Background and overview[1][2]
Adenosine triphosphate (ATP) is a nucleoside drug composed of an adenine, a ribose and a triphosphate unit, with a molecular formula of C10H16N5O13P3 and a molecular weight of 507. It is an energy converter and storage device in living organisms and plays an important role in human energy metabolism. ATP is also an important metabolic substance in organisms. As a metabolic intermediate and coenzyme, it participates in the metabolism of fats, proteins, sugars, and nucleic acids in organisms. In clinical applications, ATP can not only be used as an auxiliary drug to rescue critically ill patients, but also has good therapeutic effects on acute and chronic hepatitis, nephritis, myocarditis, coronary arteriosclerosis, progressive muscle atrophy, aplastic anemia, rheumatoid arthritis and other diseases. Therapeutic effect. This product is a coenzyme. Adenosine triphosphate is a nucleotide derivative that participates in the metabolism of fat, protein, sugar, nucleic acid and nucleotides in the body. When energy is needed for absorption, secretion, muscle contraction, and biochemical synthesis reactions in the body, adenosine triphosphate is decomposed into adenosine diphosphate and phosphate groups, and energy is released at the same time. Adenosine triphosphate disodium can penetrate the blood-cerebrospinal fluid barrier and improve nerve cell membranes. Stability and reconstruction ability of sexual structure, promoting the regrowth of neurites.
Preparation[1]
The synthesis of adenosine triphosphate mainly includes chemical synthesis, enzyme-catalyzed synthesis, photosynthetic phosphorylation and oxidative phosphorylation, and microbial enzyme-catalyzed synthesis. Considering the safety, environmental protection, production cost and other factors of drugs, using adenosine as raw material to catalyze the synthesis of adenosine triphosphate by microbial enzymes has become the main process for the current industrial production of adenosine triphosphate. However, the overall level of adenosine triphosphate production in my country is still relatively low, and the product output, recovery rate, product purity, etc. cannot reach the level of imported products.
The Chinese patent application document with application number 95100148.5 discloses a “new process for producing adenosine triphosphate (ATP) using adenosine (AR) as raw material.” The process mainly includes: beer yeast freezing treatment, fermentation enzyme extraction, centrifugal separation, adenosine fermentation to synthesize ATP, alcohol precipitation, precipitate redissolution, freezing desalination, diluted ion exchange, resin washing and elution, resin decolorization, and activated carbon removal. Heat source, crystallization, drying, etc. Due to the incomplete separation of yeast and invertase in this process, the enzyme components produced by the yeast change in the later stage of fermentation, and the activity of invertase decreases, resulting in an adenosine triphosphate conversion rate of 90%. In addition, during the fermentation process, factors such as bacterial aging and autolysis bring in higher There are many problems such as many impurities; secondly, in addition to the three antioxidants adenosine phosphate, there are also unreacted AR and side reaction products AMP and ADP, as well as nutrients dipotassium hydrogen phosphate and hydrogen phosphate required by microorganisms. Inorganic salts such as dipotassium and magnesium sulfate. The concentration of inorganic salts in the fermentation synthetic liquid is about 40,000 mg/L. During the ion exchange adsorption process, ions compete with ATP to be adsorbed onto the ion exchange resin, causing the amount of ATP adsorbed by the resin to be only about 5 to 13 grams/100 grams of resin (201×7). After using the alcohol precipitation desalting process, the amount of ATP adsorbed by the resin is increased. to 18~22g/100g resin (201×7), but it needs to consume 4~5 times the volume of ATP fermentation broth and absolute ethanol. The adenosine triphosphate recovery rate and equipment utilization rate are low, and there are many impurities in the product.
CN201510113434.7 provides a production method for adenosine triphosphate that can improve the production capacity, recovery rate, and product purity of ATP products, reduce the consumption of chemical reagents, simplify the production process, reduce pollution emissions, and significantly improve economic benefits. The object of the present invention is achieved through the following technical solution, a method for producing adenosine triphosphate (ATP), which mainly utilizes the enzyme system in yeast cells to use glucose as energy source to phosphate adenosine (AR) through the glycolysis pathway. To synthesize adenosine triphosphate, the production method includes the following steps:
a) Add deionized water to the fermentation reaction pot, raise the temperature of the water to 30~42°C, add beer yeast quantitatively, and then quantitatively add glucose, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate solution to adjust the pH The value is in the range of 6.0 to 7.0, keep the temperature at 30 to 42°C and ferment for 2.0 to 2.5 hours to extract the invertase, and obtain the fermentation enzyme liquid; the dosage of each of the above reactants is: beer yeast mud 250 to 280g/L, glucose 35 ~45 g/L, dipotassium hydrogen phosphate 25~30 g/L, potassium dihydrogen phosphate 20~25 g/L.
b) The fermentation enzyme extraction liquid needs to be filtered through plate and frame filtration to remove larger yeast mud impurities in the enzyme liquid to obtain a crude filtration enzyme extraction liquid;
c) The crude enzyme solution that has been filtered through plate and frame filtration is then processed through a microfiltration membrane with a precision of 0.1 to 10 μm to further remove fine yeast and other impurities to obtain a clear enzyme solution;
d) Add the reactant adenosine 20~40g/L to the clear enzyme solution, then add the nutrient magnesium sulfate 1.6~2.0g/L, adjust the pH value to the range of 6.0~7.0, and keep the temperature at 32~ Ferment for 4.0 to 6.0 hours at 35°C. Adenosine is provided with energy by glucose. Under the catalysis of invertase, adenosine is phosphorylated into adenosine triphosphate. The concentration of adenosine triphosphate is 36 to 72g/L. Immediately add ice water to cool. Reduce the temperature to 15~20℃;
e) Use a nanofiltration membrane of water-washed kaolin with a molecular weight cutoff of 150 to 500. Through the selective permeability of the nanofiltration membrane, the monovalent ions can permeate the membrane. More than 80% of the inorganic salts in the adenosine triphosphate solution are removed, and the adenosine triphosphate due to The larger molecules are trapped in the concentrated liquid by the nanofiltration membrane, and an adenosine triphosphate concentrated liquid is obtained;
f) By removing inorganicDilute the �� adenosine triphosphate concentrated solution with deionized water to adjust the concentration of adenosine triphosphate to 0.5~1.5%;
g) The above diluted adenosine triphosphate solution is adsorbed by 201×7 anion exchange resin with a resin particle size of 0.315~1.25mm; after the adsorption is completed, it is washed with deionized water and 0.04~0.06% sodium chloride solution to remove AR, AMP, ADP and other raw materials and synthetic by-products are then eluted with 4.0-6.0% sodium chloride solution to obtain adenosine triphosphate eluent;
h) The adenosine triphosphate eluate is processed through an ultrafiltration membrane with a molecular weight cutoff of 3000 to 10000 to remove proteins and heat sources, and colorimetric substances are removed through activated carbon adsorption;
i) The above-mentioned adenosine triphosphate eluate that has been treated by ultrafiltration membrane and adsorbed by activated carbon is purified through absolute ethanol precipitation, centrifugal separation, elution and soaking to obtain adenosine triphosphate solid. The amount of absolute ethanol used is the amount of adenosine triphosphate ultrafiltration membrane permeation. 2.5 to 3.5 times of the liquid, and then vacuum dried at 38 to 42°C to obtain the finished product of adenosine triphosphate.
The present invention uses microfiltration treatment to improve the purity of the invertase and improves the synthesis conversion rate of adenosine triphosphate; in the separation and purification process of the adenosine triphosphate fermentation synthetic liquid, a nanofiltration process is used to remove the inorganic salt nutrients added during the synthesis process. The capacity of ion exchange resin to adsorb ATP is enhanced; ultrafiltration is used to remove proteins and heat sources to improve product purity. The present invention can increase the adenosine phosphate conversion rate to more than 95%, the total recovery rate to more than 80%, and the purity to more than 98%. The invention has the characteristics of improving the production capacity, recovery rate and product purity of ATP products, reducing the consumption of chemical reagents, simplifying the production process, reducing pollution emissions, and significantly improving economic benefits.
Pharmacological effects[2]
Adenosine triphosphate is a nucleotide derivative that participates in the metabolism of fat, protein, sugar, nucleic acid and nucleotides in the body. When energy is needed for absorption, secretion, muscle contraction, and biochemical synthesis reactions in the body, adenosine triphosphate is decomposed into adenosine diphosphate and phosphate groups, and energy is released at the same time. Adenosine triphosphate disodium can penetrate the blood-cerebrospinal fluid barrier and improve nerve cell membranes. Stability and reconstruction ability of sexual structure, promoting the regrowth of neurites.
Clinical applications and indications[2]
It is used as an auxiliary treatment for progressive muscle atrophy, sequelae of cerebral hemorrhage, cardiac insufficiency, myocardial disease and hepatitis.
Notes[2]
1. It is contraindicated in patients with early stage cerebral hemorrhage.
2. It is prohibited to use the drug when its properties change.
Main reference materials
[1] CN201510113434.7 A production method of adenosine triphosphate (ATP)
[2] Instructions for Adenosine Triphosphate Disodium Tablets
