Enzyme for enzymolysis of silk fibroin as well as preparation method and application of enzyme
By preparing immobilized silk fibroin enzymes, the problems of stability and impurity introduction in the traditional enzymatic hydrolysis process were solved, realizing efficient and low-cost enzymatic hydrolysis of silk fibroin and improving the quality and safety of the hydrolysis products.
Patent Information
- Application Number
- CN202511356787.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-22
- Publication Date
- 2025-12-05
AI Technical Summary
In traditional enzymatic hydrolysis of silk fibroin, free enzymes have weak acid and alkali resistance, are prone to autolysis or self-aggregation, and are difficult to separate from the product, leading to the introduction of foreign proteins and impurities, which limits their industrial application; immobilized enzymes pose a risk of carrier components dissolving into the product.
Using silk fibroin itself as a carrier, silk fibroin microspheres are prepared and protease is immobilized to form silk fibroin immobilized enzymes, avoiding the introduction of new impurities by using other carriers. The protease is immobilized by the silk fibroin microsphere-protease binding reaction.
It achieves stability and selectivity in the enzymatic hydrolysis process, reduces the risk of introducing extraneous proteins and impurities, maintains enzyme activity above 80%, and is reusable, thereby reducing production costs and improving the quality and safety of the enzymatic hydrolysis products.
Smart Images
Figure CN121065150A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to an enzyme for hydrolyzing silk fibroin, its preparation method, and its application. Background Technology
[0002] Silk fibroin is a natural protein-based polymer extracted from animal silk such as silkworm silk. It possesses excellent biocompatibility and biodegradability, and has been widely used in clothing fabrics, electronics, medical devices, skincare, and functional foods. Silk fibroin contains 18 amino acids, including glycine, serine, and alanine, and can be hydrolyzed by proteases into small-molecule proteins or peptides, showing broad development prospects in daily chemical, food, cosmetic, pharmaceutical, and biomaterial fields. Numerous methods have been reported for preparing small-molecule silk fibroin and silk peptides, including acid hydrolysis, alkaline hydrolysis, salt hydrolysis, and enzymatic hydrolysis. Acid hydrolysis, alkaline hydrolysis, and salt hydrolysis all utilize the generalized Lewis acid-base theory to non-selectively break the amide bonds of proteins, but suffer from drawbacks such as a wide molecular weight distribution of hydrolysates and loss of activity. Enzymatic hydrolysis of silk fibroin using proteases offers advantages such as mild conditions, high selectivity, narrow molecular weight distribution of hydrolysates, and minimal impact on product activity. However, traditional enzymatic hydrolysis methods have the following drawbacks: (1) free enzymes have weak acid and alkali resistance and are prone to autolysis or self-aggregation; (2) it is not easy to separate the enzyme and silk fibroin hydrolysis products quickly and at low cost after enzymatic hydrolysis, which often leads to the introduction of impurities into small molecular weight silk fibroin or silk fibroin peptide products. The above-mentioned drawbacks limit the industrial application of enzymatic hydrolysis.
[0003] Immobilized enzyme technology involves using specific physical or chemical methods to immobilize free enzymes on a support, allowing them to be highly enriched within a defined spatial range while maintaining their unique catalytic activity. Immobilized enzymes can utilize the protective effect of the support, enabling them to maintain activity even in harsh environments such as strong acids, strong alkalis, and high temperatures, significantly improving enzyme stability. Compared to free enzymes, immobilized enzymes retain their high efficiency, specificity, and mild catalytic reaction characteristics while also offering advantages such as easy separation, recovery, and reusability, making them a current research hotspot in the field.
[0004] For the main application areas of small molecule silk fibroin or silk peptides, such as medical devices, beauty and skincare, and medical aesthetics, the introduction of extraneous proteins and impurities greatly increases the safety risks of the products. Traditional free enzyme hydrolysis methods cannot meet the needs of reducing the risk of introducing extraneous proteins, while using commonly used immobilized enzymes also carries the risk of carrier components dissolving into the product, introducing new impurities.
[0005] Based on this, the present invention addresses the high requirements for limiting impurities, especially extraneous proteins, in the main application areas of small molecule silk fibroin or silk peptides. It proposes an enzyme for enzymatic hydrolysis of silk fibroin, namely a silk fibroin immobilization enzyme with silk fibroin itself as a carrier, aiming to minimize the introduction of new impurities and extraneous proteins and maximize the control of the risk of impurity introduction in the enzymatic hydrolysis products of silk fibroin. Summary of the Invention
[0006] The purpose of this invention is to provide a method for preparing an enzyme for hydrolyzing silk fibroin and an enzyme for hydrolyzing silk fibroin obtained by the method.
[0007] Another object of the present invention is to provide the application of the above-mentioned enzyme for hydrolyzing silk fibroin in the enzymatic hydrolysis of silk fibroin and the preparation of silk fibroin enzymatic hydrolysate.
[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The method for preparing the enzyme for enzymatic hydrolysis of silk fibroin according to the present invention includes the following steps: S1. Preparation of silk fibroin microspheres: Silk fibroin was dissolved in water, polyether F-127 and genipin were added, and the mixture was stirred until dissolved. The mixture was then filtered to obtain the aqueous phase. Soybean oil was taken, polyglycerol ricinoleate was added, and the mixture was stirred until homogeneous to obtain the oil phase. The aqueous phase was slowly added dropwise to the oil phase and stirred to emulsify. After emulsification, the mixture was allowed to stand at room temperature to solidify. After solidification, the upper oil layer was discarded, and the mixture was centrifuged. The resulting precipitate was first washed with petroleum ether, then soaked in ethanol, and finally the ethanol was evaporated to obtain silk fibroin microspheres. S2. Preparation of enzymes for hydrolyzing silk fibroin: Take the silk fibroin microspheres obtained in step S1, add them to PBS buffer solution, add genipin and protease, shake in a constant temperature water bath to fix the protease; after fixation, centrifuge, wash the resulting precipitate repeatedly with distilled water and dry it to obtain the enzyme for enzymatic hydrolysis of silk fibroin.
[0009] Preferably, in the method for preparing the enzyme for enzymatic hydrolysis of silk fibroin according to the present invention, in step S1: In the aqueous phase: the concentration of silk fibroin is 40~50 mg / mL, the concentration of polyether F-127 is 20~30 mg / mL, and the concentration of genipin is 1.5~2.0 mg / mL; In the oil phase, the mass ratio of soybean oil to polyglycerol ricinoleate is 12-13:1; The volume-to-mass ratio of the aqueous phase to the oil phase is 1 mL: 25~30 g.
[0010] Preferably, in the method for preparing the enzyme for enzymatic hydrolysis of silk fibroin according to the present invention, in step S1: The filtration specifically involves passing through a 0.22μm or 0.45μm microporous filter membrane; The stirring time is 10-30 minutes; The curing time at room temperature is 24~48h.
[0011] Preferably, in the method for preparing the enzyme for enzymatic hydrolysis of silk fibroin according to the present invention, in step S2: The protease is any one of papain, alkaline protease, pepsin, trypsin, elastase, and collagenase. The amount of silk fibroin microspheres added is 5~10 mg / mL, the amount of genipin added is 5~9 mg / mL, and the amount of protease added is 3~7 mg / mL.
[0012] Preferably, in the method for preparing the enzyme for enzymatic hydrolysis of silk fibroin according to the present invention, in step S2: The pH value of the PBS buffer solution is 7.2~7.4; The temperature conditions for oscillation under the constant temperature water bath are 25~60℃, and the oscillation time is 2~12h; The drying temperature is 30~40℃.
[0013] The enzyme for hydrolyzing silk fibroin described in this invention is prepared using the method described above.
[0014] The application of the enzyme described in this invention for hydrolyzing silk fibroin in the enzymatic hydrolysis of silk fibroin and the preparation of silk fibroin enzymatic hydrolysis products.
[0015] Preferably, in the above application, the preparation method of the silk fibroin enzymatic hydrolysate is as follows: the enzyme for hydrolyzing silk fibroin is placed in a silk fibroin solution, the pH and temperature are adjusted, and the silk fibroin is hydrolyzed to obtain an enzymatic hydrolysate; the enzymatic hydrolysate is filtered through a vacuum filter, the filtrate is purified to remove impurities, and then spray-dried or freeze-dried to obtain the silk fibroin enzymatic hydrolysate.
[0016] In a further preferred embodiment, in the above application, the preparation method of the silk fibroin protein hydrolysate contains: the concentration of the silk fibroin protein solution is 20-25 mg / mL, and the amount of silk fibroin protein immobilization enzyme added is 1-4 mg / mL.
[0017] In a further preferred embodiment of the above application, the method for preparing the silk fibroin proteolytic product includes: The enzymatic hydrolysis conditions for silk fibroin are as follows: solution pH value of 2-10, hydrolysis temperature of 25-60℃, and hydrolysis time of 1-12h. The purification process specifically involves: filtering the enzymatic hydrolysate through a vacuum filter, microporous membrane filter, or membrane filtration to remove impurities; The drying method is spray drying.
[0018] The beneficial effects of this invention are: 1. This invention provides a method for preparing enzymes for enzymatic hydrolysis of silk fibroin. This method creatively uses silk fibroin, the target material to be enzymatically hydrolyzed, as a carrier. Through a special silk fibroin microsphere-protease binding reaction, the protease is immobilized on the surface of the silk fibroin microspheres, and a variety of silk fibroin immobilized enzymes are successfully prepared. When enzymatically hydrolyzing silk fibroin to prepare silk fibroin protease hydrolysate products, the risk of introducing new impurities caused by using other carriers is avoided.
[0019] 2. Enzyme activity assays revealed that the immobilized alkaline protease of silk fibroin prepared by this invention has a high enzyme activity of 3834.43 U / g. After being used repeatedly 20 times within 30 days, its enzyme activity remained above 80%, indicating that the immobilized silk fibroin enzyme provided by this invention is stable and has a high reusability, which can reduce the production cost of silk fibroin proteolytic products.
[0020] 3. The method provided by this invention for preparing silk fibroin proteolytic products offers advantages such as mild reaction conditions and low-cost, rapid separation of the enzyme and the proteolytic products, significantly reducing the risk of introducing extraneous proteins and impurities. Furthermore, the method eliminates the need for high-temperature enzyme inactivation after hydrolysis, minimizing the impact on the activity of the proteolytic products. This invention is of great significance for improving the quality of silk fibroin proteolytic products.
[0021] 4. The enzyme used in this invention to hydrolyze silk fibroin is blue, which is highly distinguishable from the white color of silk fibroin and its hydrolysate products. The presence of enzyme residue after hydrolysis can be visually identified, which is highly beneficial for controlling product quality. Attached Figure Description
[0022] Figure 1 Silk fibroin microspheres under a scanning electron microscope; Figure 2 A suspension of silk fibroin microspheres under an optical microscope; Figure 3 This is an enzyme suspension of silk fibroin obtained under an optical microscope. Detailed Implementation
[0023] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The following embodiments are for explanation and illustration only, and do not constitute a limitation on the technical solution of the present invention.
[0024] Example 1 The preparation method of the enzyme for enzymatic hydrolysis of silk fibroin (silk fibroin immobilized alkaline protease) is as follows: (1) Preparation of silk fibroin microspheres 1.47 g of silk fibroin was weighed and added to 30 mL of water. 0.6077 g of polyether F-127 and 0.0457 g of genipin were added, and the mixture was stirred until dissolved. The solution was then filtered through a 0.45 μm microporous membrane to obtain the aqueous phase. 716.10 g of soybean oil was weighed and added to 57.30 g of polyglycerol ricinoleate. The mixture was stirred for 30 min to obtain the oil phase. The aqueous phase was slowly added dropwise to the oil phase, and the mixture was stirred for 30 min to emulsify. After emulsification, the mixture was allowed to stand at room temperature for 48 h to fully solidify. After solidification, the upper oil layer was discarded, and the mixture was centrifuged. The resulting precipitate was washed first with petroleum ether, then with ethanol, and the ethanol was evaporated to obtain silk fibroin microspheres. The silk fibroin microspheres under a scanning electron microscope are shown below. Figure 1 As shown.
[0025] (2) Preparation of enzymes for hydrolyzing silk fibroin (silk fibroin immobilized alkaline protease) 0.0505 g of dried silk fibroin microspheres were weighed and added to 10 mL of PBS buffer solution (pH=7.2~7.4). Then, 0.0761 g of genipin and 0.0504 g of alkaline protease (100000 U / g) were added. The mixture was shaken in a 45℃ water bath for 2 h to immobilize the alkaline protease. After immobilization, the mixture was centrifuged, and the resulting precipitate was repeatedly washed with distilled water, drained, and dried at 40℃ to obtain silk fibroin immobilized alkaline protease. The silk fibroin microsphere suspension under an optical microscope is shown below. Figure 2 As shown, the silk fibroin immobilized enzyme suspension under an optical microscope is as follows: Figure 3 As shown.
[0026] Example 2 The enzyme (silk fibroin immobilized alkaline protease) prepared in Example 1 was used to enzymatically hydrolyze silk fibroin to prepare small molecule silk fibroin powder. The method is as follows: Weigh 1.022 g of silk fibroin powder, add 50 mg of the silk fibroin immobilized alkaline protease prepared in Example 1, add 50 mL of water, adjust the pH to 9.0, and enzymatically hydrolyze at 55°C for 4 h to obtain the enzymatic hydrolysate. Filter the enzymatic hydrolysate through a vacuum filter, repeatedly wash the obtained silk fibroin immobilized alkaline protease with distilled water, then centrifuge, drain, and dry to obtain the recovered silk fibroin immobilized alkaline protease, which is then sealed and refrigerated at 4°C for future use. Dispense the filtrate obtained after filtration of the enzymatic hydrolysate into 3000 Dalton dialysis bags, dialyze with purified water for 24 h, and then spray dry at an inlet temperature of 170°C, an outlet temperature of 90°C, and a fan speed of 5 m / s. 3 The peristaltic pump supplies liquid at a rate of 1000 mL / h to obtain small molecule silk fibroin powder.
[0027] Example 3 The preparation method of the enzyme for enzymatic hydrolysis of silk fibroin (silk fibroin immobilized papain) is as follows: (1) Preparation of silk fibroin microspheres Weigh 1.5056 g of silk fibroin and add it to 30 mL of water. Add 0.6062 g of polyether F-127 and 0.0475 g of genipin, stir until dissolved, and then filter through a 0.22 μm microporous membrane to obtain the aqueous phase. Weigh 801.78 g of soybean oil and add 64.79 g of polyglycerol ricinoleate, stir for 30 min to obtain the oil phase. Slowly add the aqueous phase dropwise to the oil phase and stir for 30 min to emulsify. After emulsification, let it stand at room temperature for 48 h to fully solidify. After solidification, discard the upper oil layer, centrifuge, and wash the resulting precipitate first with petroleum ether, then with ethanol, and then evaporate the ethanol to obtain silk fibroin microspheres.
[0028] (2) Preparation of enzymes for hydrolyzing silk fibroin (silk fibroin immobilized papain) Weigh 0.2005 g of the dried silk fibroin microspheres, add 20 mL of PBS buffer solution (pH=7.2~7.4), and add 0.1506 g of genipin and 0.1007 g of papain (200000 U / g). Incubate at 37℃ with shaking for 6 h to immobilize the papain. After immobilization, centrifuge, wash the resulting precipitate repeatedly with distilled water, drain, and dry at 35℃ to obtain silk fibroin immobilized papain.
[0029] Example 4 The enzyme (silk fibroin immobilized papain) obtained in Example 3 was used to enzymatically hydrolyze silk fibroin to prepare small molecule silk fibroin powder. The method is as follows: Weigh 1.0001 g of silk fibroin powder, add 50 mg of the silk fibroin immobilized papain prepared in Example 3, add 50 mL of water, adjust the pH to 7.0, and enzymatically hydrolyze at 55°C for 4 h to obtain the enzymatic hydrolysate. Filter the enzymatic hydrolysate through a vacuum filter, repeatedly wash the obtained silk fibroin immobilized papain with distilled water, then centrifuge, drain, and dry to obtain the recovered silk fibroin immobilized papain, which is then sealed and refrigerated at 4°C for future use. Dispense the filtrate obtained after filtration into 3000 Dalton dialysis bags, dialyze with purified water for 24 h, and then spray dry at an inlet temperature of 170°C, an outlet temperature of 90°C, and a fan speed of 5 m / s. 3 The peristaltic pump supplies liquid at a rate of 1000 mL / h to obtain small molecule silk fibroin powder.
[0030] Example 5 Silk fibroin immobilized pepsin, silk fibroin immobilized trypsin, and silk fibroin immobilized elastase were prepared using pepsin, trypsin, or elastase according to the methods in Example 1 or Example 3, respectively.
[0031] Example 6 The silk fibroin immobilized pepsin, silk fibroin immobilized trypsin, and silk fibroin immobilized elastase obtained in Example 5 were used to enzymatically hydrolyze silk fibroin to prepare small molecule silk fibroin powder, using the same method as in Example 2 or Example 4.
[0032] To further verify the reliability of the present invention, the inventors conducted enzyme activity testing on the immobilized alkaline protease from silk fibroin, as detailed below: Preparation of Folin reagent: Mix one part commercially available Folin reagent with two parts water and shake well; Preparation of sodium carbonate solution (0.0424 g / mL): Weigh 1.0596 g of anhydrous sodium carbonate and dilute to 25 mL; Preparation of trichloroacetic acid solution (0.0674 g / mL): Weigh 0.674 g of trichloroacetic acid, dissolve in water and dilute to 10 mL; Preparation of casein solution (0.01 g / mL): Weigh 0.1008 g of casein, add about 8 mL of borate buffer (pH=10.5), stir magnetically until homogeneous and free of large particles, then turn on the heating function until the casein is completely dissolved. Cool to room temperature and dilute to 10 mL in a volumetric flask; Preparation of L-tyrosine standard solution stock solution (100 μg / mL): Weigh 0.0106 g of L-tyrosine, dissolve in 1 mol / L hydrochloric acid solution and dilute to 10 mL to obtain a 1 mg / mL L-tyrosine solution. Then take 1... Prepare 1 mL of L-tyrosine solution with a concentration of mg / mL, and dilute to 10 mL with 0.1 mol / L hydrochloric acid solution. Prepare a series of L-tyrosine standard solutions according to Table 1.
[0033] ; Take 1 mL of each of the above-mentioned L-tyrosine standard solutions at different concentrations and place them in test tubes. Add 5 mL of sodium carbonate solution and 1 mL of Folin-Ciocalteu reagent sequentially. Incubate at 40℃ for 20 min. After cooling, measure the OD value at 680 nm. Adjust the OD value to 0 using the OD value of the test tube without tyrosine. Plot a standard curve of L-tyrosine concentration versus OD value. The equation of the standard curve is: A = 0.0108C - 0.0423, and the correlation coefficient is R. 2 =0.9813. Using the standard curve equation, the amount of L-tyrosine (μg) at an absorbance of 1 was calculated, which is the absorbance constant (K) value, and K = 96.50 was obtained.
[0034] Weigh out four portions of the silk fibroin immobilized alkaline protease prepared in Example 1, namely 0.0107 g, 0.0107 g, 0.0109 g, and 0.0110 g, and place them into four test tubes (one blank tube and three sample tubes), adding 1 mL of borate buffer (pH=10.5) to each. Preheat the casein solution at 40±0.2℃ for 5 min. Take the four test tubes containing the immobilized enzyme and preheat them at 40±0.2℃ for 2 min. Then add 2 mL of trichloroacetic acid to the blank tube, and then add 1 mL of casein solution to the sample tubes. Shake well, and after accurately timing for 10 min, immediately add 2 mL of trichloroacetic acid to the sample tubes and 1 mL of casein solution to the blank tubes. After thoroughly mixing all four test tubes, filter them separately using slow-speed qualitative filter paper for later use.
[0035] Accurately pipette 1 mL of the filtrate into four separate test tubes. Add 5 mL of sodium carbonate solution and 1 mL of prepared Folin-Ciocalteu reagent to each of the four test tubes, shake well, and immediately place in a constant temperature water bath at 40 ± 0.2 °C for 20 min. Measure the absorbance (OD) at 680 nm using a 10 mm cuvette. Read the concentration ρ1 of L-tyrosine in the final diluted sample solution from the standard curve, in μg / mL.
[0036] Sample enzyme activity X1 is calculated using the following formula: ; In the formula: X1: Enzyme activity of the sample, in U / g; ρ1: The concentration of L-tyrosine in the final diluted sample solution obtained from the standard curve, in μg / mL; 4: Total volume of reaction reagents, in mL; n1: The dilution factor of the sample; 10: Reaction time, in minutes.
[0037] The arithmetic mean of the absorbance values of the sample tubes measured in three parallel measurements were 0.988, 1.001, and 0.990, respectively. Substituting these values into the standard curve A=0.0108C-0.0423, the calculated concentrations ρ1 of L-tyrosine were 95.398 μg / mL, 96.602 μg / mL, and 95.583 μg / mL, respectively. Substituting these values into the enzyme activity calculation formula, the enzyme activity of the silk fibroin immobilized alkaline protease was found to be 3834.43 U / g.
[0038] The silk fibroin-immobilized alkaline protease was repeatedly used 20 times over 30 days. Enzyme activity was measured after the 20th use, and the result was 3162.75 U / g. Compared to the initial enzyme activity of 3834.43 U / g, the enzyme activity decreased by only 17.5%, indicating that the silk fibroin-immobilized enzyme prepared by this method is stable and has a high reusability. This reusable silk fibroin-immobilized enzyme can significantly reduce the production cost of silk fibroin protease hydrolysates.
[0039] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. A method for preparing an enzyme for enzymatic hydrolysis of silk fibroin, characterized in that, The preparation method includes the following steps: S1. Preparation of silk fibroin microspheres: Silk fibroin was dissolved in water, polyether F-127 and genipin were added, and the mixture was stirred until dissolved. The mixture was then filtered to obtain the aqueous phase. Soybean oil was taken, polyglycerol ricinoleate was added, and the mixture was stirred until homogeneous to obtain the oil phase. The aqueous phase was slowly added dropwise to the oil phase and stirred to emulsify. After emulsification, the mixture was allowed to stand at room temperature to solidify. After solidification, the upper oil layer was discarded, and the mixture was centrifuged. The resulting precipitate was first washed with petroleum ether, then soaked in ethanol, and finally the ethanol was evaporated to obtain silk fibroin microspheres. S2. Preparation of enzymes for hydrolyzing silk fibroin: Take the silk fibroin microspheres obtained in step S1, add them to PBS buffer solution, add genipin and protease, shake in a constant temperature water bath to fix the protease; after fixation, centrifuge, wash the resulting precipitate repeatedly with distilled water and dry it to obtain the enzyme for enzymatic hydrolysis of silk fibroin.
2. The method for preparing the enzyme for enzymatic hydrolysis of silk fibroin according to claim 1, characterized in that, In step S1: In the aqueous phase: the concentration of silk fibroin is 40~50 mg / mL, the concentration of polyether F-127 is 20~30 mg / mL, and the concentration of genipin is 1.5~2.0 mg / mL; In the oil phase, the mass ratio of soybean oil to polyglycerol ricinoleate is 12-13:1; The volume-to-mass ratio of the aqueous phase to the oil phase is 1 mL: 25~30 g.
3. The method for preparing the enzyme for enzymatic hydrolysis of silk fibroin according to claim 1, characterized in that, In step S1: The filtration specifically involves passing through a 0.22 μm or 0.45 μm microporous membrane; The stirring time is 10-30 minutes; The curing time at room temperature is 24~48 h.
4. The method for preparing the enzyme for enzymatic hydrolysis of silk fibroin according to claim 1, characterized in that, In step S2: The protease is any one of papain, alkaline protease, pepsin, trypsin, elastase, and collagenase. The amount of silk fibroin microspheres added is 5-10 mg / mL, the amount of genipin added is 5-9 mg / mL, and the amount of protease added is 3-7 mg / mL.
5. The method for preparing the enzyme for enzymatic hydrolysis of silk fibroin according to claim 1, characterized in that, In step S2: The pH value of the PBS buffer solution is 7.2~7.4; The temperature conditions for oscillation under the constant temperature water bath are 25~60℃, and the oscillation time is 2~12 h; The drying temperature is 30~40℃.
6. An enzyme for hydrolyzing silk fibroin, characterized in that, The enzyme used to hydrolyze silk fibroin is prepared by the method described in any one of claims 1-5.
7. The application of the enzyme for hydrolyzing silk fibroin as described in claim 6 in the enzymatic hydrolysis of silk fibroin and the preparation of silk fibroin enzymatic hydrolysate.
8. The application according to claim 7, characterized in that, The specific method for preparing the silk fibroin proteolytic product is as follows: the enzyme for hydrolyzing silk fibroin is placed in a silk fibroin solution, the pH and temperature are adjusted, and the silk fibroin is hydrolyzed to obtain an enzymatic hydrolysate; the enzymatic hydrolysate is filtered through a vacuum filter, the filtrate is purified to remove impurities, and then spray-dried or freeze-dried to obtain the silk fibroin proteolytic product.
9. The application according to claim 8, characterized in that, In the method for preparing the silk fibroin protein hydrolysate: the concentration of the silk fibroin solution is 20-25 mg / mL, and the amount of silk fibroin immobilization enzyme added is 1-4 mg / mL.
10. The application according to claim 8, characterized in that, In the preparation method of the silk fibroin protease hydrolysis product: The enzymatic hydrolysis conditions for silk fibroin are as follows: solution pH value of 2-10, hydrolysis temperature of 25-60℃, and hydrolysis time of 1-12 h. The purification process specifically involves: filtering the enzymatic hydrolysate through a vacuum filter, microporous membrane filter, or membrane filtration to remove impurities; The drying method is spray drying.