CN108220351B - Method for preparing L-arginine-alpha-ketoglutaric acid by biological enzyme method - Google Patents
Method for preparing L-arginine-alpha-ketoglutaric acid by biological enzyme method Download PDFInfo
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Abstract
The invention discloses a method for preparing L-arginine-alpha-ketoglutaric acid by a biological enzyme method, which comprises the steps of catalyzing glutamic acid by adopting glutamate oxidase to generate alpha-ketoglutaric acid, adding arginine to prepare L-arginine-alpha-ketoglutaric acid (2: 1), and performing ceramic membrane sterilization, ultrafiltration membrane protein and impurity removal, activated carbon decoloration, vacuum concentration and spray drying to extract and refine.
Description
Technical Field
The invention relates to a method for producing refined ketone, in particular to a method for preparing L-arginine-alpha-ketoglutaric acid by a biological enzyme method.
Background
The L-arginine-alpha-ketoglutaric acid not only has the physiological functions of arginine and alpha-ketoglutaric acid, but also can be used as a functional nutrition enhancer and a liver-protecting drug to be applied clinically, mainly applied to strengthening physique, promoting rapid growth and wound recovery of muscles, promoting absorption and metabolism of liver cells on nutrition, and enhancing energy and physical strength. At present, the quantity of the protamine as a functional food in the international market is increasing, and the protamine and the guanone are taken as important matters in the fields of medicine, nutrition and sports. Two main raw materials of the refined ketone are arginine and alpha-ketoglutaric acid, wherein the arginine is easy to obtain due to mature large-scale production process, but the alpha-ketoglutaric acid is difficult to obtain. The existing preparation method of alpha-ketoglutaric acid mainly comprises a fermentation method and a chemical synthesis method, wherein during the production of the alpha-ketoglutaric acid by the fermentation method, a lot of byproducts such as pyruvic acid, fumaric acid, malic acid and the like are generated, the fermentation period is long, and therefore, a product with high purity is difficult to obtain. At present, alpha-ketoglutaric acid is mainly produced by a chemical method, but a large amount of highly toxic substances are used in the production, so that the production pollution is large, the risk is high, the process is complex, and the cost is high.
Disclosure of Invention
In order to make up the defects of the prior art, the invention provides a method for preparing L-arginine-alpha-ketoglutaric acid (2: 1) by a biological enzyme method, the method directly adds arginine into a conversion solution to synthesize the L-arginine-alpha-ketoglutaric acid (2: 1), the process is simple, the cost is low, the produced L-arginine-alpha-ketoglutaric acid has few impurities and little pollution, and the method is very favorable for large-scale production under the condition that the current environmental protection pressure is increased day by day.
The invention is realized by the following technical scheme:
a method for preparing L-arginine-alpha-ketoglutaric acid by a biological enzyme method is characterized in that: the glutamic acid oxidase is adopted to catalyze glutamic acid to generate alpha-ketoglutaric acid, arginine is added to prepare L-arginine-alpha-ketoglutaric acid (2: 1), and the method has the advantages of simple process, good quality and small pollution.
The invention relates to a method for preparing L-arginine-alpha-ketoglutaric acid by a biological enzyme method, which comprises the following steps:
(1) preparing a reaction solution: adding glutamic acid and arginine into pure water to prepare a reaction solution, and adjusting the pH value of the reaction solution to 6.6-7;
(2) enzyme reaction: adding glutamate oxidase and catalase into the reaction solution for reaction, wherein the reaction temperature is 38-40 ℃, the reaction time is 8-10h, the reaction pH value is controlled to be 6.5-7.0, and the dissolved oxygen is controlled to be 5-30%;
(3) and (3) filtering and sterilizing by using a ceramic membrane: after the reaction is finished, filtering and sterilizing the reaction solution by a ceramic membrane, and dialyzing the trapped solution by pure water with the trapped volume being 2-4 times that of the trapped solution;
(4) filtering protein by using an ultrafiltration membrane to remove impurities: filtering with ceramic membrane to obtain clear solution, passing through ultrafiltration membrane with aperture molecular weight of 3000, and dialyzing the trapped solution with pure water with 2-4 times of trapped volume;
(5) activated carbon decolorization: decolorizing the clear liquid obtained by ultrafiltration membrane filtration with active carbon at 60-65 deg.C for 20-30min, and adding active carbon in an amount of 3-10 g/L.
(6) And (3) concentrating under reduced pressure: concentrating the decolorized product at-0.08-0.1 mpa until the concentration of L-arginine-alpha-ketoglutaric acid is 40% -60%;
(7) spray drying: spray drying to obtain L-arginine-alpha-ketoglutaric acid product.
Preferably, the concentration of glutamic acid and arginine in the reaction solution in the step (1) is 100g/l and 100g/l, respectively.
Preferably, the end point of the enzymatic reaction in step (2) is determined by determining whether or not glutamic acid remains, and the reaction is terminated after the glutamic acid has completely reacted.
Further, the content of glutamate oxidase in the reaction solution in the step (2) is 3-5U/ml, and the content of catalase is 200-400U/ml.
Preferably, the bacterial sludge filtered by the ceramic membrane in the step (3) is dried to be used as a feed additive or a raw material of feed protein.
Preferably, the retentate dialyzed in the step (4) is applied to the next batch of synthetic fluid and passes through the ceramic membrane.
Preferably, the transmittance T of the solution decolorized in the step (5)420nm≥95%。
Preferably, the concentration temperature in step (6) is 65 ℃.
Preferably, the conductivity of the pure water in the steps (1), (3) and (4) is less than or equal to 10.
The invention has the beneficial effects that:
(1) the alpha-ketoglutaric acid is prepared from glutamic acid by enzyme catalysis, so that the raw material cost is effectively reduced.
(2) No other impurities and inorganic salts are added in the synthesis process of the L-arginine-alpha-ketoglutaric acid, so that the product quality of the L-arginine-alpha-ketoglutaric acid is effectively improved; the content of L-arginine-alpha-ketoglutaric acid produced by the method reaches more than 99.0 percent, and the quality standard of the L-arginine-alpha-ketoglutaric acid is met.
(3) The ceramic membrane trapped fluid is used for preparing feed protein, and the ultrafiltration membrane trapped fluid is recycled, so that the discharge of waste water is reduced.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments thereof to assist those skilled in the art in providing a more complete, accurate and thorough understanding of the inventive concept and aspects thereof, and the scope of the present invention includes, but is not limited to, the following examples, and any modifications in the details and form of the technical aspects thereof that fall within the spirit and scope of the present application are intended to be included therein.
Example 1
A method for preparing L-arginine-alpha-ketoglutaric acid by a biological enzyme method comprises the following steps:
(1) preparation of reaction solution
Carrying out dry ethanol 25m high-pressure ethanol 50m under high-pressure ethanol, adding 3000kg glutamic acid and 3650kg arginine to adjust pH to 6.9, and carrying out dry ethanol 30m under high-pressure ethanol.
(2) Enzyme reaction
Adding 3.5U/mL glutamate oxidase and 200U/mL catalase into the reaction solution to start reaction, controlling the dissolved oxygen at 10%, controlling the temperature at 39 +/-0.5 ℃ and the pH at 6.9, detecting no glutamic acid by using a biosensor after 10 hours of reaction, and supplementing 3452kg arginine to obtain the reaction solution of L-arginine-alpha-ketoglutaric acid (2: 1).
(3) Ceramic membrane filtration sterilization
After the reaction is finished, the reaction solution firstly passes through a ceramic membrane, and the trapped solution is dialyzed by pure water with the length of 4 m; the intercepted liquid after dialysis is dried and used as feed protein, and the generation of waste water is reduced.
(4) Removing impurities by ultrafiltration membrane
Passing the clear liquid obtained by the ceramic membrane through an ultrafiltration membrane, wherein the molecular weight of the ultrafiltration membrane is 3000, and dialyzing the trapped fluid by pure water obtained by 2m high-speed cropping; the intercepted liquid after dialysis is applied to the next batch of synthetic liquid to pass through the ceramic membrane, so that the cost is saved, and the generation of waste water is avoided.
(5) Decolorizing with activated carbon
The decolorizing temperature is 65 ℃, the adding amount of the active carbon is 150kg, and the decolorizing time is 30 min. Light transmission T of decolorized solution420nm=98.5%。
(6) Concentrating under reduced pressure
Concentrating at 65 deg.C under-0.1 mpa, and concentrating to 16 m.
(7) Spray drying
Spray drying to obtain 10328kg of qualified product of L-arginine-alpha-ketoglutaric acid (2: 1), the content is 99.5%, and the yield is 95.4%.
Example 2
A method for preparing L-arginine-alpha-ketoglutaric acid by a biological enzyme method comprises the following steps:
(1) preparation of reaction solution
Carrying out dry ethanol 50m, carrying out dry ethanol 25m, carrying out dry ethanol 3640kg, carrying out dry ethanol 30m, carrying out dry ethanol solution, carrying out dry ethanol and carrying out dry ethanol solution carrying out dry ethanol carrying out thin film carrying out dry ethanol carrying out thin film carrying out dry ethanol carrying out thin film carrying out dry ethanol carrying out thin film carrying.
(2) Enzyme reaction
Adding 5U/mL glutamate oxidase and 250U/mL catalase into the reaction solution to start reaction, controlling the dissolved oxygen by 20 percent, controlling the temperature by 39.5 +/-0.5 ℃, controlling the pH value to be 6.9, detecting no glutamic acid by using a biosensor after reacting for 8 hours, and supplementing 3462kg of arginine to obtain the reaction solution of L-arginine-alpha-ketoglutaric acid (2: 1).
(3) Ceramic membrane filtration sterilization
After the reaction is finished, the reaction solution firstly passes through a ceramic membrane, and the trapped solution is dialyzed by pure water with the diameter of 6 m; drying the dialyzed trapped fluid to be used as feed protein.
(4) Removing impurities by ultrafiltration membrane
Passing the clear liquid obtained by the ceramic membrane through an ultrafiltration membrane, wherein the molecular weight of the ultrafiltration membrane is 3000, and dialyzing the trapped fluid by pure water obtained by 3m high-speed cropping; the retention solution after dialysis is applied to the next batch of synthetic fluid to pass through the ceramic membrane.
(5) Decolorizing with activated carbon
Decolorizing at 60 deg.C with addition of 180kg of activated carbon for 25min, and collecting the decolorized solution with transmittance T420nm=98.1%。
(6) Concentrating under reduced pressure
The concentration temperature was 65 ℃ and the pressure was-0.1 mpa. And (5) carrying out concentration to 15 m.
(7) Spray drying
10360kg of qualified L-arginine-alpha-ketoglutaric acid (2: 1) product is obtained after spray drying, the content is 99.4%, and the yield is 95.7%.
Example 3
A method for preparing L-arginine-alpha-ketoglutaric acid by a biological enzyme method comprises the following steps:
(1) preparation of reaction solution
Carrying out dry ethanol 25m high-speed transformation on the 50m high-speed transformation reaction kettle, then adding 3000kg of glutamic acid and 3658kg of arginine, adjusting the pH of the solution to 7.0, and carrying out dry ethanol constant volume till 30m high-speed transformation.
(2) Enzyme reaction
Adding 5U/mL glutamate oxidase and 400U/mL catalase into the reaction solution to start reaction, controlling the dissolved oxygen by 25 percent, controlling the temperature by 38.5 +/-0.5 ℃, feeding arginine to control the pH value to be 7.0, detecting no glutamic acid by using a biosensor after 10 hours of reaction, and supplementing 3444kg of arginine to obtain the L-arginine-alpha-ketoglutaric acid (2: 1) reaction solution.
(3) Ceramic membrane filtration sterilization
After the reaction is finished, the reaction solution firstly passes through a ceramic membrane, and the trapped solution is dialyzed by pure water with the length of 4 m; drying the dialyzed trapped fluid to be used as feed protein.
(4) Removing impurities by ultrafiltration membrane
Passing the clear liquid obtained by the ceramic membrane through an ultrafiltration membrane, wherein the molecular weight of the ultrafiltration membrane is 3000, and dialyzing the trapped fluid by pure water obtained by 2m high-speed cropping; the retention solution after dialysis is applied to the next batch of synthetic fluid to pass through the ceramic membrane.
(5) Decolorizing with activated carbon
Decolorizing at 60 deg.C, adding active carbon 240kg, decolorizing for 30min, and allowing the decolorized solution to pass light T420nm=97.1%。
(6) Concentrating under reduced pressure
The concentration temperature was 65 ℃ and the pressure was-0.1 mpa. And (6) carrying out concentration to 18 m.
(7) Spray drying
After spray drying, 10253kg of qualified L-arginine-alpha-ketoglutaric acid (2: 1) product is obtained, the content is 99.5%, and the yield is 94.7%.
Claims (6)
1. A method for preparing L-arginine-alpha-ketoglutaric acid by a biological enzyme method is characterized by comprising the following steps: the method comprises the following steps:
(1) preparing a reaction solution: carrying out dry top-row ethanol 50m, carrying out dry top-row ethanol precipitation, carrying out thin-row column chromatography, carrying out thin-row column chromatography, carrying out thin top-row column chromatography, carrying out thin-row chromatography, carrying out thin top-row chromatography, carrying out thin bottom-row column chromatography, carrying out thin top-row column chromatography, carrying out thin top-row chromatography, and thin bottom-row column chromatography, carrying out thin bottom-row column chromatography, carrying out thin top-row thin bottom-row thin column chromatography, carrying out thin film chromatography, carrying out thin column chromatography, and thin bottom-row thin column chromatography, carrying out thin column chromatography, and thin column chromatography, carrying out thin column chromatography, carrying out thin column chromatography, and thin column carrying out thin column chromatography, carrying out thin column;
(2) enzyme reaction: adding 5U/mL glutamate oxidase and 250U/mL catalase into the reaction solution to start reaction, controlling the dissolved oxygen by 20 percent, controlling the temperature by 39.5 +/-0.5 ℃, controlling the pH value to be 6.9, detecting no glutamic acid by using a biosensor after reacting for 8 hours, and supplementing 3462kg of arginine to obtain a reaction solution of L-arginine-alpha-ketoglutaric acid (2: 1);
(3) ceramic membrane filtration: after the reaction is finished, filtering and sterilizing the reaction solution by a ceramic membrane, and dialyzing the trapped solution by pure water with the trapped volume being 2-4 times that of the trapped solution;
(4) and (3) ultrafiltration membrane filtration: filtering with ceramic membrane to obtain clear solution, passing through ultrafiltration membrane with aperture molecular weight of 3000, and dialyzing the trapped solution with pure water with 2-4 times of trapped volume;
(5) activated carbon decolorization: decolorizing the clear liquid obtained by ultrafiltration membrane filtration with activated carbon at 60-65 deg.C for 20-30min, and adding 3-10g/L activated carbon;
(6) and (3) concentrating under reduced pressure: concentrating the decolorized product at-0.08-0.1 mpa until the concentration of L-arginine-alpha-ketoglutaric acid is 40% -60%;
(7) spray drying: spray drying to obtain L-arginine-alpha-ketoglutaric acid product.
2. The method for preparing L-arginine-alpha-ketoglutaric acid according to claim 1, wherein: and (4) drying the bacterial sludge filtered by the ceramic membrane in the step (3) to be used as a raw material of feed protein.
3. The method for preparing L-arginine-alpha-ketoglutaric acid according to claim 1, wherein: and (4) the trapped fluid after dialysis in the step (4) is applied to the next batch of synthetic fluid and passes through the ceramic membrane together.
4. The method for preparing L-arginine-alpha-ketoglutaric acid according to claim 1, wherein: light transmittance T of the solution decolorized in the step (5)420nm≥95%。
5. The method for preparing L-arginine-alpha-ketoglutaric acid according to claim 2, wherein: the concentration temperature in step (6) was 65 ℃.
6. The process for the preparation of L-arginine- α -ketoglutarate according to any one of claims 2-5, which comprises: the conductivity of the pure water in the steps (1), (3) and (4) is less than or equal to 10.
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| CN102020593A (en) * | 2010-11-29 | 2011-04-20 | 广东环西生物科技股份有限公司 | Process for preparing L-arginine-alpha-ketoglutarate (AAKG) from fermentation liquor through direct crystallization |
| CN104109698A (en) * | 2013-04-17 | 2014-10-22 | 上海工业生物技术研发中心 | Enzymic method for producing [alpha]-ketoglutaric acid |
| CN106148434A (en) * | 2016-08-27 | 2016-11-23 | 山东民强生物科技股份有限公司 | A kind of microbial enzyme method produces the method for a ketoglutaric acid |
| CN106350547A (en) * | 2016-08-24 | 2017-01-25 | 天津科技大学 | Preparation method of L-arginine-alpha-ketoglutaric acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102020593A (en) * | 2010-11-29 | 2011-04-20 | 广东环西生物科技股份有限公司 | Process for preparing L-arginine-alpha-ketoglutarate (AAKG) from fermentation liquor through direct crystallization |
| CN104109698A (en) * | 2013-04-17 | 2014-10-22 | 上海工业生物技术研发中心 | Enzymic method for producing [alpha]-ketoglutaric acid |
| CN106350547A (en) * | 2016-08-24 | 2017-01-25 | 天津科技大学 | Preparation method of L-arginine-alpha-ketoglutaric acid |
| CN106148434A (en) * | 2016-08-27 | 2016-11-23 | 山东民强生物科技股份有限公司 | A kind of microbial enzyme method produces the method for a ketoglutaric acid |
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Denomination of invention: Preparation of L-arginine by biological enzymatic method- a- Method for producing ketoglutarate Effective date of registration: 20220530 Granted publication date: 20210914 Pledgee: Shandong juancheng Rural Commercial Bank Co.,Ltd. Plaza sub branch Pledgor: SHANDONG YANGCHENG BIOTECH Co.,Ltd. Registration number: Y2022980006634 |