A trypsin inhibitor degrading enzyme mutant and use thereof

Abstract

The application belongs to the technical field of enzyme engineering, and particularly relates to a trypsin inhibitor degrading enzyme mutant and application thereof. The application provides a trypsin inhibitor degrading enzyme mutant, the SDP enzyme activity of which is increased by nearly 20% compared with that of the original protein, can tolerate a high-salt environment, and can still maintain about 90% of the activity after being treated at a 3M salt concentration for 24 hours; the degradation rate of the trypsin inhibitor can still reach nearly 50% at a 3M salt concentration, and can meet the application requirement of a high-salt scene such as food fermentation. The trypsin inhibitor degrading enzyme mutant can effectively degrade the trypsin inhibitor in soybean or soybean derivative products, improve the utilization rate of raw material protein, is suitable for the production of legume fermented products, significantly improves the ammonia nitrogen level of the products, and improves the product quality.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and particularly relates to a trypsin inhibitor degrading enzyme mutant and application thereof. Background Art

[0002] Trypsin inhibitors (STIs) generally refer to proteins or peptides that inhibit trypsin activity. They are primarily found in plants of the grass and legume families and are one of the major anti-nutritional factors in soybeans. The primary mechanism of action of trypsin inhibitors is to form stable complexes with trypsin and chymotrypsin, inactivating these proteases and significantly reducing the digestion and absorption efficiency of dietary protein. Studies have shown that STIs have a significant negative impact on animal growth performance, often leading to digestive system problems including pancreatic hypertrophy, increased diarrhea, weight loss, and excessive cholecystokinin secretion. Furthermore, excessive consumption of soy foods containing STIs may cause intestinal dysfunction, impair small intestinal proteolytic activity, and reduce free amino acid concentrations. Research into the inhibition of STI activity is crucial for improving the quality and nutritional value of soy protein and is a crucial factor to consider during the critical stages of soy product manufacturing.

[0003] Currently, the main methods for inhibiting STI activity include physical, chemical, and biological methods. Traditional physical methods, such as autoclaving and ultra-high temperature treatment, generally have limited effectiveness in degrading STIs. Furthermore, high-intensity physical processing can lead to the loss of nutrients in food, thereby reducing the bioavailability of amino acids. In recent years, emerging technologies such as ultrasound, radiation, and dielectric barrier discharge have also been explored for STI degradation. While these technologies offer advantages over traditional methods in terms of processing efficiency and time cost, their high equipment costs and technical barriers limit their large-scale application. In contrast, biological treatment methods are considered ideal for STI degradation due to their high efficiency, cost-effectiveness, and environmental friendliness. For example, in patent CN 110452833 A, Bacillus pumilus was isolated and screened, demonstrating its high efficiency in degrading soybean trypsin inhibitor. Using this strain to degrade soybean trypsin inhibitor in soybeans or soybean-derived products offers advantages such as high degradation efficiency, simplicity, and low cost. Patent CN113736765 B discovered that functional protein peptidase S8 and peptidase M84 derived from Bacillus pumilus have high trypsin inhibitor degradation capabilities. The Pichia pastoris expression system was further used to achieve large-scale expression of these two peptidases that have a degrading effect on trypsin inhibitors. The recombinant enzymes prepared using the method provided by this invention can efficiently and stably degrade trypsin inhibitors and can be used to degrade trypsin inhibitors in soybean meal.

[0004] In the food, feed and other industries, especially in the fermentation production of condiments, enzymatic hydrolysis reactions are generally required in a high-salt environment. Developing or finding a trypsin inhibitor degrading enzyme (SDP) with high salt tolerance and high catalytic efficiency can not only effectively solve the negative effects of STI, but also provide technical support for the in-depth development and efficient utilization of soybean by-products, which will be of great value and significance for expanding its scope of application. Through semi-rational design of proteins, finding key sites of enzymes for mutation and constructing protease mutants with better performance is a good enzyme improvement strategy. At present, there is still no research on the construction of trypsin inhibitor degrading enzyme mutants that can work efficiently in a high-salt environment. At the same time, in order to improve degradation efficiency and save costs, it is also crucial to screen out SDP enzymes with higher activity and use food-safe strains to heterologously express SDP enzymes. Summary of the Invention

[0005] Based on the above technical problems, the main purpose of the present invention is to overcome the shortcomings of the above-mentioned background technology. The SDP enzyme gene of halodurable Bacillus is used as a template, the SDP enzyme is modeled by Alphafold and docked with the STI molecule. The docking results are analyzed to screen out key amino acids for alanine scanning. After alanine mutation, mutants with improved STI degradation activity are screened out. The expressed SDP enzyme still maintains high activity in a high-salt environment, thereby meeting the application requirements of high-salt environments.

[0006] To achieve the above objectives, the inventors conducted in-depth research and, after repeated research and demonstration, completed the present invention, which is as follows:

[0007] In a first aspect, the present invention provides a trypsin inhibitor degrading enzyme mutant, wherein the amino acid sequence of the trypsin inhibitor degrading enzyme mutant is shown in SEQ ID NO.1.

[0008] In a second aspect, the embodiments of the present application provide a gene encoding the above-mentioned trypsin inhibitor degrading enzyme mutant.

[0009] In a third aspect, an embodiment of the present application provides a recombinant expression vector carrying a gene encoding the above-mentioned trypsin inhibitor degrading enzyme mutant.

[0010] The vector backbone of the recombinant vector can be selected by those skilled in the art according to implementation needs, and can be any vector with expression capability, disclosed or undisclosed in the art, including but not limited to eukaryotic vectors and prokaryotic vectors.

[0011] In a fourth aspect, an embodiment of the present application provides a recombinant host cell, wherein the recombinant host cell is a host cell transformed / transfected with the above-mentioned recombinant expression vector.

[0012] The host cell may be any cell type with expression capability disclosed or undisclosed in the art, including but not limited to eukaryotic cells or prokaryotic cells; preferably Bacillus, more preferably Bacillus subtilis.

[0013] In a fifth aspect, the embodiments of the present application provide the use of the above-mentioned recombinant vector or recombinant host cell in the production of trypsin inhibitor degrading enzyme.

[0014] In a sixth aspect, the embodiments of the present application provide the use of the above-mentioned trypsin inhibitor degrading enzyme mutant in the preparation of a degraded trypsin inhibitor product.

[0015] In a seventh aspect, the embodiments of the present application provide the use of the above-mentioned alkaline protease mutant in food fermentation; specifically, it can be used for the fermentation of soybean products.

[0016] In an eighth aspect, an embodiment of the present application provides an enzyme preparation comprising the above-mentioned trypsin inhibitor degrading enzyme mutant.

[0017] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

[0018] 1. The present invention provides a trypsin inhibitor degrading enzyme mutant, the SDP enzyme activity expressed by the mutant is increased by nearly 20% compared with the original protein.

[0019] 2. The present invention provides a trypsin inhibitor degrading enzyme mutant, the expressed SDP enzyme of which has excellent salt tolerance and can still maintain about 90% activity after treatment at a salt concentration of 3M for 24 hours; at a salt concentration of 3M, the degradation rate of trypsin inhibitor can still reach nearly 50%, which can meet the application requirements of high-salt scenarios such as food fermentation.

[0020] 3. The trypsin inhibitor degrading enzyme mutant provided by the present invention can effectively degrade the trypsin inhibitor in the raw materials when treating soybeans or soybean-derived products, thereby improving the utilization rate of raw material protein. When used in the production of soybean fermentation products, it can significantly increase the ammonia nitrogen level of the product and improve the product quality.

[0021] 4. The present invention provides a heterologous expression system for a trypsin inhibitor degrading enzyme mutant and constructs a Bacillus subtilis strain capable of heterologously expressing the trypsin inhibitor degrading enzyme mutant. Bacillus subtilis is a certified safe strain that has long been used in the food fermentation industry and has a mature fermentation process, making it more suitable for subsequent commercial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Figure 2 is the result of enzyme activity assay of SDP mutants at different temperatures;

[0023] Figure 2 The results of enzyme activity determination of SDP mutants at different pH values ​​are shown;

[0024] Figure 3 The results of enzyme activity assay of SDP mutants at different salt concentrations are shown;

[0025] Figure 4 This is a diagram showing the test results of the SDP mutant in a soybean high-salt fermentation scenario. DETAILED DESCRIPTION

[0026] To facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0028] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0029] The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0030] The terms and related determination methods involved in the present invention are explained as follows:

[0031] Trypsin inhibitor degrading enzyme activity assay method:

[0032] The activity of trypsin inhibitor-degrading enzymes is indirectly indicated by the residual activity of STI. The STI activity assay follows the National Food Safety Standard for Determination of Trypsin Inhibitor Activity in Soy Products (GB 5009.224-2016). The protein solution was incubated with 160 BAEE U·mL⁻¹ of STI (1 mL of STI requires 160 BAEE U) at 40°C for 30 minutes. The mixture was then reacted with the synthetic substrate Na-benzoyl-DL-arginine p-nitroanilide hydrochloride (BAPNA) at 40°C for 10 minutes. Trypsin (0.0135 mg / mL) was then added and the reaction continued at 40°C for 10 minutes. The reaction was terminated with 30% (v / v) glacial acetic acid. In the control group, the protein solution was replaced with purified water, and the remaining steps were the same as above. In the blank group, 30% (v / v) glacial acetic acid was added before the reaction, and the remaining steps were the same. Activity was measured at 410 nm using an Eppendorf BioSpectrometer. The activity of protein degradation (U·mL-1) was defined as an absorbance increase of 0.01 within 10 minutes. To ensure the accuracy of the experiment and confirm that the isolated and purified protein was an enzyme with the ability to degrade proteins, the Folin-Ciocalteu method was used to determine the activity of neutral protease (NP) as a reference throughout the experiment.

[0033] Example 1 Construction of STI degrading enzyme mutants

[0034] Using the SDP enzyme gene from a previously screened halodurable Bacillus strain as a template, the AlphaFold 2 algorithm predicted the SDP enzyme structure, resulting in a highly accurate model with a high pLDDT score and a low predicted alignment error (PAE). The average pLDDT score was 96, demonstrating high confidence in the structure prediction. The resulting SDP enzyme structure was then molecularly docked with STI (PDBID: 1AVU) using the ZDOCK 3.0.2 program. The docking results were imported into PDBe-PISAInterface, and the binding surface amino acid analysis of the two docked proteins was performed. The surface buried area (BSA) was used for screening, and finally 18 amino acids, including D88, L89, G91, G92, F93, N136, D185, E220, D240, M241, Y293, S319, H320, R94, Y95, Q137, Y292, and G294, were screened out, indicating that the SDP enzyme binds tightly to the STI.

[0035] Alanine mutations were introduced between the binding sites of STI to analyze which site had the greatest impact on the activity of SDP enzyme.

[0036] Positive clones obtained by mutation were inoculated into LB liquid medium and cultured at 37°C for 10 hours to obtain a seed solution. 50 μL of the seed solution was transferred to 40 mL of LB liquid medium and cultured at 37°C for 2 hours. IPTG was added to a final concentration of 0.2 mM and induced at 25°C for another 10 hours to obtain a fermentation broth. The fermentation broth was centrifuged at 3500 rpm at 4°C for 10 minutes, the supernatant discarded, and 5 mL of PBS buffer (pH 7.0) was added and ultrasonically disrupted to obtain a cell lysate. The cell lysate was centrifuged at 3500 rpm at 4°C for 30 minutes to obtain the cell lysate supernatant of the SDP enzyme mutant.

[0037] The cell supernatant was taken to determine the STI degrading enzyme activity, and the preferred mutants with higher activity than the wild type were screened. The obtained data are recorded in Table 1.

[0038] Table 1 Activity test of STI degrading enzyme mutants

[0039]

[0040] According to the results in Table 1, the SDP enzyme activity of the D88A mutant was increased by 18.26% compared with the WT original strain. It was selected as the target mutant, and the amino acid sequence of the SDP enzyme expressed by it is shown in SEQ ID NO: 1.

[0041] Example 2 Construction of SDP enzyme mutant expression strain

[0042] The SDP gene was amplified using mutant D88A as a template, yielding a 1038-bp PCR product. After gel extraction, the PCR product was ligated with the linearized pBE-P43 plasmid. The ligation product was then transformed into competent Bacillus subtilis SCK6. Positive clones were screened to obtain the recombinant plasmid pEB-P43-SDP, containing the gene encoding the SDP mutant, and the recombinant B. subtilis SCK6 / pEB-P43-SDP, containing the gene encoding the SDP mutant.

[0043] B. subtilis SCK6 / pEB-P43-SDP was then inoculated into LB liquid medium and cultured at 37°C for 10 hours before plasmid extraction. The plasmid was then transformed into B. subtilis WB800 and cultured at 37°C for 8-10 hours. Five transformants were picked from LB solid medium and inoculated into LB liquid medium for culture at 37°C for 10 hours before plasmid extraction. The extracted plasmid was then verified by enzyme digestion and sequencing. If confirmed, the recombinant strain B. subtilis WB800 / pEB-P43-SDP expressing the gene encoding the SDP mutant was confirmed.

[0044] The above-mentioned recombinant SDP mutant bacteria were inoculated into LB liquid medium containing Kan resistance and cultured at 37°C for 24 hours. After the bacterial liquid was centrifuged, the supernatant was taken to obtain a crude enzyme solution containing SDP. The SDP enzyme activity was determined according to the above-mentioned activity determination method, and the recombinant SDP activity was measured to be 411.94±0.37 U·L-1.

[0045] Example 3 Performance determination of SDP protease mutants

[0046] 1. Determination of the optimal temperature for SDP mutants: The activated recombinant SDP mutant strain was inoculated into LB liquid medium at a 1.5% inoculum volume and cultured at 37°C for 24 hours. After centrifugation, the supernatant was taken and incubated at 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C for 24 hours. Then, STI solution was added and incubated for 30 minutes. Samples were taken to measure the SDP protease activity and the results were recorded. Figure 1 middle.

[0047] Depend on Figure 1 The results showed that the recombinant SDP mutant exhibited enzyme activity in the temperature range of 30-65°C, with the optimal activity temperature range being between 30 and 40°C. Above 40°C, the activity gradually decreased and was almost zero at 70°C.

[0048] 2. Determination of the optimal pH of SDP mutants: The activated recombinant SDP mutant strain was inoculated into LB liquid medium at a 1.5% inoculum volume and cultured at 37°C for 24 hours with shaking. After centrifugation, the supernatant was collected to determine the enzyme activity of SDP at pH 3-12. The results were recorded in Figure 2 middle.

[0049] like Figure 2 As shown, the SDP mutants were active across the pH range of 5 to 12, with optimal activity at pH 9.0. Activity gradually decreased above pH 9. Notably, the SDP mutants remained stable within the pH range of 9.0-12.0, with relative activity exceeding 20%. Therefore, SDP can be classified as a protease with a broad pH tolerance.

[0050] 3. Determination of NaCl tolerance of SDP mutants: The activated recombinant SDP mutant strain was inoculated into LB liquid medium at a 1.5% inoculum and cultured at 37°C with shaking for 24 hours. The supernatant was collected after centrifugation. Solid NaCl was added to the supernatant and fully dissolved to make the final salt concentration 0M, 2M, and 3M, respectively. The supernatant was stored at 4°C for a certain period of time, and samples were taken every 12 hours to measure the SDP enzyme activity. The results were recorded in Figure 3 middle.

[0051] like Figure 3As shown, in a 96-hour salt tolerance experiment, the 0M NaCl control group maintained a baseline activity retention rate of 80.3±1.2%. When the salt concentration was increased to 2M, the enzyme exhibited a biphasic response characteristic of first activation and then stabilization - reaching a peak activity of 121.5±2.1% at 36 hours (an increase of 21.5% over the initial activity), and still maintained at 115.4±1.8% within 48 hours, and finally stabilized at a level of 75.6±1.5%. Under extreme 3M NaCl conditions, the salt tolerance advantage of the SDP mutant was particularly significant: even after exposure to the end of 96 hours, its activity retention rate still reached 67.4±2.3%, and it always maintained a high activity range of >70% before 60 hours.

[0052] 4. Testing the ability of SDP mutants to degrade STI at different salt concentrations: The activated recombinant SDP mutant strain was inoculated into LB liquid medium at a 1.5% inoculum and cultured at 37°C with shaking for 24 hours. The culture was centrifuged and the supernatant was collected. Solid NaCl was added to the supernatant and dissolved thoroughly to achieve final salt concentrations of 0 M, 2 M, and 3 M, respectively. The supernatant was then incubated at 4°C overnight and mixed with STI for 30 minutes. The relative residual activity of the STI was measured. The results are recorded in Table 2.

[0053] Table 2 Test of SDP's ability to degrade STI at different salt concentrations

[0054] Group Relative residual activity of STI (%) control group 98.48±0.73 0M 44.39±1.17 2M 32.56±0.46 3M 51.79±3.69

[0055] As shown in Table 2, the control group (Control) showed stable STI relative activity without the addition of SDP, and the average of three independent determinations was 98.48%, confirming that the experimental system had good stability.

[0056] When the SDP mutant was added, STI activity decreased significantly, indicating that SDP has a significant hydrolytic effect on STI. At a salt concentration of 0 M, STI activity dropped sharply to approximately 45%, a decrease of approximately 54.9% compared to the control. As the salt concentration increased to 2 M, the enzymatic hydrolysis effect further intensified, with STI activity dropping to approximately 33%, reaching its lowest level. When the salt concentration was further increased to 3 M, STI activity decreased by approximately 48% relative to 0 M, demonstrating that the SDP mutant can maintain high STI degradation efficiency even in a high-salt environment of 3 M.

[0057] Example 4 SDP protease mutant application effect test

[0058] Preparation of crude enzyme solution: The recombinant SDP mutant strain was cultured in solid tryptone glucose yeast (TGY) medium at 37°C for 16 hours to obtain a single colony. This colony was then inoculated into liquid TGY medium at 37°C for 16 hours. A 1.5% inoculum of this solution was then inoculated into liquid TGY medium and incubated at 37°C for 24 hours to obtain a seed solution. The seed solution was then inoculated into a fermenter at a 1% inoculum and fermented at 37°C for 20 hours. During this time, defoamer and sugar supplementation were added as needed. The fermenter was rotated at 200-800 rpm, the tank pressure was 0.05 MPa, and the ventilation rate was 0.54 Nm³ / h. After fermentation, diatomaceous earth was added and stirred for 30 minutes. The fermentation solution was then filtered through a plate-and-frame to obtain the supernatant, which was concentrated using a 3 kDa molecular weight cutoff fiber ultrafiltration membrane and sterilized through a 0.22 μm filter membrane. The crude enzyme solution was then assayed for protease activity.

[0059] Application effect test: After washing, soaking and steaming, the soybeans are cooled to room temperature, mixed with wheat flour at a dry material mass ratio of 7:3, and 3.042 koji essence of 1‰ of dry material is added. After incubation at 32℃ for 12h, the koji is turned over, and then transferred to 28℃ and continued to be cultivated until the surface of the soybeans is covered with yellow-green spores. Take 250g of the finished koji, put it in a 900mL sauce bottle, add 500g of 18% (M / V) salt water, mix it thoroughly, add 750U / g of SDP crude enzyme solution (the amount of crude enzyme solution added is calculated based on the mass of the koji), and at the same time add 750U / g of commercial protease as the control group. The group without enzyme solution is the blank group, and then continue to ferment in a constant temperature chamber at 30℃ as usual. The fermentation cycle is 90d, and samples are taken once at 0d, 7d, 15d, 30d, 45d, 60d, 75d, and 90d to detect the protease activity and amino acid nitrogen in the mash, and the results are recorded. Figure 4 middle.

[0060] like Figure 4 As shown in the figure, the protease activity and amino acid nitrogen content of different treatment groups showed significant differences during the fermentation process. Compared with the blank group, the protease activity of the control group was significantly improved, confirming that the addition of commercial enzymes can effectively enhance the protease activity of the fermentation system. It is worth noting that the protease activity of the experimental group was further significantly improved compared with the control group, which was attributed to the targeted degradation effect of SDP on STI. Through time series analysis, it was found that the inhibitory effect of STI on protease activity was mainly concentrated in the early stage of fermentation (0-7 days), and SDP continuously degraded

[0061] STI maintained a high level of enzyme activity throughout the fermentation period (0-90 days), indicating that the complex has stable catalytic properties.

[0062] In terms of the dynamic changes in amino acid nitrogen, no significant differences were observed among the three groups during the initial fermentation period (days 0-7). However, as fermentation progressed (days 15-90), the amino acid nitrogen content in the experimental group was significantly higher than that in both the blank and control groups, reaching approximately 1.4 times that of the control group. This suggests that the recombinant SDP mutant crude enzyme solution effectively reduces the activity of trypsin inhibitors, allowing the proteases in the fermentation system to more efficiently and fully hydrolyze soybeans, thereby improving the nutrient utilization of soybeans.

[0063] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A trypsin inhibitor degrading enzyme mutant, characterized in that: The amino acid sequence of the trypsin inhibitor degrading enzyme mutant is shown in SEQ ID NO.

1.

2. A gene, characterized in that The gene encodes the trypsin inhibitor degrading enzyme mutant according to claim 1.

3. A recombinant expression vector, characterized in that: The recombinant expression vector carries the gene according to claim 2.

4. A recombinant host cell, characterized in that The recombinant host cell is a host cell transformed / transfected with the recombinant expression vector according to claim 3.

5. The recombinant host cell according to claim 4, wherein The host cell is Bacillus subtilis.

6. Use of the recombinant expression vector according to claim 3 or the recombinant host cell according to claim 4 in producing a trypsin inhibitor degrading enzyme.

7. Use of the trypsin inhibitor degrading enzyme mutant according to claim 1 in the preparation of a degraded trypsin inhibitor product.

8. Use of the trypsin inhibitor degrading enzyme mutant according to claim 1 in food fermentation.

9. An enzyme preparation, characterized in that The enzyme preparation comprises the trypsin inhibitor degrading enzyme mutant according to claim 1.

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