An asparagine synthetase gene from chlorella variabilis and application thereof

Abstract

An ACE inhibitory peptide L1 from Asparagopsis armata has an amino acid sequence of LVLLFLFGE. A preparation method comprises the following steps: Asparagopsis armata is enzymolyzed by neutral protease to obtain an enzymolyzed product, the enzymolyzed product is filtered, centrifuged, concentrated and freeze-dried to obtain a crude product; the crude product is dissolved in water and ultrafiltrated, polypeptide liquids with different molecular weights are collected and freeze-dried to obtain polypeptide freeze-dried powders, and polypeptide freeze-dried powders with the strongest ACE inhibitory activity are screened out; the polypeptide freeze-dried powders screened out are subjected to mass spectrometry and analysis to obtain a plurality of non-repeated polypeptide sequences; the polypeptide sequences are subjected to molecular docking with ACE protein by using software to screen polypeptide sequences with strong ACE protein binding capacity; the polypeptide sequences screened out are subjected to solid-phase synthesis to screen polypeptide sequences with high activity, and the ACE inhibitory peptide L1 is obtained. The ACE inhibitory peptide L1 has good affinity with ACE and high activity, can be used for developing a therapeutic drug or a functional food for assisting in reducing blood pressure, and has the advantages of high specificity and small toxic and side effects.

Description

Technical Field

[0001] This invention relates to the field of technology, and in particular to an ACE inhibitory peptide L1 derived from *Rhodophyta rubrum*, its preparation method, and its application. Background Technology

[0002] Angiotensin-converting enzyme (ACE), as an important rate-limiting enzyme, plays a crucial role in regulating blood pressure. ACE inhibitory peptides are small-molecule polypeptides that inhibit ACE activity. They not only prevent ACE from catalyzing the hydrolysis of bradykinin, maintaining its normal vasoconstrictive function, but also prevent ACE from catalyzing the conversion of angiotensin I to angiotensin II, which has vasoconstrictive capabilities, thus achieving a good antihypertensive effect. Currently, most ACE inhibitors used to treat hypertension are chemically synthesized. Although these drugs have good efficacy, they often cause side effects such as throat swelling, kidney damage, and loss of taste after administration. Bioactive peptides with ACE inhibitory effects have become a research hotspot in recent years due to their small molecular weight, easy absorption, lack of toxic side effects, and high safety.

[0003] The ocean covers approximately 70% of the Earth's surface and is the future global food base; marine biological resources are a vital food source for humankind. With the in-depth exploration of marine biological resources, the extraction and screening of bioactive peptides from marine biological proteins holds great promise. Red hair algae, also known as hairy algae or red seaweed, belongs to the Rhodophyta phylum and is mainly produced in the Meizhou and Nanri Island areas of Putian. It grows on shallow mudflats near the low tide line and belongs to the green-banded red hair algae community. The seaweed has a slender, filamentous structure with few branches; when fresh, it is red or purplish-red, while after drying, it turns dark brown. Red hair algae is rich in protein and various vitamins, possessing excellent nutritional value and health benefits. Red beech seaweed is favored by people in southern China for its unique aroma, rich nutrition, and delicious taste. Folk applications have also shown that red beech seaweed has high medicinal value. For example, every 100g of dried red beech seaweed contains 437mg of the unsaturated fatty acid EPA (eicosapentaenoic acid), making it one of the seaweeds with the highest EPA content discovered to date. Furthermore, some literature states that red beech seaweed has nourishing and heat-clearing, blood-tonifying and blood pressure-lowering, antioxidant, anti-tumor, and cardiovascular disease-preventing effects, and can also enhance human immunity and improve physical fitness. However, no deep-processed products made from red beech seaweed are currently available on the market. Summary of the Invention

[0004] The purpose of this invention is to provide an ACE inhibitory peptide L1 derived from *Rhodotorula rubra*, its preparation method, and its application. This invention uses *Rhodotorula rubra* as raw material for deep processing to obtain a polypeptide with ACE inhibitory activity, providing a new approach for the high-value utilization of *Rhodotorula rubra* and expanding resources for natural blood pressure lowering products.

[0005] To achieve the above objectives, this invention discloses an ACE inhibitory peptide L1 derived from *Rhodophyta*, the amino acid sequence of which is: LVLLFLFGE.

[0006] Preferably, its IC 50 It was 14.22 μg / mL.

[0007] This invention also discloses a method for preparing the above-mentioned ACE inhibitory peptide L1 derived from *Rhodophyta*, which includes the following steps: Step 1: Use neutral protease to enzymatically hydrolyze red algae to obtain the enzymatic hydrolysis product; the enzymatic hydrolysis product is filtered, centrifuged, concentrated and freeze-dried to obtain the crude product; Step 2: Dissolve the crude product in distilled water and then ultrafilter it. Collect peptide solutions of different molecular weights and freeze-dry them to obtain lyophilized peptide powders of different molecular weights. Step 3: Determine the ACE inhibition rate of lyophilized peptides of different molecular weights and screen out the lyophilized peptides with the strongest ACE inhibition activity. Step 4: The selected lyophilized peptide powder was analyzed by LC-MS. The results of the mass spectrometry analysis were analyzed by mass spectrometry analysis software to obtain multiple non-repeating peptide sequences. Step 5: Use software to perform molecular docking between the polypeptide sequence and the ACE protein, and screen for polypeptide sequences with strong binding ability to the ACE protein. Step 6: The selected polypeptide sequences are subjected to solid-phase synthesis. Highly active polypeptide sequences are then selected to obtain the ACE inhibitory peptide L1.

[0008] Preferably, in step 1, the red algae are first chopped and then added to a PBS buffer solution with a concentration of 0.01–0.05 mol / L at an addition rate of 15–35 g / L, and soaked for 10–14 h to obtain a soaking solution; 1–3% of neutral protease by volume of the soaking solution is added to the soaking solution, and the solution is incubated in a water bath at 40–60 °C for 2–6 h to obtain a hydrolysate; the hydrolysate is centrifuged at 2–6 °C for 10–20 min at a centrifugation speed of 6000–9000 rpm, and the hydrolysate obtained by centrifugation is subjected to rotary evaporation to obtain a concentrated solution, which is then freeze-dried to obtain a crude product.

[0009] Preferably, in step 1, the red algae are first chopped and then added to a 0.01 mol / L PBS buffer at a dosage of 25 g / L, and soaked for 12 h to obtain a soaking solution; 2% of the volume of neutral protease is added to the soaking solution, and the solution is incubated in a water bath at 56°C for 4 h to obtain a hydrolysate; the hydrolysate is centrifuged at 4°C for 10 min at a speed of 8000 rpm.

[0010] Preferably, in step 2, after the crude product is dissolved in distilled water, it is sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 10000 Da, 2000 Da, and 800 Da for ultrafiltration separation, and the products with Mw < 800 Da and Mw < 800 Da are collected respectively. <Mw<2000 Da、2000Da<Mw <10000 Da and Mw> Four ultrafiltration components with a molecular weight of 10000 Da were freeze-dried to obtain four polypeptide solutions with different molecular weight distributions, resulting in lyophilized polypeptide powder.

[0011] Preferably, in step 4, the mass spectrometry determination conditions are as follows: the chromatographic column is a PepMap RPLC C18, 75μm id×150mm, 3μm, 100Å, cation mode, scan range: m / z 300~1500Da, emitter spray voltage 2kV; the mass spectrometry results are analyzed by de novo sequencing using analysis software to obtain multiple polypeptide sequences, duplicate polypeptide sequences are deleted, and multiple non-duplicate polypeptides are screened out.

[0012] Preferably, in step 5, the polypeptide selected in step 4 is used as the ligand file, and the ACE protein is used as the receptor, and molecular docking is performed using docking software.

[0013] Preferably, in step 5, the crystal structure of the ACE protein, which serves as the receptor, is 1O86.

[0014] Furthermore, this invention also discloses the application of the above-mentioned red algae-derived ACE inhibitory peptide L1 in the preparation of adjuvant antihypertensive drugs.

[0015] The present invention has the following beneficial effects: The ACE inhibitory peptide L1 of this invention has good affinity and high activity for ACE enzyme, and its IC50 value is [not specified]. 50 With a concentration of 14.22 μg / mL, it can be used in the development of adjuvant antihypertensive drugs or functional foods. Furthermore, since the ACE inhibitory peptide L1 of this invention is extracted from *Pleurotus erythrorhizon*, it has the advantages of high specificity and low toxicity compared to traditional antihypertensive drugs. It also provides a new approach for the high-value utilization of *Pleurotus erythrorhizon*, expanding resources for natural antihypertensive products. Attached Figure Description

[0016] Figure 1 The graph shows the ACE inhibition rate of lyophilized peptide powders with different molecular weights.

[0017] Figure 2 Mass spectrum and sequence prediction for LVLLFLFGE.

[0018] Figure 3 This is a diagram illustrating the combination pattern of LVLLFLFGE and ACE.

[0019] Figure 4Graphs showing the ACE inhibition effects of different concentrations of LVLLFLFGE (high performance liquid chromatography detection). Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0021] This invention discloses an ACE inhibitory peptide L1 derived from *Rhodophyta*, the amino acid sequence of which is: LVLLFLFGE, as shown in SEQ ID NO: 1 of the sequence listing.

[0022] The preparation method of L1, an ACE inhibitory peptide derived from *Rhodophyta*, includes the following steps: Step 1: Enzymatic hydrolysis of *Rhododendron molle* using neutral protease yields the enzymatic hydrolysis product. This product is then filtered, centrifuged, concentrated, and freeze-dried to obtain the crude product. Specifically: First, the *Rhododendron simsii* was chopped and then added to 0.01 mol / L PBS buffer at a dosage of 25 g / L. In this case, 20 g of *Rhododendron simsii* was weighed, chopped, and soaked in 800 mL of 0.01 mol / L PBS buffer for 12 hours to obtain the soaking solution. 2% (by volume) of neutral protease was added to the soaking solution, and the mixture was incubated at 56°C for 4 hours to obtain the enzymatic hydrolysate. The hydrolysate was centrifuged at 4°C for 10 minutes at 8000 rpm. The resulting hydrolysate was then subjected to rotary evaporation to obtain a concentrated solution. The concentrated solution was freeze-dried to obtain the crude product.

[0023] Step 2: Dissolve the crude product in distilled water and then ultrafilter. Collect peptide solutions of different molecular weights and freeze-dry them to obtain lyophilized peptide powders of different molecular weights. Specifically: The crude product was dissolved in distilled water and then sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 10000 Da, 2000 Da, and 800 Da for ultrafiltration separation. The products with molecular weight cutoffs of <800 Da and <800 Da were collected, respectively. <Mw<2000 Da、2000 Da<Mw <10000 Da and Mw> Four ultrafiltration components with a molecular weight of 10000 Da were named S1, S2, S3 and S4, respectively. These four polypeptide solutions with different molecular weight distributions were freeze-dried to obtain polypeptide lyophilized powder.

[0024] Step 3: Prepare solutions of the four different molecular weight lyophilized peptides at a concentration of 1 mg / mL, and measure the ACE inhibition rate of these four solutions. The results are as follows: Figure 1 As shown, the lyophilized peptide powder with the strongest ACE inhibitory activity, namely S2, was screened out, with an ACE inhibition rate of over 50%.

[0025] Step 4: The selected lyophilized peptide powder (S2) was analyzed by LC-MS. The results were then analyzed using mass spectrometry software to obtain multiple non-repetitive peptide sequences. Specifically: The mass spectrometry determination conditions were as follows: the chromatographic column was a PepMap RPLC C18, 75 μm id × 150 mm, 3 μm, 100 Å, cation mode, scan range: m / z 300~1500 Da, and emitter spray voltage was 2 kV.

[0026] The mass spectrometry results were analyzed using Peaks Studio software (Bioinformatics Solutions Inc., Canada). The de novo module of Peaks Studio was used to perform de novo sequencing analysis on the LC-MS data, obtaining multiple peptide sequences. Repeated peptide sequences were removed, and multiple non-repeating peptides were selected. The mass spectrum and predicted sequence of the LVLLFLFGE sequence are shown below. Figure 2 As shown.

[0027] Step 5: Using software, the peptide sequence is molecularly docked with the ACE protein to screen for peptide sequences with strong binding affinity to the ACE protein. Specifically: Using the peptides screened in step 4 as ligand files, the crystal structure of the ACE protein was taken as the receptor (PDB code: 1O86), and the docking site (coordinates: x:40.372, y:33.82, z:48.088, radius 10 Å) was used as the docking site for the docking experiment. Molecular docking was performed using the Ingenic Cloud Computing Platform. The results are shown in [link to results]. Figure 3 .Depend on Figure 3 It is evident that LVLLFLFGE and ACE have a total of 6 hydrogen bond interactions. The amino acid residues that form hydrogen bonds with the peptide include: G1u123, Tyr135, Trp220, Met223, Ala356, and Glu384. The hydrogen bond network formed between them is crucial for the stable binding of LVLLFLFGE to ACE. LVLLFLFGE also forms salt bridges with the amino acid residues His353 and His513 in ACE. These interactions contribute -6.1173 kcal / mol to the electrostatic force of the compound binding. Furthermore, the binding pocket contains several hydrophobic residues that can form strong hydrophobic interactions with LVLLFLFGE, contributing van der Waals forces. These strongly hydrophobic amino acid residues include: Arg124, Ile201, Asp358, Phe391, and Tyr523.

[0028] Step 6: The screened peptide sequences are subjected to solid-phase synthesis. Highly active peptide sequences are then selected to obtain the ACE repressor peptide L1. Specifically: LVLLFLFGE was prepared into five concentration gradients: 6.25, 12.5, 25, 50, and 100 μg / mL. 100 μL of hippurylhistylleucine (HHL) was added to 2 mL centrifuge tubes, and 100 μL of LVLLFLFGE solution of different concentrations was added to each tube. A blank control group containing borate buffer was also included. The centrifuge tubes were preheated at 37 °C for 5 min. After adding 50 μL of ACE, the reaction was incubated at 37 °C for 1 h. The reaction was terminated by adding 200 μL of 1 mol / L hydrochloric acid. After filtering through a 0.22 μm aqueous filter, the hippuric acid content in the solution was determined by HPLC, and the ACE inhibition rate of the peptide was calculated to obtain the IC50 of the *Rhodotorula purpurae* ACE-inhibiting peptide. 50 The concentration was 14.22 μg / mL. (See the results below.) Figure 4 .

[0029] Based on the above research, this invention also discloses the application of the above-mentioned red algae-derived ACE inhibitory peptide L1 in the preparation of adjuvant antihypertensive drugs.

[0030] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A red algae-derived ACE inhibitory peptide L1, characterized in that, Its amino acid sequence is: LVLLFLFGE.

2. The application of the red algae-derived ACE inhibitory peptide L1 as described in claim 1 in the preparation of adjuvant antihypertensive drugs.

Images