ATGL inhibitor as well as preparation method and application thereof

Abstract

The invention discloses an ATGL inhibitor as well as a preparation method and application thereof. The amino acid sequence of the ATGL inhibitor is Lys-Leu-Ile-Met-Val. The ATGL inhibitor has the remarkable effects that the ATGL inhibitor has relatively high hydrophobicity and proper size, and can be misrecognized by a hydrophobic active trench of ATGL to form a space site barrier, so that the combination of ATGL and triglyceride is prevented, and finally, the catalytic activity of ATGL is competitively inhibited.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme activity inhibition, and particularly relates to an ATGL inhibitor, a preparation method thereof, and an application thereof. Background Art

[0002] Carp (Cyprinus carpio) is an important economic freshwater fish, and its aquaculture output in China in 2024 was approximately 3 million tons. Due to its excellent meat quality and rich nutrition, carp is deeply favored by consumers and producers. However, compared with other meats, fish is more prone to spoilage during processing or storage, mainly due to factors such as its rich endogenous enzymes in muscle, high water content, relatively fragile muscle tissue, and various bacteria attached to the digestive tract or gills.

[0003] Among them, lipid decomposition caused by endogenous enzymes is one of the most important factors leading to fish spoilage. On the one hand, small molecular compounds such as alcohols, aldehydes, or ketones are produced during lipid decomposition, seriously affecting the quality of fish. On the other hand, excessive lipid decomposition accelerates protein degradation, and then accelerates the softening of fish or fish mince gel. Lipid decomposition is divided into oxidation and hydrolysis, and the two can promote each other. Lipid oxidation is divided into autoxidation, photooxidation, and enzymatic oxidation. Lipid hydrolysis is mainly dominated by three intracellular enzymes: adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoglyceride lipase (MGL).

[0004] The adipose triglyceride lipase (ATGL) is an intracellular lipase responsible for hydrolyzing triglycerides into diglycerides and fatty acids, and is widely expressed in various tissues of animals, such as muscle, skin, testis, liver, and brain tissue. In the food field, ATGL plays an important role in the first step of the lipolysis cascade reaction, that is, it can be regarded as the rate-limiting enzyme for lipid hydrolysis. Therefore, inhibiting the residual catalytic activity of ATGL in carp meat is of great significance for ensuring the quality of carp meat. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide an ATGL inhibitor, a preparation method thereof, and an application thereof, which can effectively form a spatial site barrier to prevent the binding of ATGL to triglycerides and competitively inhibit the catalytic activity of ATGL in carp meat.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows: In the first aspect, the present invention proposes an ATGL inhibitor, which is characterized in that: the amino acid sequence of the ATGL inhibitor is Lys-Leu-Ile-Met-Val.

[0007] Second aspect, the present invention provides a method for preparing the ATGL inhibitor described in the first aspect, comprising the following steps: Clone the N-terminal domain of ATGL from muscle tissue; Successfully induce the expression of ATGL using the Escherichia coli prokaryotic expression system; Analyze the enzymatic activity characteristics of ATGL at different pH values and temperatures; Perform molecular dynamics simulation to analyze the tertiary structure characteristics and catalytic mechanism of ATGL; Analyze the hydrolysis mechanism of ATGL; Design and synthesize the ATGL inhibitor.

[0008] Furthermore, the cloning of the N-terminal domain of ATGL from muscle tissue specifically includes: Extract RNA from muscle and characterize it by agarose gel electrophoresis; Use a reverse transcription kit to reverse transcribe the extracted RNA into cDNA; Use primers for PCR amplification with cDNA as the template; After PCR amplification, sequence the amplification product; Resynthesize the ATGL sequence and ligate it into an expression vector; Transfer the recombinant plasmid into the Escherichia coli prokaryotic expression system.

[0009] Furthermore, the primers are 5’-atgtttcccctcgactctccgt-3’ and 5’-taacagaccgttcttcttcag-3’.

[0010] Furthermore, when analyzing the enzymatic activity characteristics of ATGL, a high-sensitivity liquid scintillation counter is used, and the substrate consists of 0.33 mM triolein, 0.45 mM phosphatidylcholine / phosphatidylinositol (3:1), and 0.5% defatted bovine serum albumin. [9,10-3H] Triolein is used as a radioactive tracer.

[0011] Furthermore, the molecular dynamics simulation to analyze the tertiary structure characteristics and catalytic mechanism of ATGL specifically includes: Perform molecular docking of ATGL and triglyceride molecules using the Discovery Studio tool; Perform molecular dynamics simulation on the ATGL-triglyceride and ATGL-peptide complexes obtained from molecular docking. During the simulation process, the simulation file is extracted every 0.01 ns. Using a cutoff distance of 1 nm, periodic boundary conditions, and the leapfrog algorithm, calculate the radius of gyration, RMSF, root mean square distance, solvent accessible surface area, binding free energy, hydrogen bonds, and atomic distances.

[0012] Third aspect, the present invention also provides an application of the ATGL inhibitor described in the first aspect in inhibiting the hydrolysis activity of ATGL.

[0013] Fourth aspect, the present invention also provides an application of the ATGL inhibitor described in the first aspect in preparing a preservation agent for carp meat.

[0014] Fifth aspect, the present invention also provides an application of the ATGL inhibitor described in the first aspect in preparing a meat product additive.

[0015] Lipid hydrolysis caused by endogenous enzymes is one of the most important factors reducing the quality of meat products. Adipose triglyceride lipase (ATGL) is responsible for hydrolyzing triglycerides and is the initiating enzyme leading to lipid hydrolysis. The present invention first cloned the N-terminal domain of carp ATGL. Secondly, the recombinant carp ATGL was successfully expressed and purified. And the changes of its enzyme activity at different temperatures and pH values were analyzed. Thirdly, the tertiary structure of ATGL was analyzed. Carp ATGL is a monomeric protein composed of 9 α-helices and 7 β-sheets, with a radius of gyration of 1.84 nm. Its active center is a catalytic dyad composed of Ser47-Asp166, located at the bottom of the active groove, almost composed of hydrophobic amino acids. There are 3 flexible hydrophobic loops around the active groove, which is related to the binding of hydrophobic lipids. Then, the hydrolysis mechanism of carp ATGL was analyzed. Finally, an inhibitor of carp ATGL was designed and synthesized, which is a small peptide composed of hydrophobic essential amino acids (Lys-Leu-Ile-Met-Val). This inhibitor will be misrecognized by the hydrophobic active groove of ATGL, forming a steric site barrier to prevent the binding of ATGL to triglycerides.

[0016] The remarkable effects of the present invention are as follows: 1. The ATGL inhibitor described in the present invention is a small peptide composed of hydrophobic essential amino acids (Lys-Leu-Ile-Met-Val). It has high hydrophobicity and appropriate size, can be misrecognized by the hydrophobic active groove of ATGL, form a steric site barrier, prevent the binding of ATGL to triglycerides, and finally competitively inhibit the catalytic activity of ATGL. It was found that when the concentration of the small peptide reached 0.1 mg / ml, the ATGL hydrolysis activity of carp decreased by 81.5%.

[0017] 2. The interaction energy between the ATGL inhibitor described in the present invention and ATGL can reach 62.7 Kj / moL, which indicates that the complex of the small peptide and ATGL is very stable, can spontaneously prevent the binding of ATGL to triglycerides, and inhibit the activity of ATGL.

[0018] 3. In addition to having a good inhibitory effect on ATGL, the ATGL inhibitor of the present invention is composed of essential amino acids, is itself a nutritional fortifier, and has a low cost. Therefore, it is very promising as an additive for carp meat products.

[0019] 4. Since the N-terminal domain of carp ATGL is highly conserved among different species (with a similarity of up to about 80%), the ATGL inhibitor of the present invention can also be used as an additive for other meat products and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a schematic diagram of the primary structure characteristics of carp ATGL; Figure 2 is a comparison diagram of the amino acid sequences of carp ATGL and ATGL of other species; Figure 3 is a diagram of the results of prokaryotic expression and activity characteristics analysis of carp ATGL; Figure 4 is a schematic diagram of the tertiary structure characteristics of carp ATGL; Figure 5 is a diagram of the results of the hydrophilicity and hydrophobicity analysis of carp ATGL; Figure 6 is a schematic diagram of the hydrolysis mechanism and process of triglyceride by carp ATGL; Figure 7 is a schematic diagram of the secondary interaction between carp ATGL and triglyceride during the hydrolysis process; Figure 8 is a diagram of the design of the carp ATGL inhibitor and the results of the analysis of its inhibition mechanism. DETAILED DESCRIPTION OF THE INVENTION

[0021] The following further elaborates in detail on the specific embodiments and working principles of the present invention in conjunction with the accompanying drawings.

[0022] Research has found that there is an enzyme called ATGL (triglyceride hydrolase) in carp meat. It still has the activity of hydrolyzing triglycerides after the fish dies, and it still has activity at extremely low temperatures. During the storage of fish meat or fish meat products, the ATGL in them continuously hydrolyzes the lipids (triglycerides) of the fish meat, generating various small molecular compounds, which will seriously affect the quality of the fish meat products. Therefore, it is very necessary to control the activity of ATGL during storage.

[0023] Therefore, the present invention proposes a small peptide (Lys-Leu-Ile-Met-Val, lysine-leucine-isoleucine-methionine-valine) as an ATGL inhibitor. Through activity inhibition experiments, molecular docking, and molecular dynamics simulation analysis, it is found that this small peptide can efficiently inhibit the activity of carp ATGL and is expected to be used as an additive in fish products.

[0024] The reason why it can efficiently inhibit the activity of ATGL is that by analyzing the tertiary structure characteristics of ATGL, it is found that most of the residues in the active groove of ATGL are hydrophobic. And the small peptide proposed in the present invention is highly hydrophobic, so it can efficiently complex with ATGL through hydrophilic-hydrophobic interactions. Secondly, the R group of this small peptide hardly contains groups that can form hydrogen bonds, so it is not easily bound by other components in food and is not easily self-wound. The specific embodiments are as follows: Embodiment

[0025] This embodiment provides an ATGL inhibitor, and the amino acid sequence of the ATGL inhibitor is Lys-Leu-Ile-Met-Val.

[0026] The ATGL inhibitor has high hydrophobicity and a suitable size, can be misrecognized by the hydrophobic active groove of ATGL, form a spatial site barrier, prevent the binding of ATGL to triglyceride, and finally competitively inhibit the catalytic activity of ATGL. Embodiment

[0027] This embodiment provides a preparation method of the ATGL inhibitor described in Embodiment 1, and the specific steps are as follows: Step 1. Cloning of carp ATGL: As Figure 1 shown in a, researchers analyzed ATGL of various species, including humans, mice, pigs, etc., and found that ATGL contains two domains, and the N-terminal domain is the key to its catalytic function. In vitro studies have shown that ATGL still has catalytic activity in the absence of the C-terminal domain.

[0028] Therefore, in this embodiment, the N-terminal domain of carp ATGL was first cloned from carp muscle tissue, and the specific steps are as follows: Step 101. Extract RNA from the dorsal muscle of carp and characterize it by agarose gel electrophoresis; As Figure 1 shown in b, first extract carp RNA from muscle tissue, showing 3 bands: 28S, 18S, and 5S.

[0029] Step 102: Use a reverse transcription kit (Genecreate, Wuhan, China) to reverse transcribe the extracted RNA into cDNA as the template for ATGL cloning; As Figure 1 shown in c, a single band of approximately 750 bp can be observed in the agarose gel electrophoresis result, which is consistent with the size of the ATGL N-terminal domain.

[0030] Step 103: Using the cDNA as a template, perform PCR amplification with the following primers: 5’-atgtttcccctcgactctccgt-3’ and 5’-taacagaccgttcttcttcag-3’; Step 104: After PCR amplification, cut and recover the amplification product, that is, the amplification band, and sequence it by a biotechnology company (Wuhan, China) to verify its correctness; As Figure 1 shown in d, the sequencing result shows that the ATGL N-terminal domain of carp is 768 bp, encoding 256 amino acids, with a molecular weight of 27.7 KD and an isoelectric point of 7.98. The similarity of carp ATGL to ATGL of other species is approximately 80%: large yellow croaker (HQ916211) 87.11%, human (AY894804) 78.13%, pig (EF583921) 76.56%, mouse (AY894805) 79.24%, cattle (FJ897536) 78.91%, goose (HQ914789) 80.47% and Japanese quail (EU852336) 80.08% (as Figure 2 shown). In addition, compared with other genes, ATGL is highly conserved among different species. For example, the similarity between human histidase and carp histidase is only about 50% (Zhong et al., 2021). While the similarity of ATGL among different species can be as high as about 80%.

[0031] Step 105: Resynthesize the carp ATGL sequence and ligate it into an expression vector (PET-30a); Step 106: Transfer the recombinant plasmid into BL21 Escherichia coli.

[0032] Step 2: Prokaryotic expression of carp ATGL: Induce the expression of recombinant carp ATGL with 0.1 mol / L IPTG and purify the product by nickel column affinity chromatography. Specifically: As Figure 3 shown in a, a strong protein band appears at approximately 27 KD after induction, indicating the successful induction of carp ATGL expression. In addition, as can be seen from Figure 3 a, recombinant carp ATGL appears in the lysis precipitate, that is, inclusion bodies are formed. Then, the washed sample is purified by nickel column affinity chromatography. AsFigure 3 As shown in Figures a and 3b, after dialysis and concentration, a single band appeared on the SDS-PAGE electrophoresis pattern, indicating the successful purification of recombinant carp ATGL.

[0033] Step 3. Analyze the activity characteristics of carp ATGL: According to the methods of Nagy et al. and Schweiger et al., the activity characteristics of carp ATGL were analyzed using a high-sensitivity liquid scintillation counter. The substrate consisted of 0.33 mM triolein, 0.45 mM phosphatidylcholine / phosphatidylinositol (3:1), and 0.5% defatted bovine serum albumin, and [9,10-3H] triolein was used as a radioactive tracer. The concentration of purified carp recombinant ATGL was adjusted to 1 mg / ml by dialysis and concentration. During the reaction, 100 μl of the substrate was added to 100 μl of the sample, and the reaction was carried out at pH 3 to 11 (25 °C) and 10 to 50 °C (pH 7) for 30 minutes. The reaction was terminated by adding 3.25 ml of methanol / chloroform / heptane (10:9:7) and 1 ml of 0.1 M potassium carbonate and 0.1 M boric acid (pH 10.5). Finally, the mixture was centrifuged at 1000 g for 15 minutes, and the supernatant was taken for liquid scintillation counting.

[0034] As Figure 3 shown in Figures c and Figure 3 d, the activities of carp ATGL at different pH values and temperatures were analyzed. As can be seen from Figure 3 Figure c, when the pH value was 7, the activity of carp ATGL was the highest, and the increase or decrease of the pH value would affect its activity. In addition, the alkaline environment had a greater impact on the activity of carp ATGL than the acidic environment. Even when the pH value dropped to 3, the activity of carp ATGL still remained at about 26.4%. Studies have shown that the pH value of fresh carp meat is about 7.1. As storage progresses, the pH value gradually decreases and reaches a low point of 6.7 on the 4th day. Subsequently, as the storage time prolongs, the pH value gradually recovers and returns to 7.1 on the 10th day. And this pH value is the optimal pH value for ATGL activity. Therefore, in practical applications, the activity of ATGL can be inhibited by adjusting the pH value to alkaline.

[0035] In addition, the effect of temperature on the activity of carp ATGL was not as obvious as that of pH value. Between 20 and 35 °C, the activity of carp ATGL changed little. When the reaction temperature dropped to 5 °C or rose to 50 °C, the activities of carp ATGL still remained at about 73.6% and 62.8% respectively. This indicates that in practice, it is difficult to completely control the hydrolysis of triglycerides in carp meat by low-temperature storage.

[0036] Step 4. Analyze the tertiary structure of carp ATGL and its catalytic mechanism: In this embodiment, molecular dynamics simulation was used to analyze the tertiary structural characteristics of carp ATGL. The specific process is as follows: First, the molecular docking of ATGL with triglyceride molecules (or small peptides) was performed using the DiscoveryStudio tool. The water molecules in the PDB were removed and hydrogen atoms were added. Then, the model was minimized using the smart minimization algorithm. The docking results were obtained using VMD.

[0037] Then, molecular dynamics simulations were performed on the ATGL-triglyceride and ATGL-small peptide complexes obtained from molecular docking. Three parallel simulations were performed for each complex. The temperature and pressure were controlled using the Langevin and Berendsen thermostats, respectively. All simulations were performed using the Amber tool and simulated for 100 ns using the Glycam06, ff14SB, and tip5p force fields under the NPT ensemble. Before the simulation, it was equilibrated for 2 ns under the NVT ensemble and 2 ns under the NPT ensemble. In addition, a cutoff distance of 1 nm, periodic boundary conditions, and the leapfrog algorithm were used.

[0038] The simulation files were extracted every 0.01 ns, and the radius of gyration (Rg), RMSF, root mean square distance (RMSD), solvent accessible surface area (SASA), binding free energy, hydrogen bonds, and atomic distances were calculated. The binding free energy was calculated using the MM-PBSA tool. The SASA was determined using a linear combination of the pairwise overlap algorithm defined by Weiser. The presence of hydrogen bonds was determined according to the criterion that the maximum distance is equal to 0.35 nm.

[0039] As Figure 4 shown in Figure 4 a, a 100-ns simulation of carp ATGL was performed. During the simulation, the RMSD value of carp ATGL gradually increased in the first 10 ns and gradually stabilized in the later stage of the simulation. For the analysis results, please refer to

[0040] As Figure 4 shown in Figure 4As can be seen from c, the RMSF values of carp ATGL are usually low at β-sheets and α-helices, while the Loop structures show higher flexibility. There are three regions with relatively high RMSF values that require extra attention, which are the loop between α4 and α5 (Ser87-Arg95), the loop between α7 and β5 (Tyr147-Pro156), and the loop between β6 and α8 (Ser188-Asp197). From Figure 4 e, it can be seen that these three highly flexible loops are located above the active center of carp ATGL and may be closely related to substrate binding.

[0041] From Figure 4 f, it can be seen that the active center of carp ATGL is a catalytic dyad composed of Ser47-Asp166, located at the bottom of the active groove (about 1.9 nm long and about 1.7 nm wide). Hydrophobicity analysis shows that the vast majority of residues at the bottom of this active groove are hydrophobic (as Figure 5 shown), such as Pro14, Trp15, Asn16, Ile17, Asn46, Arg48, Tyr51, Asn142, Glu143, Glu144, Leu145, Thr165, Gln167, and Gly168. This is conducive to the binding of the active groove to the hydrophobic fatty acid chain. As Figure 5 shown, the vast majority of residues on the surface of carp ATGL are hydrophilic, which is consistent with the structure of the protein being hydrophilic on the outside and hydrophobic on the inside. It should be noted that among the 3 highly flexible loop structures mentioned just now, 2 flexible loops are completely hydrophobic (Ser87-Arg95, Tyr147-Pro156), while the other one is also partially hydrophobic (Ser188-Asp197). This again indicates that these loop structures may be closely related to the binding of hydrophobic fatty acid chains.

[0042] Step 5. Analyze the hydrolysis mechanism of ATGL: In this example, the interaction details between carp ATGL and triglyceride molecules were also analyzed by molecular docking. First, the interaction sites were analyzed according to the change in SASA (solvent-accessible surface area). As Figure 6 shown, after ATGL is complexed with triglyceride, its SASA decreases significantly at several positions such as Ser47, Leu90, Try151, Asp166, and Ile193. Its interaction sites are located on the active groove and the highly flexible loops. As Figure 6 shown in b, the triglyceride molecule is embedded in the active groove of carp ATGL, and the ester bond is in full contact with the catalytic dyad. As the simulation progresses, the three flexible hydrophobic loops undergo conformational changes and gradually wrap the triglyceride molecule in the cavity to ensure the occurrence of the hydrolysis reaction.

[0043] In addition, the MM-PBSA tool was used to analyze the interaction energy between carp ATGL and triglyceride molecules (negative values represent attractive forces, and positive values represent repulsive forces). As Figure 7 shown in a, the total interaction energy between carp ATGL and triglyceride molecules is approximately 85.8 kJ / mol, indicating that the interaction between carp ATGL and triglyceride molecules is very stable (the bond energy of a carbon-carbon single bond is approximately 320 kJ / mol). Among them, van der Waals forces, electrostatic forces, and hydrophobic forces all contribute to the complexation of ATGL and triglyceride molecules. The hydrophilic interaction part, however, is not conducive to the binding of ATGL and triglyceride, because triglyceride molecules have strong hydrophobicity. In addition, the negative value of the total energy also indicates that the binding of ATGL and triglyceride molecules can occur spontaneously in an aqueous solution.

[0044] In addition, the contributions of each amino acid residue to the binding of ATGL and triglyceride were analyzed. As Figure 7 shown in b, residues such as Phe17, Leo90, Ile148, and His204 are very important for the binding of ATGL and triglyceride. All these residues (including the above-mentioned flexible loop and active groove) can be used as targets for the design of ATGL inhibitors.

[0045] Step 6: Design and synthesize the ATGL inhibitor: After understanding the tertiary structure of ATGL, efficient inhibitors can be designed based on the characteristics of its tertiary structure, catalytic mechanism, and hydrolysis mechanism to precisely control the product quality of carp.

[0046] Based on the above analysis, the preliminary idea is to select some hydrophobic small peptides composed of essential amino acids as competitive inhibitors to inhibit the hydrolysis of triglyceride by ATGL. First, when the small peptides reach a certain abundance, the small peptides composed of hydrophobic amino acids can act on the active groove or hydrophobic loop of ATGL to form a steric site barrier through hydrophobic interactions. Second, the small peptides composed of essential amino acids are safe and non-toxic and are also a kind of nutritional fortifier. Third, small peptides can be efficiently expressed and produced by genetic engineering methods at low cost (designing enzyme cleavage sites, enzyme cleavage, and recovery after expression).

[0047] In previous experiments, various combinations of hydrophobic essential amino acids were synthesized and tested. Finally, it was found that the small peptide with the amino acid sequence Lys-Leu-Ile-Met-Val had a good inhibitory effect on ATGL.

[0048] In this example, the small peptide was chemically synthesized by Changzhou Mukai Biotechnology Co., Ltd. The synthesized small peptides were analyzed for molecular weight and purity by mass spectrometry (Agilent Technologies Inc., 6125B, USA) and high performance liquid chromatography (HPLC) (Agilent Technologies Inc., 1290 Infinity III, USA). HPLC test method: The chromatographic column was 4.6×250 mm, Sinochrom ODS-BP 5 μm. The mobile phase was 0.1% trifluoroacetic acid acetonitrile solution and 0.1% trifluoroacetic acid aqueous solution. The flow rate was set at 1.0 ml / min and the wavelength was set at 220 nm. Mass spectrometry test method: The probe was set to ESI, the probe bias was set to 4.5 kv, the detector was set to 1.5 kv, and the atomizing gas flow rate and T. flow rate were set to 1.5 L / min and 0.2 ml / min respectively. The mobile phase was 50% acetonitrile, the block temperature was set to 200 °C, and the CDL temperature was set to 250 °C.

[0049] As Figure 8 shown in Figures 5a and 5b, the synthesized small peptides were characterized by HPLC (high performance liquid chromatography) and MS (mass spectrometry), and it was found that the purity could reach 95.7% and the molecular weight was consistent with the design. The molecular weight of the Lys-Leu-Ile-Met-Val small peptide was about 0.6 kD and the pI was 9.70. Then the small peptide was added to the ATGL hydrolysis reaction system (taking the hydrolysis reaction product at 25 °C and pH 7 as 100%). As Figure 8 shown in Figure 5c, as the concentration of the small peptide increased, the activity of ATGL was gradually inhibited. When the concentration of the small peptide reached 0.1 mg / ml, the ATGL hydrolysis activity of carp decreased by 81.5%.

[0050] The inhibition mechanism of the Lys-Leu-Ile-Met-Val small peptide was analyzed by molecular docking and kinetic simulation. As Figure 8 shown in Figures 5d and Figure 8 5e, due to the high hydrophobicity and appropriate size of the small peptide, it was misrecognized by the hydrophobic active groove of ATGL, forming a steric site barrier, preventing the binding of ATGL to triglyceride, and finally competitively inhibiting the catalytic activity of ATGL. As Figure 8 shown in Figure 5f, the total interaction energy between the Lys-Leu-Ile-Met-Val small peptide and ATGL reached 62.7 Kj / moL, indicating that the complex between the small peptide and ATGL was very stable and could occur spontaneously. As Figure 8 shown in Figure 5g, Arg77, Leu258 and Val261 were very important for the binding of ATGL to the small peptide. The small peptide composed of essential amino acids itself was a nutritional fortifier and had a low cost. It was very promising to use the small peptide as an additive in carp meat products. Example

[0051] This embodiment provides an application of the ATGL inhibitor described in Embodiment 1 as an additive for carp meat products.

[0052] As can be seen from the above, lipid hydrolysis caused by endogenous enzymes is one of the most important factors reducing the quality of meat products. ATGL is responsible for hydrolyzing triglycerides and is the initiating enzyme leading to lipid hydrolysis. Therefore, the quality of meat products can be effectively guaranteed by inhibiting ATGL.

[0053] The present invention first cloned the N-terminal domain of carp ATGL from carp muscle tissue. Its length is 768 bp, encoding 256 amino acids, with a pI of 7.98 and a molecular weight of 27.7 kD. Secondly, the recombinant carp ATGL was successfully induced and expressed using the Escherichia coli prokaryotic expression system, and the changes in its enzyme activity at different temperatures and pH values were analyzed. Thirdly, the tertiary structure characteristics and catalytic mechanism of carp ATGL were analyzed. Then, the hydrolysis mechanism of carp ATGL was analyzed. Finally, a carp ATGL inhibitor was designed and synthesized, which is a small peptide composed of hydrophobic essential amino acids (Lys-Leu-Ile-Met-Val). This small peptide has high hydrophobicity and a suitable size, which is exactly misrecognized by the hydrophobic active groove of ATGL, forming a steric site barrier, preventing the binding of ATGL to triglycerides, and finally competitively inhibiting the catalytic activity of ATGL. At the same time, it was found that the interaction energy between the small peptide and ATGL can reach 62.7 Kj / moL, which indicates that the complex of the ATGL inhibitor and ATGL is very stable and can spontaneously prevent the binding of ATGL to triglycerides to inhibit the activity of ATGL. In addition to having a good inhibitory effect on ATGL, the ATGL inhibitor composed of essential amino acids is also a nutritional fortifier and has a low cost. It is very promising to use it as an additive for carp meat products.

[0054] In addition, the present invention can provide an important reference for the basic research of lipid hydrolases. At the same time, it can also provide a reference for quality control in the processing of carp meat.

[0055] The technical solutions provided by the present invention have been introduced in detail above. Specific examples are used herein to elaborate on the principle and implementation manner of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. An ATGL inhibitor, characterized in that: The amino acid sequence of the ATGL inhibitor is Lys-Leu-Ile-Met-Val.

2. A preparation method of the ATGL inhibitor according to claim 1, characterized in that, It includes the following steps: Clone the N-terminal domain of ATGL from muscle tissue; Successfully induce the expression of ATGL using the Escherichia coli prokaryotic expression system; Analyze the enzyme activity characteristics of ATGL at different pH values and temperatures; Analyze the tertiary structure characteristics and catalytic mechanism of ATGL by molecular dynamics simulation; Analyze the hydrolysis mechanism of ATGL; Design and synthesize the ATGL inhibitor.

3. The preparation method of the ATGL inhibitor according to claim 2, characterized in that: The specific steps of cloning the N-terminal domain of ATGL from muscle tissue include: Extract RNA from muscle and characterize it by agarose gel electrophoresis; Use a reverse transcription kit to reverse transcribe the extracted RNA into cDNA; Use primers for PCR amplification with cDNA as the template; After PCR amplification, sequence the amplification product; Resynthesize the ATGL sequence and ligate it into an expression vector; Transfer the recombinant plasmid into the Escherichia coli prokaryotic expression system.

4. The preparation method of the ATGL inhibitor according to claim 3, characterized in that: The primers are 5’-atgtttcccctcgactctccgt-3’ and 5’-taacagaccgttcttcttcag-3’.

5. The preparation method of the ATGL inhibitor according to claim 2, characterized in that: When analyzing the enzyme activity characteristics of ATGL, a high-sensitivity liquid scintillation counter is used. The substrate consists of 0.33 mM triolein, 0.45 mM phosphatidylcholine / phosphatidylinositol (3:1), and 0.5% defatted bovine serum albumin. [9,10-3H] triolein is used as a radioactive tracer.

6. The preparation method of the ATGL inhibitor according to claim 2, wherein: The specific steps of analyzing the tertiary structure characteristics and catalytic mechanism of ATGL by molecular dynamics simulation include: Use the Discovery Studio tool for molecular docking of ATGL and triglyceride molecules; Perform molecular dynamics simulations on the ATGL-triglyceride and ATGL-peptide complexes obtained from molecular docking. During the simulation process, extract the simulation files every 0.01 ns. Use a cutoff distance of 1 nm, periodic boundary conditions, and the leapfrog algorithm to calculate the radius of gyration, RMSF, root mean square distance, solvent accessible surface area, binding free energy, hydrogen bonds, and atomic distances.

7. Application of the ATGL inhibitor described in claim 1 in inhibiting the hydrolysis activity of ATGL.

8. Application of the ATGL inhibitor described in claim 1 in preparing a fresh-keeping agent for carp meat.

9. Application of the ATGL inhibitor described in claim 1 in preparing a meat product additive.

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