Preparation method of stable natural alanine aminotransferase
By using pig heart as raw material and combining ammonium sulfate precipitation and anion exchange chromatography, a highly active and stable alanine aminotransferase was prepared, solving the problems of low activity and difficulty in large-scale production in existing technologies, and realizing efficient preparation and low-cost enzyme preparation production.
Patent Information
- Application Number
- CN202510910051.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-02
- Publication Date
- 2025-10-31
AI Technical Summary
Existing technologies for preparing serum alanine aminotransferase suffer from low activity and low recovery rate, making it difficult to achieve large-scale production.
Using pig heart as raw material, impurities and large molecular weight proteins were removed by ammonium sulfate precipitation, and a highly active and stable alanine aminotransferase was prepared by combining anion exchange chromatography and liquid protective agent.
The prepared alanine aminotransferase has high activity, good stability, and low cost, making it suitable for industrial production and with broad clinical and in vitro diagnostic applications.
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Figure CN120866261A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bioengineering technology, and in particular to a method for preparing a stable natural alanine aminotransferase. Background Technology
[0002] Alanine aminotransferase (ALT), also known as glutamic-pyruvic transaminase (GPT), contains 496 amino acids, has a half-life of approximately 2 days, and is encoded by the ALT gene, located on the long arm of chromosome 8, 19. ALT is a transaminase that mainly accumulates in the cytoplasm of hepatocytes, and its function is to catalyze the aminotransfer reaction between alanine and α-keto acids.
[0003] Alanine aminotransferase (ALT) is widely found in various tissues and organs of the human body, including the liver, kidneys, myocardium, and brain, with the highest concentration in the liver. ALT plays a crucial role in maintaining normal glucose and amino acid metabolism and is closely related to diseases such as liver disease, diabetes, and coronary heart disease, thus having important indicative value in clinical practice.
[0004] Elevated serum alanine aminotransferase (ALT) is an important indicator of hepatocellular damage. ALT levels are considered a sensitive indicator of hepatocellular injury; the release of just 1 / 1000 of ALT from hepatocytes into the bloodstream is sufficient to double serum ALT levels. In acute liver injury, serum ALT levels rise sharply before the onset of clinical symptoms such as cholelithiasis and cholecystitis. Furthermore, fatigue, strenuous exercise, alcohol consumption, and even emotional factors can alter hepatocyte membrane permeability, leading to elevated serum ALT levels.
[0005] Currently, a few scholars are studying the preparation process of serum alanine aminotransferase using animal viscera. For example, they use methods such as ammonium sulfate precipitation, EDTA to remove impurities and proteins, and cation exchange chromatography for coarse separation, followed by affinity chromatography and gel filtration for fine separation to obtain enzymes with high purity. However, these methods have problems such as low activity and low recovery rate, which are not conducive to large-scale production. Summary of the Invention
[0006] The purpose of this invention is to provide a method for preparing a stable natural alanine aminotransferase, with the aim of producing an inexpensive, active, and stable alanine aminotransferase.
[0007] To achieve the above objectives, the present invention provides a method for preparing a stable natural alanine aminotransferase, comprising the following steps;
[0008] Pig heart raw material was obtained, fascia and fat were removed, a certain amount of first buffer solution was added and the mixture was crushed in a meat grinder to obtain the first enzyme extract.
[0009] Add a certain amount of ammonium sulfate to the first enzyme extract, adjust the pH value using the second buffer solution, centrifuge, and take the supernatant to obtain the second enzyme extract;
[0010] The second enzyme extract was placed in an ice-salt bath, and solid ammonium sulfate was added for salting out precipitation. The precipitate was collected by centrifugation and dialyzed with the second buffer solution to obtain the third enzyme extract.
[0011] The third enzyme extract was purified by ion exchange column, eluted sequentially by third buffer, fourth buffer and fifth buffer, and then dialyzed with sixth buffer to obtain the fourth enzyme extract.
[0012] A liquid protectant was added to the fourth enzyme extract, and the mixture was stirred to obtain a highly active and stable alanine aminotransferase.
[0013] The first buffer solution comprises sodium chloride, potassium chloride, potassium dihydrogen phosphate, and disodium hydrogen phosphate. The concentration of sodium chloride is 130-140 mmol / L, the concentration of potassium chloride is 2-3 mmol / L, the concentration of potassium dihydrogen phosphate is 7-10 mmol / L, the concentration of disodium hydrogen phosphate is 1-1.5 mmol / L, and the pH of the first buffer solution is 6.2-6.6.
[0014] The second buffer solution is a 0.5-0.6 mmol / L sodium hydroxide solution.
[0015] The third, fourth, fifth, and sixth buffer solutions comprise Tris-HCl, NaCl, and EDTA. Specifically, the third buffer solution has a Tris-HCl concentration of 10-15 mmol / L, a NaCl concentration of 5-7 mmol / L, and a pH of 7.8-8.2; the fourth buffer solution has a Tris-HCl concentration of 15-20 mmol / L, a NaCl concentration of 50-70 mmol / L, and a pH of 7.8-8.2; the fifth buffer solution has a Tris-HCl concentration of 15-20 mmol / L, a NaCl concentration of 100-120 mmol / L, and a pH of 7.8-8.2; and the sixth buffer solution has a Tris-HCl concentration of 15-20 mmol / L, a NaCl concentration of 160-180 mmol / L, and a pH of 7.8-8.2.
[0016] The ion exchange column includes DEAE and 30Q anion exchange column packing materials.
[0017] The liquid protective agent comprises sugars, salts, surfactants, polyols, and polymers. The sugars include any one of lactose, sucrose, and trehalose, with an addition amount of 3-4%. The salts include any one of sodium chloride and potassium chloride, with an addition amount of 160-180 mmol / L. The surfactants include any one of glycerol and Triton X-100, with an addition amount of 5-7%. The polyols include any one of sorbitol and mannitol, with an addition amount of 0.1-0.15%. The polymers include any one of polyvinylpyrrolidone, PEG, and polysorbate, with an addition amount of 0.08-0.12%.
[0018] This invention discloses a method for preparing a stable natural alanine aminotransferase. The method involves obtaining pig heart as raw material, removing fascia and fat, adding a certain amount of a first buffer solution, and grinding the mixture in a meat grinder to obtain a first enzyme extract. A certain amount of ammonium sulfate is added to the first enzyme extract, the pH is adjusted using a second buffer solution, centrifuged, and the supernatant is collected to obtain a second enzyme extract. The second enzyme extract is placed in an ice-salt bath, and solid ammonium sulfate is added for salting-out precipitation. The precipitate is collected by centrifugation and dialyzed against the second buffer solution to obtain a third enzyme extract. The third enzyme extract is purified by passing it sequentially through a third buffer solution, a fourth buffer solution, and a fifth buffer solution. The enzyme is eluted with buffer solution, then dialyzed with a sixth buffer solution to obtain the fourth enzyme extract. A liquid protectant is added to the fourth enzyme extract, and the mixture is stirred to obtain a highly active and stable alanine aminotransferase. This method uses pig heart as raw material, removes impurities and large molecular weight proteins by salting out with ammonium sulfate, and then performs anion exchange chromatography with the addition of a liquid protectant to obtain a highly active and stable alanine aminotransferase. The prepared alanine aminotransferase has high activity, good stability, low cost, high process reproducibility, and can be industrially produced. It has potential and wide application value in clinical diagnosis and in vitro diagnostics. Attached Figure Description
[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0020] Figure 1 This is a flowchart of a method for preparing a stable natural alanine aminotransferase provided by the present invention. Detailed Implementation
[0021] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.
[0022] Please see Figure 1 This invention provides a method for preparing a stable natural alanine aminotransferase, comprising the following steps;
[0023] S1 Obtain pig heart raw material, remove fascia and fat, add a certain amount of first buffer solution and grind in a meat grinder to obtain first enzyme extract;
[0024] In this embodiment of the invention, fresh pig heart is taken, the fascia and fat are removed, and the blood is washed away with the first buffer solution (DK1). The heart is chopped, weighed to 100g, and 200ml of DK1 is added. The heart is then placed in a meat grinder and ground. After grinding, the heart is centrifuged at 3500-5000rpm / min for 20-30min at 4℃. The supernatant is collected, the precipitate is discarded, and the first enzyme extract (crude enzyme extract A) is obtained. The activity of the crude enzyme in the supernatant is tested using a fully automated biochemical analyzer. The first buffer solution comprises sodium chloride, potassium chloride, potassium dihydrogen phosphate, and disodium hydrogen phosphate. The concentration of sodium chloride is 130-140mmol / L, the concentration of potassium chloride is 2-3mmol / L, the concentration of potassium dihydrogen phosphate is 7-10mmol / L, the concentration of disodium hydrogen phosphate is 1-1.5mmol / L, and the pH of the first buffer solution is 6.2-6.6.
[0025] S2 adds a certain amount of ammonium sulfate to the first enzyme extract, adjusts the pH value using the second buffer, centrifuges, and takes the supernatant to obtain the second enzyme extract;
[0026] In this embodiment of the invention, 32g of solid ammonium sulfate is slowly added to the first enzyme extract in small amounts multiple times until the corresponding saturation is reached. While adding, the mixture is stirred with a magnetic stirrer until completely dissolved. The pH value is adjusted using a second buffer solution (DK2). The solution is then placed in a refrigerator at 2-8℃ and allowed to stand for 2 hours. After standing, the solution is removed and centrifuged at 8000 rpm for 20 minutes at 4℃. The supernatant is discarded, and the precipitate is collected. The precipitate is redissolved with DK1, and the pH value is adjusted with DK2 solution. The solution is then placed in a water bath and heated to 55℃. After maintaining this temperature for a certain period of time, the solution is removed and quickly placed in an ice-water bath to cool for 15 minutes. Subsequently, the solution is centrifuged at 8000 rpm for 20 minutes at 4℃. The supernatant is collected to obtain the second enzyme extract (enzyme extract B). The second buffer solution is a 0.5-0.6 mmol / L sodium hydroxide solution.
[0027] S3. The second enzyme extract was placed in an ice-salt bath, and solid ammonium sulfate was added for salting out precipitation. The precipitate was collected by centrifugation and dialyzed with the second buffer solution to obtain the third enzyme extract.
[0028] In this embodiment of the invention, ammonium sulfate was slowly added to the second enzyme extract in an ice-water bath until it reached a certain saturation. The mixture was stirred with a magnetic stirrer until completely dissolved, and the pH was adjusted using DK2. The solution was then placed in a refrigerator at 2-8°C and allowed to stand for 2 hours. After standing, the solution was removed and centrifuged at 8000 r / min for 20 minutes at 4°C. The supernatant was discarded, and the precipitate was collected. The precipitate was reconstituted with 100 ml of DK3 and placed in a MW14000 dialysis bag. Dialysis was performed using 5 L of DK3. The third buffer solution (DK3) of the dialysis solution was replaced every 4 hours. This process was repeated 4 times to obtain the third enzyme extract (enriched enzyme extract C).
[0029] S4 The third enzyme extract is purified by passing it through an ion exchange column, eluted sequentially by the third buffer, the fourth buffer and the fifth buffer, and then dialyzed with the sixth buffer to obtain the fourth enzyme extract;
[0030] In this embodiment of the invention, 50 ml of anion exchange packing material was packed into the column. After packing, the column was first washed with 250 ml of 2M NaCl, then with 250 ml of purified water, and finally equilibrated with 300 ml of DK3 until the conductivity of the eluent was close to that of DK3 (within ±10%). The column was then connected to a nucleic acid and protein detector and zeroed. The dialyzed sample was slowly added to the equilibrated ion exchange column. During the sample loading process, when the protein detector reading rose, the permeate was collected in separate tubes. When the raw material solution had just entered the packing material, 3-5 times the volume of the packing material was added to continue equilibration until the detector reading was close to 0 or remained unchanged for 2 minutes. When the breakthrough peak appeared during the sample loading process, collection began. When the breakthrough peak reached its bottom, collection was stopped. After collection, 200 ml of the first solution was obtained.
[0031] First elution: Elute with 3-5 column volumes of the fourth buffer (DK4). Start collecting in separate bottles when the peak appears. Stop collecting when the detector reading is close to 0 or when there is no change for 2 minutes (the protein detector will show 3 breakthrough peaks during the entire collection process, and the main component of the third peak is ALT. A total of 100 ml of sample was collected).
[0032] Second elution: Elute with 3-5 column volumes of the fifth buffer (DK5). Start collecting when the peak appears and stop collecting when the detector reading is close to 0 or when there is no change for 2 minutes. After collection, obtain 300 ml of ALT fraction.
[0033] The ALT collected from the first and second elutions was combined to obtain 400 ml of enzyme extract. Using an ultrafiltration concentrator, the extract was centrifuged at 2000 rpm and 4°C for 20 minutes, repeated three times, to obtain 100 ml of concentrated enzyme extract. This was then transferred to an MW14000 dialysis bag and dialyzed using 5 L of sixth buffer (DK6) every 8 hours for a total of three times. After centrifugation and filtration, the fourth enzyme extract (enzyme extract D) was obtained.
[0034] The third, fourth, fifth, and sixth buffer solutions comprise Tris-HCl, NaCl, and EDTA. Specifically, the third buffer solution has a Tris-HCl concentration of 10-15 mmol / L and a NaCl concentration of 5-7 mmol / L, with a pH of 7.8-8.2; the fourth buffer solution has a Tris-HCl concentration of 15-20 mmol / L and a NaCl concentration of 50-70 mmol / L, with a pH of 7.8-8.2; the fifth buffer solution has a Tris-HCl concentration of 15-20 mmol / L and a NaCl concentration of 100-120 mmol / L, with a pH of 7.8-8.2; and the sixth buffer solution has a Tris-HCl concentration of 15-20 mmol / L and a NaCl concentration of 160-180 mmol / L, with a pH of 7.8-8.2. The ion exchange column comprises DEAE and 30Q anion exchange column packing materials.
[0035] S5. Add a liquid protectant to the fourth enzyme extract, mix, and obtain a highly active and stable alanine aminotransferase.
[0036] In this embodiment of the invention, 50 ml of the fourth enzyme extract was taken, 50 ml of liquid preservative was added, and after shaking well, the alanine aminotransferase activity was tested three times using a fully automated biochemical analyzer. The average value was recorded as the zero-time data, and the extract was aliquoted into lyophilized bottles, 1 ml per bottle, and stored in sealed containers at 2-8℃ and -20℃ respectively. The extracts were taken out on days 30, 90, 180, and 360, and the alanine aminotransferase activity was tested three times using a fully automated biochemical analyzer. The average value was recorded and compared with the zero-time test data, with a deviation of less than 10%. Detailed data are shown in Tables 1 and 2:
[0037] Table 1
[0038]
[0039] Table 2
[0040]
[0041] The liquid protective agent comprises sugars, salts, surfactants, polyols, and polymers. The sugars include any one of lactose, sucrose, and trehalose, with an addition amount of 3-4%. The salts include any one of sodium chloride and potassium chloride, with an addition amount of 160-180 mmol / L. The surfactants include any one of glycerol and Triton X-100, with an addition amount of 5-7%. The polyols include any one of sorbitol and mannitol, with an addition amount of 0.1-0.15%. The polymers include any one of polyvinylpyrrolidone, PEG, and polysorbate, with an addition amount of 0.08-0.12%.
[0042] The above-disclosed method is merely a preferred embodiment of the preparation method of a stable natural alanine aminotransferase of the present invention. Of course, it should not be construed as limiting the scope of the present invention. Those skilled in the art can understand that implementing all or part of the above-described embodiments and making equivalent changes in accordance with the claims of the present invention are still within the scope of the invention.
Claims
1. A method for preparing a stable natural alanine aminotransferase, characterized in that, Includes the following steps; Pig heart raw material was obtained, fascia and fat were removed, a certain amount of first buffer solution was added and the mixture was crushed in a meat grinder to obtain the first enzyme extract. Add a certain amount of ammonium sulfate to the first enzyme extract, adjust the pH value using the second buffer solution, centrifuge, and take the supernatant to obtain the second enzyme extract; The second enzyme extract was placed in an ice-salt bath, and solid ammonium sulfate was added for salting out precipitation. The precipitate was collected by centrifugation and dialyzed with the second buffer solution to obtain the third enzyme extract. The third enzyme extract was purified by ion exchange column, eluted sequentially by third buffer, fourth buffer and fifth buffer, and then dialyzed with sixth buffer to obtain the fourth enzyme extract. A liquid protectant was added to the fourth enzyme extract, and the mixture was stirred to obtain a highly active and stable alanine aminotransferase.
2. The method for preparing stable natural alanine aminotransferase as described in claim 1, characterized in that... ； The first buffer solution comprises sodium chloride, potassium chloride, potassium dihydrogen phosphate, and disodium hydrogen phosphate. The concentration of sodium chloride is 130-140 mmol / L, the concentration of potassium chloride is 2-3 mmol / L, the concentration of potassium dihydrogen phosphate is 7-10 mmol / L, the concentration of disodium hydrogen phosphate is 1-1.5 mmol / L, and the pH of the first buffer solution is 6.2-6.
6.
3. The method for preparing stable natural alanine aminotransferase as described in claim 1, characterized in that... ； The second buffer solution is a 0.5-0.6 mmol / L sodium hydroxide solution.
4. The method for preparing stable natural alanine aminotransferase as described in claim 1, characterized in that... ； The third, fourth, fifth, and sixth buffer solutions comprise Tris-HCl, NaCl, and EDTA. Specifically, the third buffer solution has a Tris-HCl concentration of 10-15 mmol / L, a NaCl concentration of 5-7 mmol / L, and a pH of 7.8-8.2; the fourth buffer solution has a Tris-HCl concentration of 15-20 mmol / L, a NaCl concentration of 50-70 mmol / L, and a pH of 7.8-8.2; the fifth buffer solution has a Tris-HCl concentration of 15-20 mmol / L, a NaCl concentration of 100-120 mmol / L, and a pH of 7.8-8.2; and the sixth buffer solution has a Tris-HCl concentration of 15-20 mmol / L, a NaCl concentration of 160-180 mmol / L, and a pH of 7.8-8.
2.
5. The method for preparing stable natural alanine aminotransferase as described in claim 1, characterized in that... ； The ion exchange column includes DEAE and 30Q anion exchange column packing materials.
6. The method for preparing stable natural alanine aminotransferase as described in claim 1, characterized in that... ； The liquid protective agent comprises sugars, salts, surfactants, polyols, and polymers. The sugars include any one of lactose, sucrose, and trehalose, added at 3-4%. The salts include any one of sodium chloride and potassium chloride, added at 160-180 mmol / L. The surfactants include any one of glycerol and Triton X-100, added at 5-7%. The polyols include any one of sorbitol and mannitol, added at 0.1-0.15%. The polymers include any one of polyvinylpyrrolidone, PEG, and polysorbate, added at 0.08-0.12%.