CN115819510A - Small molecular peptide and application thereof in inhibiting xanthine oxidase - Google Patents
Small molecular peptide and application thereof in inhibiting xanthine oxidase Download PDFInfo
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Abstract
The invention discloses a small molecular peptide and application thereof in inhibiting xanthine oxidase, and belongs to the technical field of enzymolysis processing products. The amino acid sequence of the small molecular peptide is shown in SEQ ID NO. 4. The small molecular peptide is used for preparing a medicine for inhibiting xanthine oxidase and a medicine for inhibiting uric acid level. The invention obtains the small molecular peptide with high inhibitory activity to xanthine oxidase through experimental research and screening. The invention has important significance for treating hyperuricemia.
Description
Technical Field
The invention relates to a small molecular peptide and application thereof in inhibiting xanthine oxidase, belonging to the technical field of enzymolysis processing products.
Background
The oyster is one of the largest cultured shellfishes in the world, is a high-quality protein bank and has rich nutritional value. At present, oysters are treated directly, the industrial value is difficult to improve, and the processing of oyster polypeptides is an important direction for deep processing.
During purine metabolism, xanthine Oxidase (XO) catalyzes the conversion of the intermediate hypoxanthine to xanthine, and then continues to catalyze the conversion of xanthine to uric acid. Hyperuricemia and its complications, including kidney stones and cardiovascular diseases, are caused by overproduction of uric acid and decreased excretion due to high purine diet and alcohol intake. At present, the medicines for treating hyperuricemia are often accompanied with serious toxic and side effects, so the food source uric acid lowering peptide which is low in development cost, small in toxic and side effects and easy to absorb has important significance for treating the hyperuricemia.
Disclosure of Invention
Aiming at the prior art, the invention provides a small molecular peptide and application thereof in inhibiting xanthine oxidase. The invention has important significance for treating hyperuricemia and improves the additional output value of the oysters.
The invention is realized by the following technical scheme:
a small molecule peptide having the amino acid sequence: VAQDSSY as shown in SEQ ID NO. 4.
The small molecular peptide is used for preparing a medicine for inhibiting xanthine oxidase and a medicine for inhibiting uric acid level.
According to the invention, experimental research shows that the oyster polypeptide has xanthine oxidase inhibition (uric acid reduction) activity, so that the oyster polypeptide has the potential of being a functional product for inhibiting the uric acid level. Further, LC-MS/MS identification shows that the small molecular peptide obtained by molecular docking screening has higher inhibitory activity on xanthine oxidase, so the small molecular peptide has the potential of being a functional product for inhibiting uric acid level.
The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art.
Drawings
FIG. 1: schematic representation of the effect of protease on xanthine oxidase inhibition.
FIG. 2: the influence of the enzymolysis time on the xanthine oxidase inhibition rate is shown in a schematic diagram.
FIG. 3: schematic diagram of the influence of enzyme addition amount on xanthine oxidase inhibition rate.
FIG. 4 is a schematic view of: the influence of feed liquid ratio on the xanthine oxidase inhibition rate is shown schematically.
FIG. 5: graph showing the effect of initial pH on xanthine oxidase inhibition.
FIG. 6: the influence of the enzymolysis temperature on the xanthine oxidase inhibition rate is shown in a schematic diagram.
FIG. 7: the separation result of the enzymolysis liquid by G-15 sephadex column chromatography is shown schematically.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The devices, reagents and materials used in the following examples are conventional devices, reagents and materials known in the art and are commercially available in normal circumstances unless otherwise specified. Unless otherwise specified, the experimental methods and detection methods described in the following examples are conventional experimental methods and detection methods known in the art.
Example 1 optimization of oyster enzymolysis Process
1. Sieve enzyme
(1) Selecting compound flavor protease, papain, bromelain, neutral protease, alkaline protease and acid protease to carry out enzymolysis on the raw materials: opening the shell of the oyster to take out the oyster, pulping to obtain oyster homogenate; water was added to 8g of the oyster homogenate at a feed-to-liquid ratio of 1. Dividing into 6 parts, respectively adding composite flavor protease, papain, bromelain, neutral protease, alkaline protease and acidic protease, wherein the enzyme addition amount is 2000U/g (enzyme activity/oyster meat homogenate), adjusting the initial pH value to the optimum pH value of each protease (the optimum pH values of the composite flavor protease, the papain, the bromelain, the neutral protease, the alkaline protease and the acidic protease are respectively 6.5, 10.5 and 3.0) by using sodium hydroxide solution or hydrochloric acid, and reacting for 4 hours under the conditions of the optimum temperature of each protease (the optimum temperatures of the composite flavor protease, the papain, the bromelain, the neutral protease, the alkaline protease and the acidic protease are respectively 50 ℃,50 ℃,55 ℃,50 ℃,50 ℃,45 ℃ and 200 rpm) to obtain an enzymolysis product. Sampling, boiling for 10min to inactivate enzyme, cooling to room temperature, centrifuging at 8000rpm for 10min, and determining xanthine oxidase inhibition rate. Each set of three parallel sets.
(2) Determination of in vitro XO inhibition: xanthine solution (1.5 mmol/l) and xanthine oxidase solution (0.1U/ml) were prepared separately with PBS buffer solution of pH = 7.4. To a 1.5ml centrifuge tube, 125. Mu.l of the sample, 150. Mu.l of PBS buffer and 100. Mu.l of xanthine oxidase solution were added, and the mixture was incubated at 37 ℃ for 15min, 125. Mu.l of xanthine solution was added, and the incubation was continued for 20min, and finally 100. Mu.l of hydrochloric acid (1 mol/l) was added to terminate the reaction. Controls were set for no sample, no xanthine oxidase and sample, respectively. Absorbance at 290nm was measured.
The in vitro XO inhibition is calculated as follows:
in vitro XO inhibition (%) = [ (1- (Abs)) 1 -Abs 3 )/(Abs 2 -Abs 4 ]×100％。
Wherein, abs 1 : absorbance of a test system comprising xanthine, xanthine oxidase and a sample; abs 2 : contains xanthine and xanthine oxidase, and does not contain the light absorption value of a living measurement system of a sample; abs 3 : absorbance of a test system containing a xanthine substrate and a sample, and not containing xanthine oxidase; abs 4 : absorbance of the assay system containing xanthine, no xanthine oxidase and no sample.
(3) The results are shown in FIG. 1. As can be seen from the figure, the in vitro XO inhibition rate of the enzymolysis product of the acid protease is the highest and can reach 67.62 percent, which is superior to other five proteases, so the acid protease is selected to carry out the following single-factor optimization experiment.
2. Optimization of enzymolysis time
Opening the shell of the oyster to take out the oyster, pulping to obtain oyster homogenate; adding water into 4g of oyster homogenate with the feed-liquid ratio of 1 to 3 (g: ml), adjusting the initial pH value to 3, adding acid protease according to the enzyme addition amount of 2000U/g, and reacting for 2-6 h at 40 ℃ and 200 rpm; sampling at 2h, 3h, 4h, 5h and 6h respectively, inactivating enzyme in boiling water bath for 10min, cooling to room temperature, centrifuging at 8000rpm for 10min, collecting supernatant, and measuring XO inhibition rate (the measurement method is the same as above), wherein each group is divided into three groups.
The effect of different enzymatic hydrolysis times on the in vitro XO inhibition of the enzymatic hydrolysate is shown in fig. 2. Along with the extension of the enzymolysis time, the XO inhibition rate of the enzymolysis product shows a trend of increasing firstly and then decreasing, and the XO inhibition rate is the highest at 4h and reaches 58.11%; compared with the 3h group, the improvement is obvious (P is less than 0.05), and the difference is not obvious (P is more than 0.05) compared with the 5h group. Therefore, the enzymolysis time is selected to be 4h for further optimization.
3. Optimization of enzyme addition
Opening the shell of the oyster to take out the oyster, pulping to obtain oyster homogenate; adding water into 4g of oyster homogenate, wherein the feed-liquid ratio is 1 (g: ml), adjusting the initial pH value to 3, dividing into 5 parts, adding acid protease according to the enzyme addition amount of 1000U/g, 2000U/g, 3000U/g, 4000U/g and 5000U/g respectively, and reacting for 4 hours at 40 ℃ and 200 rpm; sampling, inactivating enzyme in boiling water bath for 10min, cooling to room temperature, centrifuging at 8000rpm for 10min, collecting supernatant, and determining XO inhibition rate (the determination method is the same as above), wherein each group has three parallels.
The effect of different enzyme addition on the in vitro XO inhibition of the enzymatic hydrolysate is shown in FIG. 3. With the increase of the enzyme adding amount, the XO inhibition rate of the product reaches 58.74 percent when the enzyme adding amount is 3000U/g, the inhibition activity of the group is obviously higher than that of other groups (P is less than 0.05), and the XO inhibition rate tends to be reduced when the enzyme adding amount is increased. Therefore, the enzyme adding amount of the acid protease is 3000U/g for further optimization.
4. Optimization of feed-liquid ratio
Opening the shell of the oyster, taking the oyster meat, and pulping to obtain 4g of oyster meat homogenate; dividing into 5 parts, adding water into the oyster meat homogenate, adjusting the initial pH value to 3 according to the feed-liquid ratio of 1; sampling, inactivating enzyme in boiling water bath for 10min, cooling to room temperature, centrifuging at 8000rpm for 10min, collecting supernatant, and measuring XO inhibition rate (the measurement method is the same as above), wherein each group has three parallels.
The effect of different feed solutions on the in vitro XO inhibition of the enzymatic hydrolysate is shown in fig. 4. As the feed-to-liquid ratio decreased, the XO inhibition rate increased continuously, reaching a maximum of 59.14% at a feed-to-liquid ratio of 1. Therefore, the feed-liquid ratio is selected to be 1.
5. Optimization of initial pH
Opening the shell of the oyster to take out the oyster, pulping to obtain oyster homogenate; adding water into 4g of oyster homogenate, wherein the feed-liquid ratio is 1 (g: ml), dividing into 6 parts, respectively adjusting the initial pH value to be 1, 2, 3, 4, 5 and 6, adding acid protease according to the enzyme adding amount of 3000U/g, and reacting for 4 hours at 40 ℃ and 200 rpm; sampling, inactivating enzyme in boiling water bath for 10min, cooling to room temperature, centrifuging at 8000rpm for 10min, collecting supernatant, and measuring XO inhibition rate (the measurement method is the same as above), wherein each group has three parallels.
The effect of different initial pH on the in vitro XO inhibition of the enzymatic hydrolysate is shown in fig. 5. The optimum pH range of acidic proteases is 2-4, and a shift in pH from this range results in a change in protease activity. The product XO has the highest inhibition rate of 68.35% when the pH value is 2, and is obviously higher than that of other groups (P is less than 0.05); as the pH is continuously increased, the XO inhibitory activity of the product always shows a tendency to decrease. Therefore, the pH value of 2 was chosen to continue the next optimization.
6. Optimization of enzymolysis temperature
Opening the shell of the oyster to take out the oyster, pulping to obtain oyster homogenate; adding water into 4g of oyster homogenate, adjusting the initial pH value to 3 according to the feed-liquid ratio of 1 to 5 (g: ml), adding acid protease according to the enzyme addition amount of 3000U/g, dividing into 5 parts, and reacting for 4h at the conditions of 20 ℃, 30 ℃, 40 ℃,50 ℃, 60 ℃ and 200rpm respectively; sampling, inactivating enzyme in boiling water bath for 10min, cooling to room temperature, centrifuging at 8000rpm for 10min, collecting supernatant, and measuring XO inhibition rate (the measurement method is the same as above), wherein each group has three parallels.
The effect of different enzymatic temperatures on the in vitro XO inhibition of the enzymatic product is shown in fig. 6. The XO inhibition rate shows a trend of increasing first and then decreasing with the increase of the temperature, the XO inhibition activity reaches the strongest value of 68.62 percent at the temperature of 40 ℃, and the XO inhibition activity of the group can be found to be remarkably higher than that of other groups (P < 0.05). Therefore, the optimum temperature for enzymatic digestion was selected to be 40 ℃.
7. Orthogonal test optimization
According to the single-factor experiment result, determining the levels of 5 factors including enzymolysis time, enzyme addition amount, feed-liquid ratio, initial pH value and enzymolysis temperature, setting a 5-factor 4-level orthogonal test, determining the in-vitro XO inhibition rate, and obtaining the optimal enzymolysis condition. The experimental factors and levels are detailed in table 1.
Table 1 orthogonal experiment design factor level table
The results of the oyster enzymolysis orthogonal optimization experiments are shown in table 2. From the analysis of the R value, the main and secondary effects of the enzymolysis time, the enzyme adding amount, the feed-liquid ratio, the enzymolysis temperature and the initial pH on the content of the amino nitrogen in the enzymolysis liquid are as follows: a is more than C, B is more than E, D, namely the influence of enzymolysis time is the largest, and secondly, the influence of enzyme adding amount and feed-liquid ratio and the influence of initial pH on the content of amino nitrogen in the enzymolysis liquid are the smallest. Finally, the optimized combination A2B2C3D2E3 is obtained, namely the enzymolysis time is 4h, the enzyme adding amount is 3000U/g, the feed-liquid ratio is 1.
TABLE 2 orthogonal optimization Table
Example 2 Ultrafiltration, gel filtration chromatography and component Activity analysis thereof
Opening the shell of the oyster to take out the oyster, pulping to obtain oyster homogenate; adding water into 4g of oyster meat homogenate with the feed-liquid ratio of 1 (g: ml) to adjust the initial pH value to 3, adding acid protease according to the enzyme addition amount of 3000U/g, and reacting for 4h at 40 ℃ and 200rpm to obtain an enzymatic hydrolysate.
The enzymolysis solution is prepared into a solution with the concentration of 80mg/mL by using a Vivaflow 50R (Sartorius, germany) ultrafiltration membrane to obtain a component less than 10KDa and then obtain a component less than 5KDa, freeze-drying, and adding water to obtain a sample to be used as a sample loading sample. After the ultrafiltration membrane package is used, the ultrafiltration membrane package is washed by NaOH solution with the concentration of 0.5mol/l and sealed by 10 percent ethanol solution and placed in an environment with the temperature of 4 ℃.
G-15
Adding appropriate amount of ultrapure water into the sephadex G-15, boiling for 2h, and removing froth and impurities; adding ultrapure water, stirring and standing. Removing impurities on the upper layer, and repeating the above operations until the upper layer is free of impurities. Absorbing excessive water on the upper layer of the gel, draining the filler to a chromatography column with the size of 26mm multiplied by 80cm by a glass rod to avoid layering and air bubbles, and washing the column with pure water after the column is filled until the height of the chromatography column does not change any more.
Loading: the sample loading volume is 3ml; the mobile phase is ultrapure water, and the elution speed is 2ml/min; the peak fractions were collected and lyophilized to determine the activity of the different fractions.
The results of the separation of the enzymatic hydrolysate on the gel column are shown in FIG. 7, and 5 elution peaks are shown, which are designated as X1, X2, X3, X4 and X5. The eluents corresponding to the elution peaks X1 to X5 were collected in sequence, lyophilized and then mixed with water to form a solution with a concentration of 20mg/ml, and the XO inhibition ratios of the components were measured, with the results shown in Table 3. The inhibition rate of the eluent corresponding to the elution peak X3 is the highest,
the eluent corresponding to the elution peak X3 is selected for the next experiment.
TABLE 3 XO inhibition of different components
Example 3 identification of oyster polypeptide X3 peptide fragment composition by LC-MS/MS Mass Spectrometry
1. Polypeptide extraction: the eluent corresponding to the elution peak X3 in the example 2 is lyophilized, redissolved by 0.15% acetic acid solution, filtered by a 10K ultrafiltration tube, washed for 2 times by 5% acetic acid solution, and the obtained filtrate is desalted by C18 StageTip and dried in vacuum to obtain peptide fragments. After drying, redissolving with 0.1% TFA, measuring absorbance at 260nm, and determining peptide fragment concentration for LC-MS/MS analysis.
LC-MS/MS analysis
An appropriate amount of peptide fragment was taken from each sample and chromatographed using a nano liter flow rate Easy nLC 1200 chromatographic system (Thermo Scientific). Buffer solution: the solution A is 0.1% formic acid aqueous solution, and the solution B is a mixed solution of formic acid, acetonitrile and water (acetonitrile volume concentration is 80%, formic acid concentration is 0.1%). The column was equilibrated with 100% of liquid A. Samples were injected into a Trap Column (100 μm × 20mm,5 μm, C18, dr. Maisch GmbH) and subjected to gradient separation through a chromatography Column (75 μm × 150mm,3 μm, C18, dr. Maisch GmbH) at a flow rate of 300nl/min.
The liquid phase separation gradient was as follows: the linear gradient of the liquid B is from 3 percent to 5 percent from 0 minute to 2 minutes; 2-42 min, the linear gradient of the liquid B is from 5% to 25%; 42-52 min, the linear gradient of the B liquid is from 25% to 45%; 52-55 minutes, and the linear gradient of the liquid B is from 45% to 90%; the time is 55-70 minutes, and the liquid B is maintained at 90%.
The peptide fragments were separated and analyzed by DDA (data dependent acquisition) mass spectrometry using a Q-exact Plus mass spectrometer (Thermo Scientific). The analysis time is 70min, and the detection mode is as follows: positive ion, parent ion scan range: 300-1800 m/z, first-order mass spectrum resolution: 70,000@ m/z 200, AGC target, 3e6, first order Maximum IT, 30ms.
Peptide fragment secondary mass spectrometry was collected as follows: triggering acquisition of secondary mass spectra (MS 2 scan) of 20 highest intensity parent ions after each full scan (full scan), secondary mass resolution: 17,500@ m/z 200, AGC target 2e5, secondary Maximum IT:60ms, MS2 Activation type, HCD, isolation window:1.6m/z, normalized collisionenergy:30.
3. Database retrieval
Adopting mass spectrum database retrieval software as pfind; the following protein databases were used: derived from the Uniprot Protein Databas. Wherein the analysis parameter settings of the MaxQuant library search software are shown in Table 4.
Table 4pFind analysis parameter settings
And after the X3 component is subjected to mass spectrum data retrieval, PSM FDR (pulse position modulation) is less than or equal to 0.05 and Protein FDR is less than or equal to 0.05 are respectively used as screening standards for peptide fragment and Protein identification to obtain 19 peptide fragment sequences and 14 Protein sequences.
Example 4 peptide fragment screening, synthesis and validation
1. Treatment of ligands
The molecular structural formula of amino acid and polypeptide is drawn by adopting Chemdraw 19.0 and Chem3D Pro 14.0 software, and is stored as mo12 through MM2 force field optimization.
2. For treating a receptor
Downloading the crystal structure 1N5X of XO from the PDB database, deleting the B chain in the 1N5X by adopting PyMOL software, and extracting the ligand febuxostat (TEI) to store the ligand febuxostat (TEI) in a pdbqt format for later use. Dehydration and hydrogenation are carried out on an acceptor 1N5X, gastiger charge is calculated, all atoms are classified into AD4 types, and the atoms are stored in a pdbqt format for later use.
3. Molecular docking
Molecular docking is carried out by using the vina function of PyRx software to simulate the interaction of amino acid, polypeptide micromolecule and macromolecular protein. Based on the position of the primary ligand febuxostat in 1N5X, docking center coordinates were set to (96, 54, 39) (X, y, z), the box size was 25 × 25 × 25, and other parameters were taken as defaults. Then, the amino acids and polypeptide ligands are butted one by one with the XO crystal structure.
As a result: the 19 peptides of example 3 were each subjected to Vina-mimetic docking with XO, and six peptides of Gly-Gly-Tyr-Gly-Ile-Phe (GGYGIF) (shown in SEQ ID NO. 1), ile-Ala-Ser-Gly-Phe-Phe (IASGFF) (shown in SEQ ID NO. 2), ala-Leu-Ser-Gly-Ser-Trp (ALSGSW) (shown in SEQ ID NO. 3), val-Ala-Gln-Asp-Ser-Ser-Tyr (VAQDSSY) (shown in SEQ ID NO. 4), leu-His-Cys-Ser-Thr-Leu (LHCSTL) (shown in SEQ ID NO. 5) and Met-Ala-Gly-Leu-Trp (MAGGLW) (shown in SEQ ID NO. 6) were selected based on Vina score and synthesized.
Example 5 peptide fragment Synthesis and Activity verification
1. Peptide fragment synthesis
Peptide fragments GGYGIF, IASGFF, ALSGSW, VAQDSSY, LHCSTL and MAIGLW were synthesized by Fmoc solid phase synthesis method from Biotechnology engineering (Shanghai) GmbH.
2. Activity verification
Measurement of XO inhibition: the polypeptides were prepared as 3mg/ml solutions and the in vitro XO inhibition of the synthesized polypeptides was determined according to the method in example 1. IC (integrated circuit) 50 Values were calculated from regression lines of percent inhibition of XO activity versus substance concentration.
The results of the peptide fragment correlation and activity verification are shown in table 5.
Table 5 results of verification of related properties and activities of peptide fragments
Note: (1) Since the molecular docking results were random, vina scores shown in the table were the average of the highest values of 10 docking results (negative values); (2)' - "means no XO inhibitory activity.
(2) Peptide hydrophobicity and molecular weight were obtained by http:// www. Ulane.edu/. Biochem/WW/PepTracw/.
As can be seen from table 5, all of the six peptide fragments have higher XO inhibitory activity, and among them, IASGFF, VAQDSSY and LHCSTL exhibit higher XO inhibitory activity.
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.
Claims (3)
1. A small molecule peptide, characterized by: the amino acid sequence is VAQDSSY, and is shown as SEQ ID NO. 4.
2. Use of the small molecule peptide of claim 1 for the manufacture of a medicament for inhibiting xanthine oxidase.
3. Use of the small molecule peptide of claim 1 for the preparation of a medicament having an effect of inhibiting uric acid levels.
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