shaking speed of 120 rpm for 30 min. 100 μL of the reacted ALP enzyme activity detection solution was taken and the absorbance was measured at 405 nm in a 96-well plate to evaluate the ALP enzyme activity of different samples.

[0173] The results of ALP enzyme activity assays for titanium sheet samples with different coatings are as follows: Figure 8As shown, the results indicate that the ALP enzyme activity of the coating solution samples with a final silk fibroin concentration of 20 mg / mL and a final protocatechuic aldehyde concentration of 1.25–250 mM was superior to that of other samples, with the coating solution having the highest enzyme activity at a protocatechuic aldehyde concentration of 125 mM.

[0174] Example 7

[0175] Following the method described in Example 1, ALP@SF / PA coating solution and ALP@SF coating solution were prepared. 50 μL of ALP@SF / PA coating solution and ALP@SF coating solution were respectively added to polytetrafluoroethylene (PTFE, 1cm×1cm) that had been polished with 2000-grit sandpaper, and air-dried at room temperature to obtain PTFE-ALP@SF / PA sample and PTFE-ALP@SF sample.

[0176] Appearance images of PTFE-ALP@SF / PA and PTFE-ALP@SF samples are shown below. Figure 9 As shown, the results indicate that, compared to the ALP@SF / PA coating, the ALP@SF coating is more wrinkled and cannot adhere to the PTFE material, while the ALP@SF / PA coating can adhere smoothly to the PTFE material.

[0177] Example 8: Preparation of silk fibroin / protocatechuic aldehyde microspheres

[0178] I. Solution Preparation

[0179] (1) 276.24 g (2 mol) protocatechuic aldehyde was dissolved in 1 L of pure water by heating in an 80℃ oven to prepare a 276.24 mg / mL (2 mol / L) protocatechuic aldehyde solution.

[0180] II. Preparation of Microspheres

[0181] Add 100 μL of silk fibroin solution (35 mg / mL) dropwise to 100 μL of protocatechuic aldehyde solution (276.24 mg / mL), and gently shake the solution to mix well to obtain silk fibroin / protocatechuic aldehyde microspheres.

[0182] The silk fibroin / protocatechuic aldehyde microsphere solution was observed under an optical microscope (magnification 400x), and the results are as follows. Figure 10 As shown, the results indicate that silk fibroin and protocatechuic aldehyde can form a microsphere structure, proving that samples with microsphere morphology can be obtained by changing the ratio of silk fibroin and protocatechuic aldehyde.

[0183] Example 9

[0184] I. Solution Preparation

[0185] Silk fibroin solution and protocatechuic aldehyde solution (27.624 mg / mL, i.e. 0.2 mol / L) were prepared according to the method in Example 1. The silk fibroin solution was diluted with pure water to 30 mg / mL for later use.

[0186] II. Preparation of Hydrogels

[0187] Two mixed solutions were prepared according to schemes (i) and (ii), as follows:

[0188] (i) Add 150 μL of glycerol, 350 μL of silk fibroin solution (30 mg / mL), and 8.75 μL of pure water to a centrifuge tube, mix well, and obtain a mixed solution (denoted as glycerol + SF).

[0189] (ii) Add 150 μL of glycerol, 350 μL of silk fibroin solution (30 mg / mL), and 8.75 μL of protocatechuic aldehyde solution (27.624 mg / mL) to a centrifuge tube, mix well, and obtain a mixed solution (denoted as glycerol + SF / PA).

[0190] Two mixed solutions, prepared according to schemes (i) and (ii), were dropped into different wells of a 24-well plate and left to stand at room temperature overnight to obtain different hydrogels.

[0191] Photographs were taken of the glycerol + SF hydrogel and the glycerol + SF / PA hydrogel, as shown in the images. Figure 11 As shown, the results indicate that the addition of protocatechuic aldehyde does not affect the crosslinking and light transmittance of the silk fibroin / glycerol hydrogel.

[0192] Example 10

[0193] I. Solution Preparation

[0194] Silk fibroin solution and protocatechuic aldehyde solution (27.624 mg / mL, i.e. 0.2 mol / L) were prepared according to the method in Example 1. The silk fibroin solution was diluted with pure water to 30 mg / mL for later use.

[0195] II. Preparation of Hydrogels

[0196] Two hydrogel solutions were prepared according to schemes (i) and (ii), as follows:

[0197] (i) Mix the silk fibroin solution, glycerol and pure water evenly to obtain a mixed solution with a final concentration of 21 mg / mL of silk fibroin and a volume fraction of 30% of glycerol in the mixed solution; add alkaline phosphatase stock solution to the mixed solution (1 μL of alkaline phosphatase stock solution per 100 μL of mixed solution) to obtain an enzyme-containing hydrogel solution (ALP@SF).

[0198] (ii) Mix the silk fibroin solution, protocatechuic aldehyde solution, glycerol and pure water evenly to obtain a mixed solution. The final concentration of silk fibroin in the mixed solution is 21 mg / mL, the final concentration of protocatechuic aldehyde is 0.05 mol / L, and the volume fraction of glycerol in the mixed solution is 30%. Add alkaline phosphatase stock solution to the mixed solution (1 μL of alkaline phosphatase stock solution is added per 100 μL of mixed solution) to obtain an enzyme-containing hydrogel solution (ALP@SF / PA).

[0199] 50 μL of the hydrogel solutions prepared by schemes (i) and (ii) were added to different 24-well plates and left to stand overnight at room temperature to obtain hydrogels.

[0200] II. Experimental Methods and Results

[0201] (1) Add 2 mL of pure water to each well and wash the hydrogel by shaking at 120 rpm at room temperature. Change the pure water every 12 hours and collect the used washing solution. Continue washing for a total of 48 hours. After 48 hours of washing, the ALP@SF hydrogel and ALP@SF / PA hydrogel are as follows: Figure 12 As shown in Figure a, the results indicate that the two hydrogels have approximately the same volume.

[0202] (2) The ALP enzyme activity detection solution was prepared according to the method in Example 1.

[0203] Add 250 μL of washing buffer at different time intervals and 500 μL of ALP enzyme activity assay solution to each well of a 24-well plate. Incubate the plates at 37°C and shake at 120 rpm to test the enzyme activity of the washing buffer at different time intervals. After the reaction, transfer 100 μL of the reaction solution to a 96-well plate and measure the absorbance at 405 nm (higher absorbance indicates higher enzyme activity).

[0204] The ALP enzyme activity assay results of the hydrogel washing solution at different time points are as follows: Figure 12 Figures b, c, d, and e show that the enzyme activity in the washing solution of ALP@SF / PA hydrogel was lower during the washing process, indicating that it released less enzyme than ALP@SF hydrogel. This suggests that the introduction of protocatechuic aldehyde can also enhance the enzyme fixation effect of silk fibroin-based enzyme fixation media under wet conditions.

[0205] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A medium for immobilizing enzymes, characterized in that, It includes silk fibroin and protocatechuic aldehyde; the mass ratio of the silk fibroin to protocatechuic aldehyde is (10~30):(0.06906~172.65).

2. A solution for immobilizing biological enzymes, characterized in that, It includes silk fibroin, protocatechuic aldehyde and solvent; the mass ratio of silk fibroin to protocatechuic aldehyde is (10~30):(0.06906~34.53); the final concentration of silk fibroin is 10~30 mg / mL and the final concentration of protocatechuic aldehyde is 0.5~250 mmol / L.

3. A microsphere for immobilizing an enzyme, characterized in that, It includes silk fibroin, protocatechuic aldehyde and solvent; the mass ratio of silk fibroin to protocatechuic aldehyde is (10~20):(69.06~172.65); the final concentration of silk fibroin is 10~20 mg / mL and the final concentration of protocatechuic aldehyde is 0.5~1.25 mol / L.

4. The microspheres according to claim 3, characterized in that, A 30-40 mg / mL silk fibroin solution was added to a 1.5-2.5 mol / L protocatechuic aldehyde solution and mixed to obtain a microsphere solution; the volume ratio of the silk fibroin solution to the protocatechuic aldehyde solution was (1-2):(1-2).

5. A hydrogel solution for immobilizing enzymes, characterized in that, The solution includes silk fibroin, protocatechuic aldehyde, glycerol, and solvent; the mass ratio of silk fibroin to protocatechuic aldehyde is (10~30):(0.469~6.906); the final concentration of silk fibroin in the hydrogel solution is 10~30 mg / mL, and the final concentration of protocatechuic aldehyde is 3.4~50 mmol / L.

6. The solution according to claim 2, the microspheres according to claim 3, or the hydrogel solution according to claim 5, characterized in that, The solvent is water.

7. The use of the medium of claim 1, the solution of claim 2, the microspheres of claim 3, or the hydrogel solution of claim 5 as an enzyme immobilization medium or in the preparation of immobilized enzymes.

8. A method for enzyme immobilization, characterized in that, The enzyme solution is mixed with the immobilization medium; the immobilization medium is the medium of claim 1, the solution of claim 2, the microspheres of claim 3, or the hydrogel solution of claim 5.

9. An immobilized enzyme, characterized in that, It is prepared by the method described in claim 8.

10. The immobilized enzyme according to claim 9, characterized in that, The enzyme solution was mixed with the immobilization medium and then dried to obtain the immobilized enzyme.

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