A Kunitz-type potato protease inhibitor mutant, its preparation method and application

Abstract

This invention relates to a Kunitz-type potato trypsin inhibitor mutant, its preparation method, and its applications, belonging to the fields of genetic engineering and protein engineering modification technology. Based on a wild-type potato trypsin inhibitor, this invention constructs the mutant shown in SEQ ID NO.3. This mutant exhibits 5.67 times higher trypsin inhibitory activity than the wild type, and in animal models, it increases cholecystokinin (CCK) secretion levels by 1.94 times and reduces food intake by 44.58%. The Kunitz-type potato trypsin inhibitor mutant provided by this invention can serve as an active ingredient and has significant application potential in biomedicine and food health fields, particularly in regulating appetite and alleviating obesity.

Description

Technical Field

[0001] This invention relates to a Kunitz-type potato protease inhibitor mutant, its preparation method, and its application, belonging to the fields of genetic engineering and protein engineering modification technology. Background Technology

[0002] According to a recent research report published in *The Lancet*, the global obese population reached over 1 billion in 2022. In China, based on Body Mass Index (BMI), approximately 34.8% of the population is overweight, and 14.1% is obese. Furthermore, the incidence of obesity-related complications such as fatty liver, prediabetes, dyslipidemia, and hypertension is increasing, seriously impacting national health. Currently, there are two main methods for weight loss: accelerating fat metabolism and enhancing and prolonging satiety. In recent years, the utilization of dietary fiber has primarily focused on enhancing satiety. However, excessive dietary fiber can hinder the absorption of nutrients such as iron and amino acids. Studies have found that dietary fiber that evades digestion and absorption in the small intestine leads to delayed gastric emptying and slower transit through the small intestine, resulting in a reduced rate of nutrient absorption. Therefore, dietary fiber may not be suitable for long-term consumption. Recent research indicates that amino acids have a stronger ability to promote fatty acid synthesis in the liver than glucose, making them the primary carbon source for liver fat production. This research also proposes a new strategy of limiting dietary protein to improve lipid accumulation.

[0003] Plant protease inhibitors (PIs) are proteins widely distributed in plant seeds and tubers. Generally, PIs form stable complexes with target proteases, blocking the enzyme's active site and thus inhibiting protease activity; they are considered anti-nutritional factors. Increasing research indicates that PIs regulate proteolytic activity in the body, affecting protein digestion and absorption, delaying gastric emptying, and consequently regulating appetite, making them a focus of attention in healthy weight loss. Potatoes (Solanum tuberosum L.) are a nutrient-rich and widely adaptable crop, a staple food for over 1.3 billion people in more than 120 countries. Potato protease inhibitors (PIs) can penetrate deep into the substrate pocket of target enzymes and form stable complexes, thereby blocking the catalytic center of the protease and inhibiting the activity of different types of proteases, exhibiting anti-nutritional functions. Currently, potato protease inhibitor II (PI-II) components are widely used in dietary supplements for weight control in many developed countries worldwide; European and American brands offer gummies and slimming soups, Japanese brands offer jellies, and Korean brands offer meal replacement products. Potato Kunitz-type protease inhibitors (PKPIs) are a class of resistant peptides formed during long-term evolution. They are the most abundant type of potato PIs and typically contain two disulfide bonds, making them biheaded inhibitors. PKPIs bind to serine proteases in a substrate-like manner via a tight, non-covalent binding, directly blocking the active site of the serine protease without altering its conformation, thus inhibiting enzyme activity. They hold great promise for applications in healthy weight loss. The potential satiety effect resulting from their strong protease inhibitory activity has become a research hotspot, making PKPIs an excellent carrier for developing highly effective anti-obesity factors and potentially leading to their widespread use in meal replacement foods.

[0004] However, the activity of currently discovered potato protease inhibitors is limited; studies have shown that a 2 mg / mL potato protein extract inhibits trypsin by only 75%. Therefore, screening and purifying highly active protease inhibitors (PIs) has been a key research focus in this field. Furthermore, the diverse inhibition modes of PIs (e.g., competitive and non-competitive mixed inhibition), their dynamic conformation, and thermal stability (e.g., stable within the pH range of 3.0-7.5, but potentially denatured at high temperatures) place high demands on production processes and formulation technologies. In addition, the process of recovering active proteins from potato processing wastewater is complex; traditional methods (acid-heat flocculation) easily lead to denaturation, while novel technologies (e.g., expanded bed adsorption), although able to retain activity, are costly, limiting large-scale application. Summary of the Invention

[0005] To address the aforementioned shortcomings of existing technologies, this invention utilizes artificial intelligence and molecular biology techniques to screen for proteins with high trypsin inhibitory activity from potato protein inhibitors (PIs). Furthermore, it enhances these proteins' activity and thermostability through directed evolution and protein engineering, and achieves expression and acquisition from microorganisms, significantly reducing costs and providing a new functional protein reference for the anti-obesity market.

[0006] This invention provides a novel potato Kunitz-type protease inhibitor mutant (PI-6130-M3) having the amino acid sequence shown in SEQ ID NO.3.

[0007] The present invention also provides a gene encoding the potato Kunitz protease inhibitor mutant (PI-6130-M3).

[0008] In one embodiment, the nucleotide sequence of the gene is shown in SEQ ID NO.4.

[0009] This invention also provides a genetically engineered bacterium that produces a potato Kunitz-type protease inhibitor.

[0010] In one embodiment, the genetically engineered bacteria express a potato Kunitz protease inhibitor with the amino acid sequence shown in SEQ ID NO.1.

[0011] In one embodiment, the genetically engineered bacteria expresses a potato Kunitz-type protease inhibitor mutant with the amino acid sequence shown in SEQ ID NO.3.

[0012] In one embodiment, the genetically engineered bacteria uses Escherichia coli BL21 as a host.

[0013] In one embodiment, pMAL-C5X is used as a vector to express the gene shown in SEQ ID NO.2 or SEQ ID NO.4.

[0014] The present invention also provides a method for constructing the genetically engineered bacteria, comprising the following steps: chemically synthesizing the gene sequence of the fragment shown in SEQ ID NO.4, fusing this sequence to the pMAL-C5X vector through restriction enzyme sites to obtain a recombinant; transforming the recombinant into Escherichia coli BL21, and screening to obtain the engineered bacteria pMAL-C5X-(PI-6130-M3) / BL21.

[0015] The present invention also provides a method for preparing potato Kunitz-type protease inhibitor mutants, wherein the genetically engineered bacteria are cultured in a culture medium, bacterial cells are collected, and potato Kunitz-type protease inhibitor mutants are broken and collected.

[0016] In one embodiment, the culture medium includes, but is not limited to, LB medium.

[0017] In one embodiment, the culture is carried out in a shaker at 210-230 rpm, at 15-17°C, and induced with IPTG at a final concentration of 40-60 mg / L for 16-18 h.

[0018] The present invention also provides a composition containing the potato Kunitz protease inhibitor mutant PI-6130-M3.

[0019] In one embodiment, the composition includes, but is not limited to, food, health products, or pharmaceuticals.

[0020] In one embodiment, the composition is a functional food additive.

[0021] This invention also provides the use of the potato Kunitz protease inhibitor mutant PI-6130-M3 in the preparation of products that suppress appetite and / or alleviate obesity.

[0022] In one embodiment, the product includes, but is not limited to, health supplements or medicines.

[0023] In one embodiment, the suppression of appetite and / or relief of obesity includes:

[0024] (a) Promotes the secretion of satiety factors;

[0025] (b) Slows gastric emptying;

[0026] (c) Suppress food intake;

[0027] (d) Prevention or adjunctive treatment of obesity.

[0028] In one embodiment, the drug further comprises a pharmaceutically acceptable carrier.

[0029] Beneficial effects:

[0030] 1. This invention successfully expressed the Kunitz-type potato protease inhibitor PI-6130 (amino acid sequence as shown in SEQ ID NO.1) and its mutant PI-6130-M3 (amino acid sequence as shown in SEQ ID NO.3) in Escherichia coli. Protein interaction analysis revealed that the mutant PI-6130-M3 can form more hydrogen bonds and other interactions with trypsin, and the root mean square deviation (RMSD) of the complex is significantly lower than that of wild-type PI-6130, indicating higher conformational stability.

[0031] 2. The IC50 of the mutant PI-6130-M3 prepared in this invention inhibits trypsin activity.50 =0.61μM, the inhibitory activity was 5.67 times higher than that of the wild type; after treatment at 60℃ for 15 min, the activity could be maintained at more than 92%, and the thermal stability was significantly improved compared with the wild type (the activity was only 50%).

[0032] 3. The mutant PI-6130-M3 prepared in this invention significantly increases the secretion level of CCK in mouse serum. Two hours after gavage, the CCK secretion level was 1.94 times that of the control group and 1.10 times that of wild-type PI-6130. Monitoring of mouse food intake revealed that mice gavaged with PI-6130-M3 showed the most significant reduction in food intake. Within 2 hours after gavage, the total food intake was reduced by 43.97% compared to the control group and by 14.89% compared to wild-type PI-6130. This mutant can be applied to the development of drugs or health products that suppress appetite.

[0033] 4. The Kunitz-type trypsin inhibitor mutant of this invention, which has an appetite-suppressing effect, is colorless and odorless and can be used to prepare food, health products or medicines, providing new possibilities for limiting excessive food intake and alleviating or preventing obesity. Attached Figure Description

[0034] Figure 1 The figure shows the results of the docking interaction analysis between the Kunitz-type potato trypsin inhibitor (PI-6130) and the Kunitz-type potato trypsin inhibitor mutant (PI-6130-M3) and trypsin molecules.

[0035] Figure 2 This is an SDS-PAGE analysis of Kunitz-type potato trypsin inhibitor (PI-6130) and its mutant (PI-6130-M3) expressed in Escherichia coli.

[0036] Figure 3 The graph shows the inhibition results of the Kunitz-type potato trypsin inhibitor (PI-6130) and the Kunitz-type potato trypsin inhibitor mutant (PI-6130-M3) on trypsin activity.

[0037] Figure 4 The graph shows the effect of temperature on the trypsin inhibitory activity of the Kunitz-type potato trypsin inhibitor (PI-6130) and the Kunitz-type potato trypsin inhibitor mutant (PI-6130-M3).

[0038] Figure 5The effects of the Kunitz-type potato trypsin inhibitor (PI-6130) and the Kunitz-type potato trypsin inhibitor mutant (PI-6130-M3) on the promotion of CCK secretion in mice (different letters represent significant differences, p<0.05).

[0039] Figure 6 The effects of the Kunitz-type potato trypsin inhibitor (PI-6130) and the Kunitz-type potato trypsin inhibitor mutant (PI-6130-M3) on food intake in mice (* indicates a significant difference compared with the control group under the same conditions, p<0.05). Detailed Implementation

[0040] The present invention will be described below in conjunction with biochemical and cell experimental data. Unless otherwise specified, the reagents used in the examples are all commercially available products or reagents prepared by conventional methods. Unless otherwise specified, the methods in the examples are all conventional experimental methods.

[0041] The PCR enzymes and ligases used in the following examples were purchased from Vazyme, restriction endonucleases from New England Biolabs, plasmid extraction kits and gel purification kits from Tiangen Biotech (Beijing) Co., Ltd., Escherichia coli BL21 and DH5α from Shanghai Weidi Biotechnology Co., Ltd., pMAL-c5x vector, primers, gene synthesis, and gene sequencing were all performed by Qingke Biotechnology, IPTG from BioFroxx, trypsin from Simga, benzoyl-DL-arginine p-nitroaniline from Shanghai Yuanye Biotechnology Co., Ltd., mice from the Experimental Animal Center of Kunming Medical University, and mouse cholecystokinin kits from Hangzhou Lianke Biotechnology Co., Ltd.

[0042] Example 1: Preparation of Kunitz-type potato trypsin inhibitor PI-6130 and its mutants

[0043] (1) Preparation of Kunitz-type potato trypsin inhibitor PI-6130:

[0044] Based on the amino acid sequence of potato PI-6130 (SEQ ID NO.1), the gene sequence shown in SEQ ID NO.2 was synthesized by Qingke Biotechnology using chemical methods. The recombinant expression plasmid pMAL-C5X-(PI-6130) was obtained by double digestion and fusion with BamHI and HindIII into the pMAL-c5X vector. Sequencing confirmed the successful construction of the recombinant plasmid.

[0045] The recombinant expression plasmid pMAL-C5X-(PI-6130) was transformed into Escherichia coli BL21. Positive clones were screened, and single colonies were picked and inoculated into 5 mL of LB medium containing ampicillin and cultured overnight. Then, the culture was transferred to 1 L of medium and cultured at 37°C with shaking until A 600 Once the saturation point (nm) reaches 0.5–0.7, the temperature is adjusted to 16°C, and 50 mg / L IPTG is added. The cells are then cultured with shaking for 15–17 hours before collection. The cells are resuspended in PBS buffer and sonicated to disrupt the protein. The supernatant is collected. The supernatant is purified using MBP affinity chromatography to obtain the target protein.

[0046] (2) Construction of the Kunitz-type potato trypsin inhibitor PI-6130 mutant:

[0047] The most similar sequences to PI-6130 from different wild-type and cultivated potato sources were screened from existing technologies. Through sequence evolution analysis, conserved amino acids were retained, and amino acids that mutated during evolution were set as variable amino acids. Protein design optimization was performed using ProteinMPNN, and a total of 100 mutants were obtained. The mutant PI-6130(M3) shown in SEQ ID NO.3 was obtained by mutual screening using AlphaFold 3.

[0048] Following the method in step (1), the recombinant expression plasmid pMAL-C5X-(PI-6130-M3) and the corresponding recombinant bacteria were constructed, and the mutant PI-6130-M3 protein was prepared by fermentation.

[0049] Example 2: Interaction analysis of Kunitz-type potato trypsin inhibitor PI-6130 and its mutant PI-6130-M3 with trypsin

[0050] The interaction between Kunitz-type potato trypsin inhibitor (PI-6130) and its mutant PI-6130-M3 and trypsin was predicted online using AlphaFold 3, an artificial intelligence (AI) method developed by DeepMind. The results were visualized using PyMol software. In addition, the complex was kineticly simulated for 100 ns using Gromacs 2022.

[0051] The results are as follows Figure 1As shown, both wild-type PI-6130 and the mutant PI-6130-M3 exhibit strong binding to trypsin. Wild-type PI-6130 forms 18 interactions with 14 amino acids in trypsin, including Ser195 and Gly219, while the mutant PI-6130-M3 forms 29 interactions with 19 amino acids in trypsin, including His63 and Ser195, with significantly stronger binding than the wild-type. During kinetic simulations, the Trypsin / PI-6130-M3 complex reached equilibrium after 20 nm, and the root mean square deviation (RMSD) was within [value missing]. Fluctuating up and down, this value is significantly lower than that of the Trypsin / PI-6130 complex. Furthermore, the PI-6130-M3 complex with Trypsin exhibits a lower and more stable radius of gyration (Rg), indicating that the mutant PI-6130-M3 binds more tightly to trypsin, has higher conformational stability, and can form a more stable complex.

[0052] Example 3: Preparation of Kunitz-type potato trypsin inhibitor PI-6130

[0053] Following the method described in Example 1, the gene sequence fragment shown in SEQ ID NO.4 was synthesized by Qingke Biotechnology using chemical methods. This sequence was then fused to the pMAL-c5X vector via double digestion with BamHI and HindIII to obtain the recombinant expression plasmid pMAL-C5X-(PI-6130-M3). The successful construction of the recombinant plasmid was confirmed by sequencing.

[0054] The recombinant expression plasmids pMAL-C5X-(PI-6130) and pMAL-C5X-(PI-6130-M3) were transformed into Escherichia coli BL21, respectively. Positive clones were screened, and single colonies were picked and inoculated into 5 mL of LB medium containing ampicillin and cultured overnight. Then, the culture was transferred to 1 L of medium and cultured at 37°C with shaking until A 600 Once the saturation point (nm) reaches 0.5–0.7, the temperature is adjusted to 16°C, and 50 mg / L IPTG is added. The cells are then cultured with shaking for 15–17 hours before collection. The cells are resuspended in PBS buffer and sonicated to disrupt the protein. The supernatant is collected. The supernatant is purified using MBP affinity chromatography to obtain the target protein.

[0055] The PI-6130 protein prepared in Example 1 and the mutant PI-6130-M3 protein prepared in this example were identified by SDS-PAGE, and the results are as follows: Figure 2As shown in the figure. During protein expression, MBP (Maltose Binding Protein) tags were used for purification, therefore all the resulting proteins contained MBP-tagged proteins.

[0056] Example 4: Evaluation of the trypsin inhibitory activity of Kunitz-type potato trypsin inhibitor PI-6130 and its mutant PI-6130-M3

[0057] The proteins obtained in Examples 1 and 3 were used as substrates, and the release of p-nitroaniline was measured using a microplate reader to analyze the inhibitory activity of Kunitz-type potato trypsin inhibitor (PI-6130) and its mutant PI-6130-M3. Due to the presence of the MBP-tagged protein, it was used as a negative control in all activity assessment experiments. Specific reaction conditions are shown in Table 1.

[0058] Table 1. Enzyme inhibitory activity reaction conditions (unit: μL):

[0059]

[0060] The formula for calculating trypsin inhibitory activity is as follows:

[0061]

[0062] The results are as follows Figure 3 As shown, MBP protein had no inhibitory activity against trypsin (purchased from Sigma, 1000–2000 U / mg), while PI-6130 and PI-6130-M3 both showed significant inhibitory effects on trypsin activity. The mutant PI-6130-M3 exhibited a significantly higher IC50 value than the target protein. 50 =0.61 μM, its inhibitory activity is similar to that of wild-type PI-6130 (IC50). 50 This represents a 5.67-fold improvement compared to (3.46 μM).

[0063] Example 5: Thermal stability analysis of Kunitz-type potato trypsin inhibitor PI-6130 and its mutant PI-6130-M3

[0064] After adjusting the concentrations of wild-type PI-6130 and mutant PI-6130-M3 to 10 μM, they were dispensed into different eptubs and treated at 40, 50, 60, 70, 80 and 90 °C for 20 min, respectively. Then, the trypsin inhibitory activity was measured according to the method described in Example 3.

[0065] The results are as follows Figure 4As shown, treatment at 40 and 50 °C had no significant effect on the activity of PI-6130 and PI-6130-M3. After treatment at 60 °C, the activity of wild-type PI-6130 was reduced by nearly 50%, while the activity of PI-6130-M3 remained at 92%, indicating that the thermal stability of PI-6130-M3 was significantly improved compared to PI-6130.

[0066] Example 6: Evaluation of the CCK-promoting activity of Kunitz-type potato trypsin inhibitor PI-6130 and its mutant PI-6130-M3

[0067] PI-6130 and PI-6130-M3 prepared in Example 2 were administered via gavage to mice, and the level of CCK secretion was measured to evaluate their satiety-promoting effects. Specifically, SPF-grade C57BL / 6 mice were housed in a standard animal room. After a 7-day acclimatization period, the mice were randomly divided into 4 groups (n=8). After a 12-hour fast, the mice were administered PBS (Control group), MBP (negative control group, 50 mg / kg bw), PI-6130 (50 mg / kg bw), and PI-6130-M3 (50 mg / kg bw) via gavage, respectively. Blood was collected from the orbital sinus at 30, 60, 90, and 120 minutes after gavage. After centrifugation at 4°C to obtain plasma, the changes in CCK concentration in the plasma of each group of mice were measured according to the ELISA kit instructions.

[0068] like Figure 5 As shown, throughout the experiment, the plasma CCK level in untreated mice (Control) remained around 40 pg / mL. In contrast, gavage administration of MBP, PI-6130, and PI-6130-M3 all rapidly increased plasma CCK levels within 30 minutes. Among them, PI-6130-M3 treatment showed the most significant increase in plasma CCK, reaching 1.94, 1.33, and 1.10 times that of the Control, MBP, and PI-6130 groups, respectively, at 120 minutes. CCK has a significant effect on satiety at both physiological and pharmacological levels; therefore, it is speculated that the ingestion of PI-6130 and PI-6130-M3 can increase satiety through their CCK secretion-stimulating activity.

[0069] Example 7: Inhibitory effect of Kunitz-type potato trypsin inhibitor (PI-6130) and its mutant PI-6130-M3 on food intake in mice

[0070] The PI-6130 prepared in Example 1 and the PI-6130-M3 prepared in Example 3 were used in an animal model to verify their inhibitory effect on food intake in mice.

[0071] After acclimatization, SPF-grade C57BL / 6 mice were randomly assigned to 4 groups (n=8), as follows:

[0072] Control group: PBS buffer administered by gavage

[0073] MBP control group: MBP protein administered by gavage (50 mg / kg bw)

[0074] PI-6130 group: PI-6130 (50 mg / kg bw) administered by gavage.

[0075] PI-6130-M3 group: PI-6130-M3 (50 mg / kg bw) administered by gavage.

[0076] Mice in each group were fasted from 21:00 to 9:00 and administered physiological saline, MBP, PI-6130, and PI-6130-M3 by gavage at 9:00 AM daily, along with a fixed amount of food. Food consumption was monitored and recorded at 1, 2, 6, and 12 hours after feeding. This gavage and monitoring continued for 7 days. At the end of the experiment, the average daily diet of each mouse was calculated, and their appetite was analyzed.

[0077] like Figure 6 As shown, compared with the Control group, the food intake of mice administered MBP, PI-6130, and PI-6130-M3 via gavage was reduced to varying degrees. Within 2 hours after gavage, the average food intake per mouse in the Control, MBP, PI-6130, and PI-6130-M3 groups was 0.83, 0.74, 0.54, and 0.46 g, respectively, indicating that PI-6130 and its mutants can produce a strong feeling of satiety and effectively reduce food intake in mice. As a more effective trypsin inhibitor, PI-6130-M3 has potential application value in the development of foods / drugs that provide satiety and alleviate / prevent obesity.

[0078] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A potato Kunitz-type protease inhibitor mutant, characterized in that, The amino acid sequence is shown in SEQ ID NO.

3.

2. The gene encoding the mutant of claim 1.

3. Genetically engineered bacteria, characterized in that, This describes the potato Kunitz-type protease inhibitor mutant as described in claim 1.

4. The genetically engineered bacteria according to claim 3, characterized in that, Escherichia coli BL21 is used as the host.

5. The genetically engineered bacterium according to claim 4, characterized in that, The gene shown in SEQ ID NO.4 was expressed using pMAL-C5X as a vector.

6. A method for preparing potato Kunitz-type protease inhibitors, characterized in that, The genetically engineered bacteria according to any one of claims 3 to 5 are cultured in a culture medium, bacterial cells are collected, and potato Kunitz protease inhibitors are broken and collected.

7. The method according to claim 6, characterized in that, The culture was carried out in a shaker at 210-230 rpm, at 15-17°C, and induced with IPTG at a final concentration of 49-51 mg / L for 16-18 h.

8. A composition containing the potato Kunitz protease inhibitor mutant of claim 1.

9. The use of the potato Kunitz protease inhibitor mutant of claim 1 in the preparation of a medicament for suppressing appetite and / or alleviating obesity.

10. The application according to claim 9, characterized in that, The appetite suppression and / or obesity relief include at least one of the functions of (a) to (d): (a) Promotes the secretion of satiety factors; (b) Slows gastric emptying; (c) Suppress food intake; (d) Prevention or adjunctive treatment of obesity.

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