Method for preparing gamma-aminobutyric acid through whole-cell catalysis

Abstract

The invention discloses a method for preparing gamma-aminobutyric acid through whole-cell catalysis, and relates to the technical field of gamma-aminobutyric acid preparation, and the method comprises the following steps: culture medium preparation, microbial culture, thallus preparation, catalytic conversion, reaction liquid pretreatment, vacuum concentration, crystallization purification and drying. Concentration treatment is carried out in a gradient temperature setting mode in the vacuum concentration step, on one hand, low energy consumption is facilitated, on the other hand, the situation that a small amount of impurities in a sample are changed due to the temperature is prevented, the solubility of aminobutyric acid is reduced after cooling, crystals are separated out more easily, and the quality of the product is improved. In addition, through temperature gradient setting in the microbial culture step and temperature reduction after induction, correct folding of protease can be promoted, then the activity of unit protease is increased, the reaction effect is improved, and therefore the purity and the finished product yield of gamma-aminobutyric acid are improved.

Description

Technical Field

[0001] The invention relates to the technical field of gamma-aminobutyric acid preparation, and in particular to a method for preparing gamma-aminobutyric acid by whole-cell catalysis. Background Art

[0002] γ-Aminobutyric acid is a non-protein natural amino acid widely distributed in animals, plants and microorganisms. It is the most important inhibitory neurotransmitter in the central nervous system of mammals. γ-Aminobutyric acid not only has analgesic, anti-anxiety, anticonvulsant, blood pressure lowering, anti-epileptic, hormone secretion regulating and nerve cell nutrition effects on humans, but also has the effects of promoting animal feeding, regulating endocrine, regulating hormone secretion and its reproductive physiological effects, and improving animal's own immunity.

[0003] The whole-cell catalytic method is a common method for preparing γ-aminobutyric acid. It mainly uses enzymes in microbial cells for catalysis, and has the advantages of high conversion efficiency and simple separation and purification process. However, when preparing γ-aminobutyric acid using the existing whole-cell catalytic method, the purity and finished product yield of γ-aminobutyric acid products are both around 90%, and the impurities and aminobutyric acid losses generated during the reaction are large, and the conversion effect needs to be improved. Therefore, a method for preparing γ-aminobutyric acid using whole-cell catalysis is needed to solve the above problems. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing γ-aminobutyric acid by whole-cell catalysis, so as to solve the problems existing in the prior art mentioned in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for preparing γ-aminobutyric acid by whole-cell catalysis comprises the following steps:

[0007] S1: Culture medium preparation: The culture medium used for culturing microorganisms is formulated with tryptone, yeast extract, lactose, K2HPO4, KH2PO4, and glycerol, prepared with ultrapure water and sterilized;

[0008] S2: Microbial culture: inoculate the seed solution into the culture medium prepared in S1 at a 4-8% inoculum, set an appropriate culture temperature, culture on a shaker for 4-6 hours, add an inducer to induce the expression of amino acid decarboxylase, lower the culture temperature, and continue to culture on a shaker for 4-6 hours;

[0009] S3: Bacteria preparation: measure the OD600 of the fermentation broth in S2, collect the microbial cells by centrifugation or membrane filtration, wash the cells twice with phosphate buffer solution to remove senescent and broken cells;

[0010] S4: catalytic conversion: the substrate glutamate or sodium glutamate is mixed with ultrapure water to prepare a raw material solution, the pH of the solution is adjusted to 6.0-7.0 with 25-30% ammonia water or 1-5% acetic acid, adenine nucleoside triphosphate, reduced nicotinamide adenine dinucleotide phosphate and the microbial cells collected in S3 are added to the solution to carry out a catalytic reaction, and the solution is cultured on a shaker at an appropriate temperature for 12-24 hours to generate γ-aminobutyric acid;

[0011] S5: Pretreatment of reaction solution: centrifuge or filter the reaction solution after the reaction in S4 to remove the bacteria, collect the filtrate and heat it at 80-100℃ for 20-30min, add sodium hydroxide to adjust the pH to 5-5.8, add chitosan, stir evenly, let stand for 30-60min, filter and collect the filtrate;

[0012] S6: Vacuum concentration: at a certain temperature, the filtrate collected in S5 is vacuum concentrated until the liquid volume is 1 / 2 of the original volume, the temperature is lowered, the filtrate is vacuum concentrated until the liquid volume is 1 / 3 of the original volume, the temperature is lowered again, the filtrate is vacuum concentrated until it is viscous with no obvious liquid, and the concentration is terminated;

[0013] S7: Crystallization purification: add 3-4 times of anhydrous ethanol to the viscous substance obtained in S6, stir and mix, let stand for 2-3 hours, filter and retain the solid matter, add 3-4 times of anhydrous ethanol again, stir and mix, let stand for 2-3 hours, filter and retain the solid matter;

[0014] S8: Drying: Dry the solid matter in S7 to obtain purified γ-aminobutyric acid crystals.

[0015] Preferably, in S1, the microorganism is any one of Corynebacterium glutamicum, lactic acid bacteria, and Escherichia coli; tryptone is 12 g / L, yeast extract is 24 g / L, lactose is 2 g / L, K2HPO4 is 10 g / L, KH2PO4 is 2 g / L, and glycerol is 4 mol / L, wherein the mass ratio of tryptone, yeast extract and lactose is 6:12:1; and the sterilization treatment parameters are sterilization at 115°C for 20 min.

[0016] Preferably, in S2, the culture temperature is set to 35-45°C, the shaking speed is 250 rpm, and after the induction agent is added, the temperature is reduced to 25-30°C, and the shaking speed is 200 rpm.

[0017] Preferably, in S4, the concentration of adenosine triphosphate is 0.02-0.05 g / L, the catalytic reaction temperature is set to 37° C., and the shaking speed is set to 200 rpm.

[0018] Preferably, in S4, the concentration of the substrate glutamate or sodium glutamate is 500-1000 g / L.

[0019] Preferably, in S5, the concentration of the flocculant chitosan is 0.01-0.1 g / L.

[0020] Preferably, in S6, the temperature is set to 75-80°C at the beginning, and when the filtrate is vacuum concentrated to a liquid volume of 1 / 2 of the original volume, the temperature is lowered to 65-70°C, and when the filtrate is vacuum concentrated to a liquid volume of 1 / 3 of the original volume, the temperature is lowered again to 55-60°C.

[0021] Preferably, in S8, the solid matter is dried at a drying temperature of 50-60°C.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention performs concentration treatment by adopting a gradient temperature setting in the vacuum concentration step. The selection of the concentration temperature depends on the water content in the sample. At the beginning, there is more water in the sample, and evaporation will take away heat, so a higher temperature is used. As the sample concentration increases, the temperature is continuously lowered. On the one hand, it is conducive to lower energy consumption, and on the other hand, it prevents the temperature from causing a small amount of impurities in the sample to change due to temperature. After cooling, the solubility of aminobutyric acid decreases, and crystals are more easily precipitated. In addition, the loss of aminobutyric acid caused by long-term high-temperature heating is avoided, thereby improving the purity and finished product yield of γ-aminobutyric acid.

[0024] 2. The present invention sets a temperature gradient in the microbial culture step. Lowering the temperature after induction can promote the correct folding of the protease, thereby increasing the activity of the unit protease and improving the reaction effect, thereby improving the preparation efficiency and finished product yield of γ-aminobutyric acid.

[0025] 3. The present invention adds adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the catalytic conversion step. ATP is the energy currency in the cell, which provides energy through hydrolysis to support various life activities. NADPH is a reducing force carrier, which participates in various reduction reactions, protects cells from oxidative damage, and supports various biochemical reactions by providing energy and reducing force, thereby improving the activity of cells and promoting catalytic reactions. DETAILED DESCRIPTION

[0026] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0027] The present invention provides the following technical solutions:

[0028] Example 1

[0029] A method for preparing γ-aminobutyric acid by whole-cell catalysis comprises the following steps:

[0030] S1: Culture medium preparation: The culture medium formula for culturing Corynebacterium glutamicum is 12 g / L tryptone, 24 g / L yeast extract, 2 g / L lactose, 10 g / L K2HPO4, 2 g / L KH2PO4, and 4 mol / L glycerol, wherein the mass ratio of tryptone, yeast extract, and lactose is 6:12:1; it is prepared with ultrapure water and sterilized at 115°C for 20 min; the phosphate solution is sterilized separately and added when used.

[0031] S2: Microbial culture: Inoculate the seed liquid into the culture medium prepared in S1 at a 6% inoculum size, set the culture temperature to 37°C, and culture on a shaker at a speed of 250 rpm for 5 h. Add an inducer to induce the expression of amino acid decarboxylase, lower the culture temperature to 26°C, and continue to culture on a shaker at a speed of 200 rpm for 5 h.

[0032] S3: Bacterial cell preparation: Measure the OD600 of the fermentation broth in S2, collect the microbial cells by centrifugation or membrane filtration, and wash the cells twice with phosphate buffer solution to remove senescent and broken cells.

[0033] S4: Catalytic conversion: Mix the substrate sodium glutamate with ultrapure water to prepare a raw material solution, the concentration of sodium glutamate is 700 g / L, and adjust the pH of the solution to 6.5 with 2% acetic acid. Add adenine nucleoside triphosphate, reduced nicotinamide adenine dinucleotide phosphate and the microbial cells collected in S3 to the solution for catalytic reaction. Set the temperature to 37°C and culture on a shaker at a speed of 200 rpm for 20 hours to generate γ-aminobutyric acid.

[0034] S5: Pretreatment of reaction liquid: centrifuge or filter the reaction liquid after the reaction in S4 to remove the bacteria, collect the filtrate and heat it at 85℃ for 25min, add sodium hydroxide to adjust the pH to 5.5, add chitosan with a concentration of 0.01-0.1g / L, stir evenly, let it stand for 50min, filter and collect the filtrate.

[0035] S6: Vacuum concentration: At a temperature of 75°C, the filtrate collected in S5 is vacuum concentrated to a liquid volume of 1 / 2 of the original volume, the temperature is lowered to 65°C, the filtrate is vacuum concentrated to a liquid volume of 1 / 3 of the original volume, the temperature is lowered to 55°C again, the filtrate is vacuum concentrated to a viscous state with no obvious liquid, and the concentration is terminated.

[0036] S7: Crystallization purification: add 3 times of anhydrous ethanol to the viscous substance obtained in S6, stir and mix, let stand for 2 hours, filter and retain the solid matter, add 3 times of anhydrous ethanol again, stir and mix, let stand for 2 hours, filter and retain the solid matter;

[0037] S8: Drying: Dry the solid matter in S7 at a drying temperature of 50° C. to obtain purified γ-aminobutyric acid crystals.

[0038] Liquid phase detection of the γ-aminobutyric acid crystals obtained in Example 1 showed a purity of 99.5% and a finished product yield of 98.8%.

[0039] Example 2

[0040] The difference between Example 2 and Example 1 is that the reaction temperature in S6 is set to 80°C at the beginning, the temperature is reduced to 70°C when the filtrate is vacuum concentrated to a liquid volume of 1 / 2 of the original volume, and the temperature is reduced again to 60°C when the filtrate is vacuum concentrated to a liquid volume of 1 / 3 of the original volume. The other steps and parameters are the same as those in Example 1. The γ-aminobutyric acid crystals obtained in Example 2 are subjected to liquid phase detection, and the purity is 99.2%, and the finished product yield is 99.1%.

[0041] Comparative Example 1

[0042] The difference between Comparative Example 1 and Example 1 is that the vacuum concentration temperature in S6 is maintained at 75° C., and the other steps and parameters are the same as those in Example 1; the γ-aminobutyric acid crystals obtained in Comparative Example 1 are subjected to liquid phase detection, and the purity is 91.5%, and the finished product yield is 89.8%.

[0043] Comparative Example 2

[0044] The difference between Comparative Example 2 and Example 1 is that the microbial culture temperature in S2 is maintained at 37° C., and the other steps and parameters are the same as those in Example 1. The γ-aminobutyric acid crystals obtained in Comparative Example 2 were subjected to liquid phase detection, and the purity was 94.3%, and the finished product yield was 93.8%.

[0045] It can be analyzed from the above examples 1-2 and comparative examples 1-2 that the use of gradient temperature concentration in the vacuum concentration step can effectively improve the purity and finished product yield of γ-aminobutyric acid crystals. The principle is: the choice of concentration temperature depends on the water content in the sample. At the beginning, there is more water in the sample, and evaporation will take away heat, so a higher temperature is used. As the sample concentration increases, the temperature is continuously reduced. On the one hand, it is conducive to lower energy consumption, and on the other hand, it prevents the temperature from causing a small amount of impurities in the sample to change due to temperature, and the solubility of aminobutyric acid decreases after cooling, and the crystal is more likely to precipitate, and avoids the loss of aminobutyric acid caused by long-term high temperature heating; in addition, the temperature gradient setting in the microbial culture step can also improve the finished product yield of γ-aminobutyric acid crystals. The principle is: lowering the temperature after induction can promote the correct folding of the protease, thereby increasing the activity of the unit protease and improving the effect of the reaction. The method of the present invention is used to prepare γ-aminobutyric acid, which can effectively improve the purity and finished product yield of γ-aminobutyric acid, and has high preparation efficiency and low energy consumption.

[0046] 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 method for preparing γ-aminobutyric acid by whole-cell catalysis, characterized in that: The following steps are involved: S1: Culture medium preparation: The culture medium used for culturing microorganisms is formulated with tryptone, yeast extract, lactose, K2HPO4, KH2PO4, and glycerol, prepared with ultrapure water and sterilized; S2: Microbial culture: inoculate the seed solution into the culture medium prepared in S1 at a 4-8% inoculum, set an appropriate culture temperature, culture on a shaker for 4-6 hours, add an inducer to induce the expression of amino acid decarboxylase, lower the culture temperature, and continue to culture on a shaker for 4-6 hours; S3: Bacteria preparation: measure the OD600 of the fermentation broth in S2, collect the microbial cells by centrifugation or membrane filtration, wash the cells twice with phosphate buffer solution to remove senescent and broken cells; S4: catalytic conversion: the substrate glutamate or sodium glutamate is mixed with ultrapure water to prepare a raw material solution, the pH of the solution is adjusted to 6.0-7.0 with 25-30% ammonia water or 1-5% acetic acid, adenine nucleoside triphosphate, reduced nicotinamide adenine dinucleotide phosphate and the microbial cells collected in S3 are added to the solution to carry out a catalytic reaction, and the solution is cultured on a shaker at an appropriate temperature for 12-24 hours to generate γ-aminobutyric acid; S5: Pretreatment of reaction solution: centrifuge or filter the reaction solution after the reaction in S4 to remove the bacteria, collect the filtrate and heat it at 80-100℃ for 20-30min, add sodium hydroxide to adjust the pH to 5-5.8, add chitosan, stir evenly, let stand for 30-60min, filter and collect the filtrate; S6: Vacuum concentration: at a certain temperature, the filtrate collected in S5 is vacuum concentrated until the liquid volume is 1 / 2 of the original volume, the temperature is lowered, the filtrate is vacuum concentrated until the liquid volume is 1 / 3 of the original volume, the temperature is lowered again, the filtrate is vacuum concentrated until it is viscous with no obvious liquid, and the concentration is terminated; S7: Crystallization purification: add 3-4 times of anhydrous ethanol to the viscous substance obtained in S6, stir and mix, let stand for 2-3 hours, filter and retain the solid matter, add 3-4 times of anhydrous ethanol again, stir and mix, let stand for 2-3 hours, filter and retain the solid matter; S8: Drying: Dry the solid matter in S7 to obtain purified γ-aminobutyric acid crystals.

2. The method for preparing γ-aminobutyric acid by whole-cell catalysis according to claim 1, characterized in that: In the S1, the microorganism is any one of Corynebacterium glutamicum, lactic acid bacteria, and Escherichia coli; the tryptone is 12 g / L, the yeast extract is 24 g / L, the lactose is 2 g / L, the K2HPO4 is 10 g / L, the KH2PO4 is 2 g / L, and the glycerol is 4 mol / L, wherein the mass ratio of tryptone, yeast extract and lactose is 6:12:1; and the sterilization treatment parameters are sterilization at 115°C for 20 min.

3. The method for preparing γ-aminobutyric acid by whole-cell catalysis according to claim 1, characterized in that: In S2, the culture temperature is set at 35-45°C, and the shaking speed is 250 rpm. After the induction agent is added, the temperature is reduced to 25-30°C, and the shaking speed is 200 rpm.

4. The method for preparing γ-aminobutyric acid by whole-cell catalysis according to claim 1, characterized in that: In the S4, the concentration of adenosine triphosphate is 0.02-0.05 g / L, the catalytic reaction temperature is set to 37° C., and the shaking speed is set to 200 rpm.

5. The method for preparing γ-aminobutyric acid by whole-cell catalysis according to claim 1, characterized in that: In the S4, the concentration of the substrate glutamate or sodium glutamate is 500-1000 g / L.

6. The method for preparing γ-aminobutyric acid by whole-cell catalysis according to claim 1, characterized in that: In the S5, the concentration of the flocculant chitosan is 0.01-0.1 g / L.

7. The method for preparing γ-aminobutyric acid by whole-cell catalysis according to claim 1, characterized in that: In S6, the temperature is set to 75-80°C at the beginning, and the temperature is lowered to 65-70°C when the filtrate is vacuum concentrated to a liquid volume of 1 / 2 of the original volume. When the filtrate is vacuum concentrated to a liquid volume of 1 / 3 of the original volume, the temperature is lowered again to 55-60°C.

8. The method for preparing γ-aminobutyric acid by whole-cell catalysis according to claim 1, characterized in that: In S8, the solid matter is dried at a drying temperature of 50-60°C.