CN110923276B - Preparation method for synthesizing calcium aspartate by biological enzyme catalysis - Google Patents
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12P13/20—Aspartic acid; Asparagine
Abstract
The invention discloses a preparation method for synthesizing calcium aspartate by biological enzyme catalysis, which comprises the following steps: (1) culturing and fermenting escherichia coli in a fermentation medium to obtain wet thalli; (2) preparing the wet thalli prepared in the step (1) into a suspension, adding 1g/L calcium chloride solution, and incubating for 2h at 30 ℃; then refrigerating at 4 ℃ for at least 2h to obtain pretreated transformed bacteria; (3) adding the transformed thallus pretreated in the step (2) into a transformation liquid, and carrying out enzymatic reaction for 6 hours; after the reaction is finished, removing somatic cells by a high-speed centrifuge; (4) heating the transformation liquid after removing the somatic cells to 80-100 ℃, adding activated carbon for decolorization, and filtering to obtain a filtrate; (5) purifying the filtrate to obtain the product of calcium aspartate. The method adopts the biological enzyme method to prepare the calcium aspartate, has mild conditions, and the obtained product has the characteristics of high bioavailability, high purity and the like, and is suitable for industrial production and application in the high-end biological medicine field.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a preparation method for synthesizing calcium aspartate by biological enzyme catalysis.
Background
The major and trace mineral elements are indispensable elements in plant and human life. Their earliest development was mainly in the form of inorganic salts to meet nutritional requirements. Because of the reasons of low mineral absorption rate, poor effect, more side effects and the like, some simple organic salts appear in the market gradually, which are improved in comparison with the first generation, the dosage can be less, and the absorption utilization rate is improved. At present, the amino acid complex and the salt thereof are the latest generation nutrition enhancer, and compared with the previous two generations, the amino acid complex and the salt thereof have the advantages of high absorption and utilization rate, no antagonism, stable structure and environmental protection. The reaction of amino acid, metal element and organic acid is the hot research of experts in both chemical theory research and practical application of animals and plants.
Aspartic acid is an amino acid with stronger acidity, and has wide application in the aspects of medicines, foods, feeds and the like. The method has the advantages of strong acidity and easy reaction with metal salt. In the existing synthesis of aspartic acid metal complex, the extraction process conditions of most of the synthesized products are harsh. Organic solvent harmful to human body is mostly added for extraction in the extraction process, organic solvent is easy to remain in the product, the dosage is large, the environment is polluted, and the development of the asparagus series complex and the salt thereof is greatly restricted.
Calcium aspartate is one of the amino acid calcium salts, which is an organic acid chelated calcium with a bioactive structure. It has stable chemical structure, good water solubility and high absorption rate. Therefore, there is a need to develop a process for preparing calcium aspartate which is environmentally friendly and has high extraction efficiency.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method for synthesizing calcium aspartate by biological enzyme catalysis. The method adopts the biological enzyme method to prepare the calcium aspartate, has mild conditions, obtains the product with high purity, and is suitable for industrial production.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a preparation method for synthesizing calcium aspartate by biological enzyme catalysis is characterized by comprising the following steps:
(1) preparation of aspartase-containing transformant bacteria: culturing and fermenting escherichia coli in a fermentation medium, and centrifuging fermentation liquor to obtain wet thalli;
(2) pretreatment of the transformed bacteria: preparing the wet thalli prepared in the step (1) into a suspension, adding 1g/L calcium chloride solution, and incubating for 2h at 30 ℃; then refrigerating at 4 ℃ for at least 2 h; obtaining pretreated transformed thalli;
(3) catalytic conversion of calcium L-aspartate: adding the transformed thallus pretreated in the step (2) into the transformation liquid, and carrying out enzymatic reaction at the pH of 6.5 and the temperature of 40 ℃ for 6 hours; wherein the conversion solution comprises 50-80 g/L of fumaric acid, 5-8 g/L of ammonia, 0.03-0.1 g/L of calcium hydroxide and 32.5-37.5g g/L of calcium chloride; after the reaction is finished, removing somatic cells;
(4) and (3) decoloring: heating the transformation liquid without the somatic cells to 80-100 ℃, and adding activated carbon for decolorization; filtering to obtain filtrate;
(5) purifying the filtrate to obtain the product of calcium aspartate.
In the above technical solution, the method for purifying the filtrate in step (5) comprises step (5a) or step (5 b);
the step (5a) is as follows: spray drying the filtrate prepared in the step (4) to obtain a calcium aspartate product; the spray drying parameters were: the air inlet temperature is 160-200 ℃, and the air outlet temperature is 80-100 ℃;
the step (5b) comprises the steps of pumping the filtrate obtained in the step (4) into a concentration kettle, and carrying out vacuum reduced pressure concentration for 3.5-5 h at the temperature of 70 ℃ to obtain a concentrated solution; and cooling the obtained concentrated solution, stirring and crystallizing for 5-7 h, centrifuging, and drying to obtain the calcium aspartate product.
In the above technical solution, in the step (1), the preparation method of the fermentation medium comprises: 10g/L of ammonium fumarate, 6g/L of soluble starch, 7g/L of peptone, 5g/L of sodium chloride and 1g/L of monopotassium phosphate; magnesium sulfate 0.2g/L, dissolving the above raw materials with appropriate amount of water, and adjusting ph to 7.0-7.2 with ammonia water.
In the above technical scheme, in the step (3), the concentration of fumaric acid in the conversion solution is 50 g/L; 5g/L of ammonia, 0.03g/L of calcium hydroxide and 37.5g/L of calcium chloride.
In the above technical solution, in the step (3), the specific method for removing the somatic cells is as follows: the conversion solution after the enzymatic reaction is centrifuged for 15min at the rotating speed of 4000r/min and the temperature of 4 ℃.
In the above technical scheme, in the step (3), the mass-to-volume ratio of the wet cells to the conversion solution is: the amount of the cells was calculated by adding 1L of the transformation solution to 30g of the cells.
The product prepared by the invention has the following advantages:
(1) the method for preparing the calcium aspartate by the biological enzyme one-step method simplifies the process steps, has mild preparation conditions, and is more suitable for industrial production.
(2) The purity of the calcium aspartate prepared by the method is more than 99%, and the conversion rate is more than 95%.
Drawings
FIGS. 1 and 2 are graphs comparing the IR spectra of the calcium aspartate product of example 1 and a calcium aspartate standard.
Detailed Description
Embodiment 1 a preparation method for synthesizing calcium aspartate by biological enzyme catalysis, comprising the following steps:
(1) preparation of aspartase-containing transformant bacteria: coli (E.coli) in a fermentation medium (10 g/L ammonium fumarate; 6g/L soluble starch, 7g/L peptone, 5g/L sodium chloride; 1g/L potassium dihydrogen phosphate; 0.2g/L magnesium sulfate), dissolving the above materials with appropriate amount of water, adjusting ph to 7.0-7.2 with ammonia water, fermenting, and centrifuging the fermentation broth to obtain 30g wet thallus;
(2) pretreatment of the transformed bacteria: preparing the wet thalli prepared in the step (1) into a suspension, adding 1g/L calcium chloride solution, and incubating for 2h at 30 ℃; then refrigerating at 4 ℃ for at least 2 h; obtaining pretreated transformed thalli;
(3) catalytic conversion of calcium L-aspartate: adding the transformed thallus pretreated in the step (2) into 1L of transformation liquid (containing 50g/L fumaric acid, 5g/L ammonia, 0.03g/L calcium hydroxide and 37.5g/L calcium chloride), and performing enzymatic reaction at 40 ℃ at pH6.5 for 6 h; after the reaction is finished, centrifuging the conversion solution after the enzymatic reaction for 15min at the rotating speed of 4000r/min and the temperature of 4 ℃ to remove thallus cells;
(4) and (3) decoloring: heating the transformation liquid without the somatic cells to 80 ℃, and adding activated carbon for decolorization; filtering to obtain filtrate;
(5b) concentration: putting the filtrate obtained in the step (4) into a concentration kettle, and carrying out vacuum reduced pressure concentration for 3.5h at the temperature of 70 ℃ to obtain a concentrated solution;
cooling and crystallizing: cooling the obtained concentrated solution, stirring and crystallizing for 7h, centrifuging, and drying to obtain the product of calcium aspartate.
The product purity of the calcium aspartate product in example 1 is 101.2%; infrared spectroscopic analysis is shown in fig. 1 and 2; wherein the conversion rate of converting fumaric acid into calcium aspartate is 96%.
FIG. 1 and graph represent comparison plots of infrared spectra of the calcium aspartate product of example 1 and a calcium aspartate standard.
The results of the tests in FIGS. 1 and 2 show that:
similarity: 99.80 percent
Standard limit values: 95.00 percent
Test samples: 66102019012701
Comparing the standard substance: c2743252018-321-01-13
The detection method comprises the following steps: YQ-SOP-ZL-4032-01 (2019/03/1708: 35:22 AM)
The purity of the product of calcium aspartate catalyzed by a biological enzyme in example 1 was 99.7%.
Example 2
A method for synthesizing calcium aspartate based on biological enzyme comprises the following steps:
(1) preparation of aspartase-containing transformant bacteria: coli (E.coli) in a fermentation medium (10 g/L ammonium fumarate; 6g/L soluble starch, 7g/L peptone, 5g/L sodium chloride; 1g/L potassium dihydrogen phosphate; 0.2g/L magnesium sulfate), dissolving the above materials with appropriate amount of water, adjusting ph to 7.0-7.2 with ammonia water, fermenting, and centrifuging the fermentation broth to obtain 30g wet thallus;
(2) pretreatment of the transformed bacteria: preparing the wet thalli prepared in the step (1) into a suspension, adding 1g/L calcium chloride solution, and incubating for 2h at 30 ℃; then refrigerating at 4 ℃ for at least 2 h; obtaining pretreated transformed thalli;
(3) catalytic conversion of calcium L-aspartate: adding the transformed thallus pretreated in the step (2) into 1L of transformation liquid (containing 50g/L fumaric acid, 5g/L ammonia, 0.03g/L calcium hydroxide and 37.5g/L calcium chloride), and performing enzymatic reaction at 40 ℃ at pH6.5 for 6 h; after the reaction is finished, centrifuging the conversion solution after the enzymatic reaction for 15min at the rotating speed of 4000r/min and the temperature of 4 ℃ to remove thallus cells;
(4) and (3) decoloring: heating the transformation liquid without the somatic cells to 80 ℃, and adding activated carbon for decolorization; filtering to obtain filtrate;
(5a) spray drying the filtrate prepared in the step (4) to obtain a calcium aspartate product; the spray drying parameters were: the air inlet temperature is 160-180 ℃, and the air outlet temperature is 80-90 ℃; .
The purity of the product calcium aspartate product in example 1 was 99%; wherein the conversion rate of converting fumaric acid into calcium aspartate is 98%.
Comparative example 1
A method for synthesizing calcium aspartate based on biological enzyme comprises the following steps:
(1) preparation of aspartase-containing transformant bacteria: coli (E.coli) in a fermentation medium (10 g/L ammonium fumarate; 6g/L soluble starch, 7g/L peptone, 5g/L sodium chloride; 1g/L potassium dihydrogen phosphate; 0.2g/L magnesium sulfate), dissolving the above materials with appropriate amount of water, adjusting ph to 7.0-7.2 with ammonia water, fermenting, and centrifuging the fermentation broth to obtain 30g wet thallus;
(2) catalytic conversion of calcium L-aspartate: adding the transformed thallus pretreated in the step (1) into 1L of transformation liquid (containing 50g/L fumaric acid, 5g/L ammonia, 0.03g/L calcium hydroxide and 37.5g/L calcium chloride), and performing enzymatic reaction at 40 ℃ at pH6.5 for 6 h; after the reaction is finished, centrifuging the conversion solution after the enzymatic reaction for 15min at the rotating speed of 4000r/min and the temperature of 4 ℃ to remove thallus cells;
(3) and (3) decoloring: heating the transformation liquid without the somatic cells to 80 ℃, and adding activated carbon for decolorization; filtering to obtain filtrate;
(4) concentration: putting the filtrate obtained in the step (3) into a concentration kettle, and carrying out vacuum reduced pressure concentration for 3.5h at the temperature of 70 ℃ to obtain a concentrated solution; cooling and crystallizing: cooling the obtained concentrated solution, stirring and crystallizing for 7h, centrifuging, and drying to obtain the product of calcium aspartate.
In comparative example 1 the purity of the product calcium aspartate product was 95%; wherein the conversion rate of converting fumaric acid into calcium aspartate is 97%.
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.
Claims (5)
1. A preparation method for synthesizing calcium aspartate by biological enzyme catalysis is characterized by comprising the following steps:
(1) preparation of aspartase-containing transformant bacteria: culturing and fermenting escherichia coli in a fermentation medium, and centrifuging fermentation liquor to obtain wet thalli;
(2) pretreatment of the transformed bacteria: preparing the wet thalli prepared in the step (1) into a suspension, adding 1g/L calcium chloride solution, and incubating for 2h at 30 ℃; then refrigerating at 4 ℃ for at least 2 h; obtaining pretreated transformed thalli;
(3) catalytic conversion of calcium L-aspartate: adding the transformed thallus pretreated in the step (2) into the transformation liquid, and carrying out enzymatic reaction at the pH of 6.5 and the temperature of 40 ℃ for 6 hours; (ii) a After the reaction is finished, removing somatic cells; wherein in the step (3), the concentration of fumaric acid in the conversion solution is 50 g/L; 5g/L of ammonia, 0.03g/L of calcium hydroxide and 37.5g/L of calcium chloride.
(4) And (3) decoloring: heating the transformation liquid without the somatic cells to 80-100 ℃, and adding activated carbon for decolorization; filtering to obtain filtrate;
(5) purifying the filtrate to obtain the product of calcium aspartate.
2. The process for preparing calcium aspartate by bio-enzyme catalysis according to claim 1, wherein the method for purifying the filtrate in the step (5) comprises the steps (5a) or (5 b);
the step (5a) is as follows: spray drying the filtrate prepared in the step (4) to obtain a calcium aspartate product; the spray drying parameters were: the air inlet temperature is 160-200 ℃, and the air outlet temperature is 80-100 ℃;
the step (5b) comprises the steps of pumping the filtrate obtained in the step (4) into a concentration kettle, and carrying out vacuum reduced pressure concentration for 3.5-5 h at the temperature of 70 ℃ to obtain a concentrated solution; and cooling the obtained concentrated solution, stirring and crystallizing for 5-7 h, centrifuging, and drying to obtain the calcium aspartate product.
3. The method for preparing calcium aspartate through biological enzyme catalysis in claim 1, wherein in the step (1), the fermentation medium is prepared by the following steps: 10g/L of ammonium fumarate; 6g/L of soluble starch, 7g/L of peptone and 5g/L of sodium chloride; 1g/L potassium dihydrogen phosphate; magnesium sulfate 0.2g/L, dissolving the above raw materials with appropriate amount of water, and adjusting ph to 7.0-7.2 with ammonia water.
4. The method for preparing calcium aspartate through biological enzyme catalysis and synthesis according to claim 1, wherein in the step (3), the specific method for removing the somatic cells is as follows: the conversion solution after the enzymatic reaction is centrifuged for 15min at the rotating speed of 4000r/min and the temperature of 4 ℃.
5. The method for preparing calcium aspartate through biological enzyme catalysis in claim 1, wherein in the step (3), the mass-to-volume ratio of the wet thallus to the conversion solution is as follows: the amount of the cells was calculated by adding 1L of the transformation solution to 30g of the cells.
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