Process for preparing L-norvaline by enzyme method
Through the enzymatic preparation process, the reaction and treatment of complex enzymes is solved, and the problems of low yield, low purity and high cost in the existing L-nonvaline synthesis methods are achieved, and efficient and economical preparation of L-nonvaline is achieved.
Patent Information
- Application Number
- CN202510391724.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-31
- Publication Date
- 2025-05-23
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The existing L-nonvaline synthesis methods have low yields, cumbersome processes, and introduce more by-products, resulting in relatively low purity and high cost.
The process of preparing L-nonvaline by enzymatic method is used, and the reaction is carried out by preparing the base liquid, adding the first complex enzyme and the second complex enzyme, and after ultrafiltration, concentration, decolorization and crystallization, high-purity L-nonvaline is finally obtained.
The yield and purity of L-nonvaline are improved, the generation of side reaction products is reduced, the separation process is simplified, the production efficiency is improved, and the cost is reduced.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of L-norvaline, in particular to a process for preparing L-norvaline by an enzymatic method. Background Art
[0002] Hypertension refers to a clinical syndrome characterized by increased systemic arterial blood pressure (systolic and / or diastolic blood pressure) (systolic blood pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg), which may be accompanied by functional or organic damage to organs such as the heart, brain, and kidney. Hypertension is the most common chronic disease and the most important risk factor for cardiovascular and cerebrovascular diseases. The blood pressure of a normal person fluctuates within a certain range with changes in the internal and external environment. In the overall population, blood pressure levels gradually increase with age, with systolic blood pressure being more obvious, but diastolic blood pressure after the age of 50 shows a downward trend, and pulse pressure also increases accordingly. With the irregular life of modern people, hypertension has become an increasingly common chronic cardiovascular disease. Nearly 7 million patients die from hypertension and related cardiovascular diseases every year worldwide, and perdalpril is a common and relatively effective antihypertensive drug with a large market demand. L-norvaline is a key intermediate for the synthesis of perdalpril.
[0003] L-norvaline, also known as L-2-aminovaleric acid, has a molecular formula of C5H11NO2. It is an isomer of valine and a non-protein branched-chain amino acid. The finished product of L-norvaline is a white crystalline powder with a water solubility of 0.60 mol / L (30°C) and is soluble in ether and ethanol. Early reports found that L-norvaline is an amino acid component of natural antimicrobial peptides in Bacillus subtilis. Currently, large-scale industrial preparation mainly relies on chemical synthesis methods.
[0004] However, the existing synthesis methods have low yields, the synthesis process is relatively complicated, many by-products are introduced, and the separation is relatively troublesome, resulting in relatively low purity and significantly increased costs.
[0005] Based on this, we proposed a process for preparing L-norvaline by enzymatic method, hoping to solve the shortcomings of the existing technology. Summary of the invention
[0006] 1. Technical issues to be resolved In view of the deficiencies in the prior art, the present invention provides a process for preparing L-norvaline by an enzymatic method.
[0007] (II) Technical solution To achieve the above-mentioned purpose, the present invention provides the following technical solutions: A process for preparing L-norvaline by an enzymatic method comprises the following steps: (1) First, clean the reactor; (2) Preparation of base solution: Add DL-norvaline to water and stir at 500 r / min for 15-20 min to obtain DL-norvaline solution; (3) Add 120 g of DL-norvaline solution into the reactor, adjust the temperature in the reactor to 30-32°C, and keep warm for 10 minutes; (4) adding 18 g of the first complex enzyme into the reaction kettle, keeping the temperature, and stirring the reaction at 200 r / min for 24-26 h to obtain a first reaction solution; (5) Add 15 g of the second complex enzyme to the 100 g first reaction solution obtained above, keep warm, and stir to react for 10-14 hours to obtain a second reaction solution; (6) ultrafiltration treatment is performed on the second reaction liquid, followed by concentration to obtain crude L-norvaline; (7) Add the crude L-norvaline to water, adjust the temperature to 70-80°C, keep warm and stir for 20 minutes to obtain a mixed solution, and then decolorize it. After decolorization, concentrate and cool it to 22°C, crystallize it for 2 hours, centrifuge it, and filter it to obtain the initial L-norvaline product; (8) The L-norvaline obtained above is vacuum dried, and after drying, it is tested and packaged after passing the test.
[0008] As a further technical solution, the reactor cleaning in step (1) is as follows: Use high-pressure water through the nozzle to rinse the inner wall of the reactor for 20-30 minutes, and then dry it.
[0009] As a further technical solution, the pressure of the high-pressure water is 5-5.5 MPa.
[0010] As a further technical solution: the mass fraction of the DL-norvaline solution in step (2) is 20%.
[0011] As a further technical solution: in step (4), the first complex enzyme includes D-amino acid oxidase and catalase; The mixed mass ratio of D-amino acid oxidase and catalase is 1:2.
[0012] As a further technical solution, in step (5), the second complex enzyme includes formate dehydrogenase and NADPH; Among them, the mixed mass ratio of formate dehydrogenase and NADPH is 1:1.
[0013] As a further technical solution, in step (7), the crude L-norvaline and water are mixed in a mass ratio of 1-2:30.
[0014] As a further technical solution: the decolorization treatment in step (7) is performed using activated carbon, specifically: 45 g of activated carbon is added to 80 g of the mixed solution, the decolorization treatment is performed for 4 hours, and then the mixture is filtered.
[0015] The synthetic method of the present invention has the following route:
[0016] (III) Beneficial effects
[0017] Compared with the prior art, the present invention provides a process for preparing L-norvaline by enzymatic method, which has the following beneficial effects: Compared with the existing L-norvaline preparation method, the L-norvaline preparation process of the present invention has the characteristics of higher yield and better purity. The purity is improved by more than 10% compared with the existing L-norvaline preparation method, and it has more economic benefits.
[0018] The present invention adopts the first composite enzyme and the second composite enzyme to carry out auxiliary reaction, which can not only effectively improve the yield and conversion rate of L-norvaline, but also does not produce a large number of side reaction products, thereby reducing the separation process. Compared with the complex separation process introduced in the existing preparation method, the process of the present invention not only has higher production efficiency, but also can shorten the production time and save production costs.
[0019] In summary, the process of the present invention has the characteristics of high product purity, less environmental pollution, high conversion efficiency, and no accumulation of by-products. DETAILED DESCRIPTION
[0020] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0021] The following are specific embodiments: Example 1
[0022] A process for preparing L-norvaline by an enzymatic method comprises the following steps: (1) First, clean the reactor. The reactor cleaning is as follows: Use high-pressure water through a nozzle to rinse the inner wall of the reactor for 20 minutes, and then dry it; the pressure of the high-pressure water is 5MPa.
[0023] (2) Preparation of base solution: Add DL-norvaline to water and stir at 500 r / min for 15 min to obtain a DL-norvaline solution; the mass fraction of the DL-norvaline solution is 20%.
[0024] (3) Add 120 g of DL-norvaline solution into the reactor, adjust the temperature in the reactor to 30 °C, and keep it warm for 10 min; (4) adding 18 g of the first complex enzyme into the reaction kettle, keeping the temperature, and stirring the reaction at a speed of 200 r / min for 24 hours to obtain a first reaction solution; the first complex enzyme includes D-amino acid oxidase and catalase; Among them, the mixed mass ratio of D-amino acid oxidase and catalase is 1:2; (5) adding 15 g of the second complex enzyme to the 100 g first reaction solution obtained above, keeping warm, stirring and reacting for 10 hours to obtain a second reaction solution; the second complex enzyme comprises formate dehydrogenase and NADPH; Among them, the mixed mass ratio of formate dehydrogenase and NADPH is 1:1; (6) ultrafiltration treatment is performed on the second reaction liquid, followed by concentration to obtain crude L-norvaline; (7) Add crude L-norvaline to water, adjust the temperature to 70°C, keep warm and stir for 20 minutes to obtain a mixed solution, and then perform decolorization. After decolorization, concentrate and cool to 22°C, crystallize for 2 hours, centrifuge and filter to obtain L-norvaline. The mass ratio of crude L-norvaline to water is 1:30. The decolorization is performed by using activated carbon, specifically: adding 45g of activated carbon to 80g of the mixed solution, performing decolorization for 4 hours, and then filtering. (8) The L-norvaline obtained above is vacuum dried, and after drying, it is tested and packaged after passing the test. Example 2
[0025] A process for preparing L-norvaline by an enzymatic method comprises the following steps: (1) First, clean the reactor. The reactor cleaning is as follows: Use high-pressure water through a nozzle to rinse the inner wall of the reactor for 25 minutes, and then dry it; the pressure of the high-pressure water is 5.2MPa.
[0026] (2) Preparation of base solution: Add DL-norvaline to water and stir at 500 r / min for 15 min to obtain a DL-norvaline solution; the mass fraction of the DL-norvaline solution is 20%.
[0027] (3) Add 120 g of DL-norvaline solution into the reactor, adjust the temperature in the reactor to 31 °C, and keep it warm for 10 min; (4) adding 18 g of the first complex enzyme into the reaction kettle, keeping the temperature, and stirring the reaction at a speed of 200 r / min for 25 h to obtain a first reaction solution; the first complex enzyme includes D-amino acid oxidase and catalase; Among them, the mixed mass ratio of D-amino acid oxidase and catalase is 1:2; (5) adding 15 g of the second complex enzyme to the 100 g of the first reaction solution obtained above, keeping warm, stirring and reacting for 12 hours to obtain a second reaction solution; the second complex enzyme comprises formate dehydrogenase and NADPH; Among them, the mixed mass ratio of formate dehydrogenase and NADPH is 1:1; (6) ultrafiltration treatment is performed on the second reaction liquid, followed by concentration to obtain crude L-norvaline; (7) Add crude L-norvaline to water, adjust the temperature to 72°C, keep warm and stir for 20 minutes to obtain a mixed solution, and then perform decolorization. After decolorization, concentrate and cool to 22°C, crystallize for 2 hours, centrifuge and filter to obtain L-norvaline. The mass ratio of crude L-norvaline to water is 1.2:30. The decolorization is performed by using activated carbon, specifically: adding 45g of activated carbon to 80g of the mixed solution, performing decolorization for 4 hours, and then filtering. (8) The L-norvaline obtained above is vacuum dried, and after drying, it is tested and packaged after passing the test. Example 3
[0028] A process for preparing L-norvaline by an enzymatic method comprises the following steps: (1) First, clean the reactor. The reactor cleaning is as follows: Use high-pressure water through a nozzle to rinse the inner wall of the reactor for 25 minutes, and then dry it; the pressure of the high-pressure water is 5.4MPa.
[0029] (2) Preparation of base solution: Add DL-norvaline to water and stir at 500 r / min for 18 min to obtain a DL-norvaline solution; the mass fraction of the DL-norvaline solution is 20%.
[0030] (3) Add 120 g of DL-norvaline solution into the reactor, adjust the temperature in the reactor to 31 °C, and keep it warm for 10 min; (4) adding 18 g of the first complex enzyme into the reaction kettle, keeping the temperature, and stirring the reaction at a speed of 200 r / min for 25 h to obtain a first reaction solution; the first complex enzyme includes D-amino acid oxidase and catalase; Among them, the mixed mass ratio of D-amino acid oxidase and catalase is 1:2; (5) adding 15 g of the second complex enzyme to the 100 g of the first reaction solution obtained above, keeping warm, stirring and reacting for 13 hours to obtain a second reaction solution; the second complex enzyme comprises formate dehydrogenase and NADPH; Among them, the mixed mass ratio of formate dehydrogenase and NADPH is 1:1; (6) ultrafiltration treatment is performed on the second reaction liquid, followed by concentration to obtain crude L-norvaline; (7) Add crude L-norvaline to water, adjust the temperature to 75°C, keep warm and stir for 20 minutes to obtain a mixed solution, and then perform decolorization. After decolorization, concentrate and cool to 22°C, crystallize for 2 hours, centrifuge and filter to obtain L-norvaline. The mass ratio of crude L-norvaline to water is 1.6:30. The decolorization is performed by using activated carbon, specifically: adding 45g of activated carbon to 80g of the mixed solution, performing decolorization for 4 hours, and then filtering. (8) The L-norvaline obtained above is vacuum dried, and after drying, it is tested and packaged after passing the test. Example 4
[0031] A process for preparing L-norvaline by an enzymatic method comprises the following steps: (1) First, clean the reactor. The reactor cleaning is as follows: Use high-pressure water through a nozzle to rinse the inner wall of the reactor for 30 minutes, and then dry it; the pressure of the high-pressure water is 5.5MPa.
[0032] (2) Preparation of base solution: Add DL-norvaline to water and stir at 500 r / min for 20 min to obtain a DL-norvaline solution; the mass fraction of the DL-norvaline solution is 20%.
[0033] (3) Add 120 g of DL-norvaline solution into the reactor, adjust the temperature in the reactor to 32 °C, and keep it warm for 10 min; (4) adding 18 g of a first complex enzyme into the reaction kettle, keeping the temperature, and stirring the reaction at a speed of 200 r / min for 26 h to obtain a first reaction solution; the first complex enzyme includes D-amino acid oxidase and catalase; Among them, the mixed mass ratio of D-amino acid oxidase and catalase is 1:2; (5) adding 15 g of the second complex enzyme to the 100 g of the first reaction solution obtained above, keeping warm, stirring and reacting for 14 hours to obtain a second reaction solution; the second complex enzyme comprises formate dehydrogenase and NADPH; Among them, the mixed mass ratio of formate dehydrogenase and NADPH is 1:1; (6) ultrafiltration treatment is performed on the second reaction liquid, followed by concentration to obtain crude L-norvaline; (7) Add crude L-norvaline to water, adjust the temperature to 80°C, keep warm and stir for 20 minutes to obtain a mixed solution, and then perform decolorization. After decolorization, concentrate and cool to 22°C, crystallize for 2 hours, centrifuge and filter to obtain L-norvaline. The mass ratio of crude L-norvaline to water is 2:30. The decolorization is performed by using activated carbon, specifically: adding 45g of activated carbon to 80g of the mixed solution, performing decolorization for 4 hours, and then filtering. (8) The L-norvaline obtained above is vacuum dried, and after drying, it is tested and packaged after passing the test.
[0034] test: The enzymatic activity of each enzyme in the embodiment was detected: Table 1
[0035] It can be seen from Table 1 that the enzyme activity of each enzyme in the present invention is relatively high, which can effectively improve the reaction efficiency.
[0036] The conversion rates of DL-norvaline in the example methods are compared, as shown in Table 2: Table 2
[0037] It can be seen from Table 2 that the method of the present invention can significantly increase the conversion rate of DL-norvaline to L-norvaline.
[0038] The L-norvaline yield of the embodiment is compared: Table 3
[0039] As can be seen from Table 3, the method of the present invention has a higher L-norvaline yield.
[0040] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A process for preparing L-norvaline by enzymatic method, characterized in that: The following steps are involved: (1) First, clean the reactor; (2) Preparation of base solution: Add DL-norvaline to water and stir at 500 r / min for 15-20 min to obtain DL-norvaline solution; (3) Add 120 g of DL-norvaline solution into the reactor, adjust the temperature in the reactor to 30-32°C, and keep warm for 10 minutes; (4) adding 18 g of the first complex enzyme into the reaction kettle, keeping the temperature, and stirring the reaction at 200 r / min for 24-26 h to obtain a first reaction solution; (5) Add 15 g of the second complex enzyme to the 100 g first reaction solution obtained above, keep warm, and stir to react for 10-14 hours to obtain a second reaction solution; (6) ultrafiltration treatment is performed on the second reaction liquid, followed by concentration to obtain crude L-norvaline; (7) Add the crude L-norvaline to water, adjust the temperature to 70-80°C, keep warm and stir for 20 minutes to obtain a mixed solution, and then decolorize it. After decolorization, concentrate and cool it to 22°C, crystallize it for 2 hours, centrifuge it, and filter it to obtain the initial L-norvaline product; (8) The L-norvaline obtained above is vacuum dried, and after drying, it is tested and packaged after passing the test.
2. The process for preparing L-norvaline by enzymatic method according to claim 1, characterized in that: The cleaning of the reactor in step (1) is as follows: Use high-pressure water through the nozzle to rinse the inner wall of the reactor for 20-30 minutes, and then dry it.
3. The process for preparing L-norvaline by enzymatic method according to claim 2, characterized in that: The pressure of the high-pressure water is 5-5.5 MPa.
4. The process for preparing L-norvaline by enzymatic method according to claim 1, characterized in that: The mass fraction of the DL-norvaline solution in step (2) is 20%.
5. The process for preparing L-norvaline by enzymatic method according to claim 1, characterized in that: Step (4) the first complex enzyme includes D-amino acid oxidase and catalase; The mixed mass ratio of D-amino acid oxidase and catalase is 1:
2.
6. The process for preparing L-norvaline by enzymatic method according to claim 1, characterized in that: Step (5) the second complex enzyme comprises formate dehydrogenase and NADPH; Among them, the mixed mass ratio of formate dehydrogenase and NADPH is 1:
1.
7. The process for preparing L-norvaline by enzymatic method according to claim 1, characterized in that: In step (7), the crude L-norvaline and water are mixed in a mass ratio of 1-2:
30.
8. The process for preparing L-norvaline by enzymatic method according to claim 1, characterized in that: The decolorization treatment in step (7) is performed using activated carbon, specifically, 45 g of activated carbon is added to 80 g of the mixed solution, the decolorization treatment is performed for 4 hours, and then filtering is performed.