Method for extracting effective active ingredients of radix puerariae by using modified enzyme and application

Abstract

The invention relates to the technical field of biology, and discloses a method for extracting active ingredients of radix puerariae by using modified enzyme and application, the method comprises the following steps: performing homologous modeling on papain, including model construction, model refinement and model evaluation, to obtain a papain receptor model; performing molecular docking on the papain receptor model and the active ingredients of the radix puerariae, and determining key amino acid sites of the papain receptor model; by comprehensively analyzing different molecular docking results correspondingly formed by the key amino acid sites, determining target amino acid sites and modifying the target amino acid sites to obtain the modified papain. According to the method, the papain is subjected to site-directed mutagenesis by combining a computer method and an experimental method, and the enzyme is precisely designed and modified, so that the catalytic efficiency, stability and selectivity of the enzyme are fundamentally improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for extracting effective active ingredients of kudzu root by utilizing modified enzymes and application thereof. Background Art

[0002] Pueraria root, as an important part of traditional Chinese medicine, has a long history of medicinal use and is widely used in many therapeutic fields such as clearing away heat and detoxification, promoting body fluid and quenching thirst, and raising yang and stopping diarrhea. Modern scientific research has revealed that Pueraria root is rich in a variety of ingredients with significant biological activity, such as isoflavone compounds (such as puerarin, daidzein, etc.), polysaccharides, flavonoid glycosides, and a variety of minerals and vitamins. These ingredients show good pharmacological effects in anti-oxidation, anti-inflammation, anti-cardiovascular disease, improving diabetes symptoms, and regulating the immune system. However, the extraction and purification of effective active ingredients in Pueraria root has always been a technical bottleneck restricting its in-depth development and utilization. Traditional extraction methods, such as solvent extraction, water extraction, and ultrasound-assisted extraction, although they can achieve the extraction of active ingredients to a certain extent, often have problems such as low extraction efficiency, high energy consumption, poor selectivity, and easy destruction of active ingredients during the extraction process. In addition, these methods may also cause environmental pollution, which is not in line with the concepts of modern green chemistry and sustainable development.

[0003] In recent years, enzymatic hydrolysis has gradually gained attention in the field of natural product extraction due to its mild conditions, strong specificity, and environmental friendliness. As a biocatalyst, enzymes can act specifically on target substances, improve extraction efficiency, and reduce the generation of by-products. However, enzymes existing in nature are often difficult to directly meet the needs of efficient extraction of active ingredients from Pueraria lobata under specific conditions. Therefore, it has become a hot research direction to transform enzymes through genetic engineering, protein engineering, etc. to improve their catalytic efficiency, stability, and selectivity. At present, some studies have attempted to improve the extraction efficiency of active ingredients from Pueraria lobata by means of enzyme immobilization, construction of complex enzyme systems, and optimization of enzymatic hydrolysis conditions. However, most of these methods remain at the adjustment of enzyme application conditions, and have not fundamentally solved the problem of insufficient performance of the enzyme itself. Summary of the invention

[0004] (I) Purpose of the invention

[0005] The purpose of the present invention is to provide a method and application of extracting effective active ingredients from Pueraria lobata using modified enzymes. The method accurately designs and modifies the enzymes, uses the modified enzymes to improve the extraction efficiency of specific active ingredients in Pueraria lobata, while maintaining or improving their stability and selectivity, thereby providing a new solution for the green and efficient extraction of active ingredients from Pueraria lobata.

[0006] (II) Technical solution

[0007] To solve the above problems, the first aspect of the present invention provides a method for extracting effective components of Pueraria lobata using a modified enzyme, the method comprising:

[0008] Conduct homology modeling of papain: including model construction, model refinement and model evaluation, and obtain the papain receptor model;

[0009] Molecular docking is performed between the papain receptor model and the effective components of Pueraria lobata to determine the key amino acid sites of the papain receptor model;

[0010] By comprehensively analyzing the different molecular docking results corresponding to the key amino acid sites, the target amino acid sites are determined and modified to obtain the modified papain.

[0011] Further, the homology modeling of papain comprises:

[0012] The homology model of papain was constructed using the SWISS-MODEL website;

[0013] Download the homology template of papain from the UniProtKB database to refine the constructed homology model of papain;

[0014] The refined papain homology model was evaluated using the Ramachandran plot to obtain the papain receptor model.

[0015] Furthermore, the molecular docking of the papain receptor model and the effective components of Pueraria lobata to determine the key amino acid sites of the papain receptor model includes: using AutoDock Vina software to dock the effective components of Pueraria lobata into the papain receptor model, and determining the key amino acid sites of the papain receptor model according to the docking binding energy results.

[0016] Furthermore, determining the key amino acid sites of the papain receptor model according to the docking binding energy results includes: sorting the docking binding energies according to energy levels, and using PyMOL to perform a visual analysis on the docking configuration corresponding to the lowest-ranked binding energy to determine the key amino acid sites of the papain receptor model.

[0017] Furthermore, the key amino acid sites include CYS56, ARG58, ASP57, GLN53 and SER70.

[0018] Furthermore, the molecular docking results include binding energy analysis and geometric configuration analysis.

[0019] Furthermore, the target amino acid sites include SER70 and ARG58.

[0020] Furthermore, the modification includes: replacing the SER70 site with CYS or TYR, and replacing the ARG58 site with CYS or SER.

[0021] Furthermore, the effective ingredients of the kudzu root include puerarin, soybean extract, genistein and formononetin.

[0022] Furthermore, the enzymatic hydrolysis time of the modified papain is 2.5 to 3.5 hours.

[0023] Another aspect of the present invention provides a method for extracting effective components of Pueraria root using modified enzymes and its application in the field of cosmetics.

[0024] (III) Beneficial effects

[0025] The above technical scheme of the present invention has the following beneficial technical effects: the present invention provides a method and application for extracting effective active ingredients of Pueraria root by using modified enzymes. The method performs site-directed mutagenesis on papain by combining computer methods and experimental methods, and accurately designs and modifies the enzyme to improve the extraction efficiency of papain on effective ingredients in Pueraria root. The process is as follows: first, homology modeling is performed on papain to obtain a papain receptor model; then, molecular docking is performed on the papain receptor model and the effective ingredients of Pueraria root to determine the key amino acid sites of the papain receptor model; finally, the target amino acid sites are determined and modified by comprehensively analyzing the different molecular docking results corresponding to the key amino acid sites to obtain the modified papain. The present invention aims to address the technical bottlenecks existing in the current extraction and purification process of effective active ingredients in Pueraria lobata, especially the problems of low efficiency, high energy consumption, poor selectivity, easy destruction of active ingredients and environmental pollution in traditional extraction methods, as well as the challenge of insufficient performance of enzymes themselves in the extraction of active ingredients in Pueraria lobata by existing enzymatic hydrolysis methods. An innovative solution is proposed, which uses cutting-edge biotechnology means such as homology modeling, molecular docking, and amino acid residue modification to perform precise structural design and functional modification of the enzyme, aiming to fundamentally improve the catalytic efficiency, stability and selectivity of the enzyme, so as to achieve efficient, green and specific extraction of specific active ingredients in Pueraria lobata. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The papain receptor model obtained by evaluating the Ramachandran Plot diagram in the present invention;

[0027] Figure 2 is a comparison chart of the effect of temperature on enzyme activity in various embodiments of the present invention;

[0028] Figure 3 It is a comparison chart of the effect of pH value on enzyme activity in various embodiments of the present invention. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0030] 1. The first aspect of the present invention provides a method for extracting effective components from Pueraria root using a modified enzyme, the method comprising:

[0031] S1, homology modeling of papain: including model construction, model refinement and model evaluation, to obtain a papain receptor model. In this step, homology modeling is used to establish the 3D structure of papain, specifically including: (1) using the SWISS-MODEL website to build a papain receptor model, the SWISS-MODEL website address is https: / / swissmodel.expasy.org / ; (2) downloading the amino acid sequence of papain (9PAP) from the UniProtKB (https: / / www.rcsb.org / ) database as a template for homology modeling to refine the constructed papain homology model; (3) using the Ramachandran graph to evaluate the refined papain homology model to obtain a papain receptor model, and the final papain receptor model structure is named PAP_N. Figure 1 As shown, the Ramachandran plot results show that 91.3% of the points in the papain receptor model are located in the fully allowed region, 8.7% are located in the additional allowed region, and there is no part located in the unallowed region. These structures show that the homology model is reliable and can be used for subsequent molecular docking.

[0032] S2, molecular docking the papain receptor model and the effective components of Pueraria root to determine the key amino acid sites of the papain receptor model. This step specifically includes: using AutoDock Vina software to dock the effective components of Pueraria root into the papain receptor model, the grid size is 102×94×126 (XYZ), the coordinate values ​​of the active center are X=-5.963, Y=-3.561, Z=5.263, and the key amino acid sites of the papain receptor model are determined according to the docking binding energy results. The docking binding energy is sorted according to the energy level, and the configuration with the lowest docking energy is selected for further analysis. PyMOL is used to visualize the docking configuration corresponding to the lowest ranking binding energy (docking binding energy) to determine the key amino acid sites of the papain receptor model. The effective components of Pueraria root include puerarin, soybean glycosides, genistein and formononetin. The molecular docking results of PAP_N and different Pueraria root effective components before modification are shown in Table 1. The results showed that the key amino acid sites of the main active ingredients of Pueraria lobata and papain were CYS56, ARG58, ASP57, GLN53 and SER70.

[0033] Table 1 Molecular docking results of PAP_N before modification and different active ingredients of Pueraria root

[0034]

[0035] S3, by comprehensively analyzing the different molecular docking results corresponding to the key amino acid sites, the target amino acid sites are determined and modified to obtain the modified papain. This step specifically includes: by comparing and analyzing the energy (binding energy) and geometric configuration of different docking conformations, SER70 and ARG58 are identified as key amino acid sites that affect the binding efficiency of PAP_N and the effective components of Pueraria lobata, so SER70 and ARG58 are used as target amino acid sites. These two sites may directly participate in the key steps in substrate recognition, binding or catalysis, and thus become the target of subsequent modification. Based on the above identification results, a modification scheme for SER70 and ARG58 is designed. Considering that cysteine ​​(CYS) can often form disulfide bonds in proteins, thereby enhancing the stability and structural rigidity of proteins, and may introduce new interaction sites, it is selected to replace the two sites of SER70 and ARG58 in Example 1 with CYS, SER70 and ARG58 are replaced with TYR and SER respectively in Example 2, and ARG58 is replaced with SER in Example 3. After the modification, the same molecular docking tool was used again to simulate the docking of the modified PAP_N (denoted as PAP_N_mod) with the four active ingredients in Pueraria. The results are shown in Table 2. In Example 1, it was observed that the binding energy after docking with the four active ingredients showed a significant downward trend. This result strongly indicates that the modified papain (i.e., PAP_N_mod1) obtained by Example 1 and the active ingredients in Pueraria show a tighter binding state, and their interaction is more advantageous and favorable. This significant reduction in binding energy directly reflects the higher affinity between PAP_N_mod1 and the Pueraria components, indicating that in actual application scenarios, the modified enzyme can more accurately and efficiently identify and catalyze the target components in Pueraria, thereby achieving a significant improvement in extraction efficiency. In addition, compared with the unmodified original papain, Example 2 (i.e., PAP_N_mod2) and Example 3 (i.e., PAP_N_mod3) also showed a certain degree of change in the binding energy with the four active ingredients. Although these changes were relatively small, they still had a certain degree of influence on the enzymatic activity of papain, see Table 3 and Figure 2 , Figure 3 .

[0036] Table 2 Molecular docking results of PAP_N_mod and different active ingredients of Pueraria root

[0037]

[0038] 2. Determination results of modified papain (PAP_N_mod)

[0039] 1. Extraction of puerarin and total flavonoids from Pueraria root

[0040] After slicing, drying and crushing the kudzu root, accurately weigh 1g of kudzu root powder, add 2.5% PAP_N_mod and 4mL Tris-HCl buffer, and perform enzymolysis at 55°C for different times (2h, 3h, 4h, 5h), add 50% ethanol, extract under water bath reflux for 2h, filter, and set the filtrate to a 50mL volumetric flask for later use. The control group was added with the same mass fraction of original papain (papain before modification), and the test results are shown in Table 3.

[0041] Table 3 Effects of papain before and after modification on the yields of puerarin and total flavonoids from pueraria root

[0042]

[0043] The results showed that as the hydrolysis time of Pueraria lobata with papain was prolonged, the yield of the effective ingredients in Pueraria lobata first increased and then decreased, and the modified papain could shorten the hydrolysis time to 2.5 to 3.5 hours, thereby improving the efficiency of extracting the effective ingredients from Pueraria lobata.

[0044] 2. Determination of Papain Stability Before and After Modification

[0045] By dilution or concentration, papain before and after modification with an enzyme concentration of 100 μg / mL was obtained, and subsequent temperature stability and kinetic parameters were determined at this concentration.

[0046] 2.1 Determination of temperature stability of papain before and after modification

[0047] The modified papain (PAP_N_mod) and the modified papain (PAP_N) were incubated at 50°C, and samples were taken every 10 minutes until the incubation lasted for 1 hour. During this period, the residual enzyme activity of the samples taken at each time point was evaluated by sodium hydroxide titration, wherein the enzyme activity without heat incubation was defined as 100% of the baseline activity.

[0048] Wherein, the sodium hydroxide titration method specifically comprises the following steps:

[0049] (1) Solution preparation

[0050] Substrate solution: Weigh 1.42 g of N-benzoyl-L-arginine ethyl ester hydrochloride, add 10 mmol / LEDTA, 2.6 mL of 50 mmol / L cysteine ​​solution, add appropriate amount of water, adjust the pH to 6.2 with 0.1 mol / L NaOH solution, and finally make up to 50 mL.

[0051] Enzyme solution to be tested: Adjust the previously prepared enzyme solution to pH 6.2.

[0052] (2) Determination: Place a 25 ml beaker on a pH automatic titrator at a constant temperature of 50°C. Add 3.75 mL of substrate solution, 2.50 mL of 3 mol / L sodium chloride solution and 1.25 mL of double distilled water to the beaker. After mixing evenly, immediately add the enzyme solution to be tested and start timing. Under the action of electromagnetic stirring, use 0.1 mol / L sodium hydroxide solution to continuously titrate to keep the pH value of the solution stable at 6.2. Record the volume of 0.01 mol / L sodium hydroxide solution consumed per minute and perform blank correction.

[0053] The activity P (unit / mg) of papain can be calculated by the following formula:

[0054]

[0055] In formula (1), c represents the concentration of sodium hydroxide solution (mol / L), ΔV represents the volume of sodium hydroxide solution consumed per minute minus the blank value, and W represents the mass of papain in the reaction solution (mg).

[0056] The results of the test are as follows Figure 2 As shown, the enzyme activity of papain, whether before or after modification, shows a significant improvement trend with the increase of temperature. Specifically, except for the modified papain in Example 1, which reaches its optimum temperature at 70°C, the optimum temperatures of all other experimental groups (including unmodified enzymes and other modified enzyme examples) are 80°C. It is worth noting that when the temperature is further increased to 90°C, the enzyme activity exhibited by the modified papain in Example 1 is significantly higher than that of the unmodified papain. The results show that the thermal stability of papain under specific modification conditions has been enhanced, especially in high temperature environments, and its practical value has been significantly improved. Since different mutations were made to SER70 and ARG5 in the papain sequence in Examples 2 and 3, the optimum temperatures are both 80°C.

[0057] 2.2 pH stability of papain before and after modification

[0058] In order to compare the changes in the activity of papain before and after modification, 0.1 mL of each of the two enzyme solutions (modified and unmodified) were taken and their activities were measured in a series of 0.05 mol / L Tris-HCl buffers with different pH values ​​according to the experimental method described in Section 2.1.

[0059] The results are as follows Figure 3As shown, the experimental results show that, in the unmodified papain system, when the pH value of the solution exceeds 6, its enzyme activity significantly decreases. In contrast, the papain treated with specific modification can maintain a high level of enzyme activity in a wide range of pH values ​​between 5 and 11. Specifically, in Example 1, Example 2 and Example 3, the optimum pH values ​​shown by the modified papain are 8, 7 and 9, respectively. It is worth noting that the optimum pH values ​​of the three modified enzymes all show a trend of shifting to the alkaline interval, which clearly indicates that this modification strategy effectively enhances the alkali resistance of papain. This feature has significant potential advantages for broadening the application range of papain in the industrial field, particularly in the production process requiring alkaline conditions. Through comprehensive analysis of the above-mentioned experimental results, it is verified that Example 1 is the best embodiment.

[0060] 3. Animal Experiments

[0061] This experiment is to verify the effect of total flavonoids from Pueraria lobata obtained by enzymatic hydrolysis with modified papain.

[0062] Sixty 2-month-old female rats (200±20g) with intact ovaries were selected and raised under standard conditions (23-25℃, 12h dark-light cycle, 50-55% relative humidity, ventilation every 16h) and randomly divided into 3 groups, namely the experimental group, the control group and the blank group, with 20 rats in each group. They were given Pueraria lobata flavonoids and 17β-estradiol at a dose of 800mg / kg, respectively.

[0063] After 28 days of feeding, the rats were killed and blood was collected from the trunk, centrifuged at 3000rpm for 20min to obtain serum samples, and immediately stored at -20℃ for serum analysis. The levels of serum follicle-stimulating estrogen (FSH) and luteinizing hormone (LH) were detected using the German Roche E601 chemiluminescence kit. The level of prolactin in rat serum was detected using a rat prolactin (PRL) ELISA kit. The level of E2 in rat serum was determined using a rat estradiol (E2) ELISA kit. The levels of HDL-C and LDL-C were detected using a high-density lipoprotein cholesterol (HDL-C) detection kit and a low-density lipoprotein cholesterol (LDL-C) detection kit, respectively. The level of ALP was detected using a rat alkaline phosphatase (ALP) enzyme-linked immunosorbent assay (ELISA). The level of TSH was detected using a rat thyroid stimulating hormone (TSH) ELISA kit.

[0064] Table 4 Body weight and serum levels of reproductive hormones in rats of different groups

[0065]

[0066] The results in Table 4 show that the intake of pueraria lobata flavonoids and 17β-estradiol significantly reduced the levels of LH and FSH in serum, indicating that both substances can play a certain role in the regulation of the menstrual cycle. At the same time, the intake of 17β-estradiol significantly increased the level of PRL, while pueraria lobata flavonoids had almost no effect on this. In addition, after 28 days of intake of 17β-estradiol, the weight of the rats' liver increased.

[0067] Table 5 Blood lipid parameters, ALP and TSH levels in rats in different groups

[0068]

[0069] The results in Table 5 show that the intake of pueraria flavonoids and estradiol both reduced the levels of high-density lipoprotein cholesterol and low-density lipoprotein cholesterol, while ALP and TSH levels increased. This shows that the intake of both causes some damage to the liver, but in comparison, the damage caused by estradiol is greater. The results of this experiment show that pueraria flavonoids has a similar mechanism to estradiol in regulating lipids and thyroid, and at the same time causes less damage to the liver.

[0070] Another aspect of the present invention provides a method for extracting effective components of kudzu root using modified enzymes for use in the cosmetics field. The effective components of kudzu root extracted by any of the above-described methods for extracting effective components of kudzu root using modified enzymes can be used in the cosmetics field.

[0071] It should be understood that the above specific embodiments of the present invention are only used to illustrate or explain the principles of the present invention, and do not constitute a limitation of the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modifications that fall within the scope and boundaries of the appended claims, or the equivalent forms of such scope and boundaries.

Claims

1. A method for extracting effective ingredients from Pueraria root using modified enzymes, characterized in that: The method comprises: Conduct homology modeling of papain: including model construction, model refinement and model evaluation, and obtain the papain receptor model; Molecular docking is performed between the papain receptor model and the effective components of Pueraria lobata to determine the key amino acid sites of the papain receptor model; By comprehensively analyzing the different molecular docking results corresponding to the key amino acid sites, the target amino acid sites are determined and modified to obtain the modified papain.

2. The method for extracting effective components from Pueraria root using modified enzymes according to claim 1, characterized in that: The homology modeling of papain comprises: The homology model of papain was constructed using the SWISS-MODEL website; Download the homology template of papain from the UniProtKB database to refine the constructed homology model of papain; The refined papain homology model was evaluated using the Ramachandran plot to obtain the papain receptor model.

3. The method for extracting effective components from Pueraria root using modified enzymes according to claim 1, characterized in that: The method of performing molecular docking on the papain receptor model and the effective components of Pueraria root to determine the key amino acid sites of the papain receptor model comprises: using AutoDock Vina software to dock the effective components of Pueraria root into the papain receptor model, and determining the key amino acid sites of the papain receptor model according to the docking binding energy results.

4. The method for extracting effective components from Pueraria root using modified enzymes according to claim 3, characterized in that: Determining the key amino acid sites of the papain receptor model according to the docking binding energy results includes: sorting the docking binding energies according to energy levels, and using PyMOL to perform a visual analysis on the docking configuration corresponding to the lowest-ranked binding energy to determine the key amino acid sites of the papain receptor model.

5. The method for extracting effective components from Pueraria root using modified enzymes according to claim 1, characterized in that: The key amino acid sites include CYS56, ARG58, ASP57, GLN53 and SER70.

6. The method for extracting effective components from Pueraria root using modified enzymes according to claim 1, characterized in that: The molecular docking results include binding energy analysis and geometric configuration analysis.

7. The method for extracting effective components from Pueraria root using modified enzymes according to claim 1, characterized in that: The target amino acid sites include SER70 and ARG58.

8. The method for extracting effective components from Pueraria root using modified enzymes according to claim 7, characterized in that: The modification includes: replacing the SER70 site with CYS or TYR, and replacing the ARG58 site with CYS or SER.

9. The method for extracting effective components from Pueraria root using modified enzymes according to claim 1, characterized in that: The enzymatic hydrolysis time of the modified papain is 2.5 to 3.5 hours.

10. Application of a method for extracting effective ingredients from Pueraria root using modified enzymes in the field of cosmetics.

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