Method for preparing fructose 1, 6-diphosphate
By adding aldehyde compounds to yeast culture medium to promote enzyme activity and catalyze the synthesis of fructose 1,6-bisphosphate, the problem of low yield in existing technologies has been solved, and efficient and low-cost preparation of fructose 1,6-bisphosphate has been achieved.
Patent Information
- Application Number
- CN202511407517.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-29
- Publication Date
- 2026-01-06
AI Technical Summary
Existing methods for producing fructose 1,6-bisphosphate suffer from low yields.
Aldehyde compounds such as formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, or glyoxal are added to the culture medium of yeast. Glucose is converted into fructose 1,6-bisphosphate by yeast catalysis. Fructose 1,6-bisphosphate powder is obtained by purification through solid-liquid separation, ultrafiltration, nanofiltration, and spray drying.
It significantly improved the catalytic synthesis yield of fructose 1,6-bisphosphate from 60 g/L to 85 g/L, shortened the catalytic synthesis time, reduced production costs, and simplified the operation.
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial catalytic synthesis technology, specifically relating to a method for preparing fructose-1,6-bisphosphate. Background Technology
[0002] Fructose-1,6-bisphosphate (FDP) is a natural high-energy compound widely found in human, animal, plant, and microbial cells. It is an intermediate product of glycolysis and has the functions of rapidly providing energy to cells, regulating the activity of several enzymes in glycolysis, activating cells, and improving the body's adaptability. Since the 1980s, FDP has been widely used in medicine, food, health products, and cosmetics. Especially in clinical practice, it is used for the emergency treatment of ischemic and hypoxic diseases such as acute myocardial infarction, severe myocardial ischemia, peripheral vascular disease, and various types of shock. It is also used as an important adjuvant drug in various surgical procedures and is effective in treating deep jaundice, elevated transaminases, and hypoalbuminemia caused by various types of hepatitis.
[0003] Currently, the production and preparation methods of fructose 1,6-bisphosphate mainly revolve around bio-enzymatic conversion technology, using yeast cells (such as brewer's yeast residue or highly active dry yeast) to catalyze the conversion of the substrate into fructose 1,6-bisphosphate in a reaction solution. However, existing technologies still suffer from low yields. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to provide a method for preparing fructose 1,6-bisphosphate, which addresses the shortcomings of the prior art.
[0005] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:
[0006] A method for preparing fructose 1,6-bisphosphate involves inoculating yeast into a culture medium, reacting with glucose as a substrate, separating the resulting reaction solution into solid and liquid components, collecting the supernatant, ultrafiltration, collecting the permeate, nanofiltration, collecting the retentate, and drying to obtain fructose 1,6-bisphosphate powder.
[0007] The culture medium contains aldehyde compounds.
[0008] The aldehyde compounds are any one or a combination of formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, glyoxal, and malondialdehyde.
[0009] Preferably, the aldehyde compound is formaldehyde.
[0010] The concentration of aldehyde compounds in the culture medium is 0.1–1 g / L, preferably 0.1–0.6 g / L, more preferably 0.1–0.3 g / L, and most preferably 0.2 g / L.
[0011] Aldehydes can promote the activity of key enzymes in FDP synthesis.
[0012] The yeast is brewer's yeast; the yeast is wet yeast; the inoculation amount of the yeast is 50-500 g / L, preferably 100-300 g / L.
[0013] The brewing yeast mentioned is commercially available common brewing yeast.
[0014] The reaction conditions are: temperature of 20–40°C and time of 1–10 h.
[0015] The culture medium comprises the following components: glucose 20-200 g / L, sodium dihydrogen phosphate 10-100 g / L, magnesium chloride 2-20 g / L, ammonium chloride 1-10 g / L, and potassium chloride 1-10 g / L; the pH of the culture medium is 4.0-8.0.
[0016] The solid-liquid separation is centrifugation at 5000-10000 rpm for 10-30 min; the ultrafiltration membrane used for ultrafiltration has a molecular weight cutoff of 4000-8000 Da; the nanofiltration membrane used for nanofiltration has a molecular weight cutoff of 100-300 Da; and the drying is spray drying.
[0017] Beneficial effects:
[0018] Aldehydes can enhance the activity of key enzymes in FDP synthesis. This invention increases the single-batch catalytic yield from 60 g / L to 85 g / L by adding aldehydes for catalytic synthesis. This method significantly shortens the catalytic synthesis time, increases the catalytic synthesis yield, is low-cost, simple to operate, and has obvious effects, thus reducing production costs. Detailed Implementation
[0019] The present invention will be further described below with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.
[0020] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.
[0021] The brewing yeast used in the following examples was purchased from Nanjing Jinling Brewery and obtained through plate and frame pressing.
[0022] Example 1
[0023] Preparation of catalytic synthesis medium: glucose 120 g / L, sodium dihydrogen phosphate 60 g / L, magnesium chloride 8 g / L, ammonium chloride 5 g / L, potassium chloride 2 g / L, solvent is water, pH = 6.0.
[0024] Catalytic synthesis of fructose 1,6-bisphosphate: 6L of culture medium was added to a 10L stirred tank, and 200g / L of wet brewer's yeast was inoculated for catalytic synthesis culture. After 5 hours of catalytic synthesis at 30℃ in the catalytic synthesis tank, the yield stabilized at 60g / L, and the catalytic synthesis was stopped.
[0025] Example 2
[0026] Preparation of catalytic synthesis medium: glucose 120 g / L, sodium dihydrogen phosphate 60 g / L, magnesium chloride 8 g / L, ammonium chloride 5 g / L, potassium chloride 2 g / L, formaldehyde 0.2 g / L, solvent is water, pH = 6.0.
[0027] Catalytic synthesis of fructose 1,6-bisphosphate: 6L of culture medium was added to a 10L stirred tank, and 200g / L of Saccharomyces cerevisiae was inoculated for catalytic synthesis culture. After 5 hours of catalytic synthesis at a temperature of 30℃ in the catalytic synthesis tank, the yield stabilized at 85g / L, and the catalytic synthesis was stopped.
[0028] Example 3
[0029] Preparation of catalytic synthesis medium: glucose 120 g / L, sodium dihydrogen phosphate 60 g / L, magnesium chloride 8 g / L, ammonium chloride 5 g / L, potassium chloride 2 g / L, acetaldehyde 0.2 g / L, solvent is water, pH = 6.0.
[0030] Catalytic synthesis of fructose 1,6-bisphosphate: 6L of culture medium was added to a 10L stirred tank, and 200g / L of Saccharomyces cerevisiae was inoculated for catalytic synthesis culture. After 5 hours of catalytic synthesis at a temperature of 30℃ in the catalytic synthesis tank, the yield stabilized at 76g / L, and the catalytic synthesis was stopped.
[0031] Example 4
[0032] Preparation of catalytic synthesis medium: glucose 120 g / L, sodium dihydrogen phosphate 60 g / L, magnesium chloride 8 g / L, ammonium chloride 5 g / L, potassium chloride 2 g / L, propionaldehyde 0.2 g / L, solvent is water, pH = 6.0.
[0033] Catalytic synthesis of fructose 1,6-bisphosphate: 6L of culture medium was added to a 10L stirred tank, and 200g / L of Saccharomyces cerevisiae was inoculated for catalytic synthesis culture. After 5 hours of catalytic synthesis at 30℃ in the catalytic synthesis tank, the yield stabilized at 75g / L, and the catalytic synthesis was stopped.
[0034] Example 5
[0035] Preparation of catalytic synthesis medium: glucose 120 g / L, sodium dihydrogen phosphate 60 g / L, magnesium chloride 8 g / L, ammonium chloride 5 g / L, potassium chloride 2 g / L, butyraldehyde 0.2 g / L, solvent is water, pH=6.0.
[0036] Catalytic synthesis of fructose 1,6-bisphosphate: 6L of culture medium was added to a 10L stirred tank, and 200g / L of Saccharomyces cerevisiae was inoculated for catalytic synthesis culture. After 5 hours of catalytic synthesis at 30℃ in the catalytic synthesis tank, the yield stabilized at 56g / L, and the catalytic synthesis was stopped.
[0037] Example 6
[0038] Preparation of catalytic synthesis medium: glucose 120 g / L, sodium dihydrogen phosphate 60 g / L, magnesium chloride 8 g / L, ammonium chloride 5 g / L, potassium chloride 2 g / L, glyoxal 0.2 g / L, solvent is water, pH = 6.0.
[0039] Catalytic synthesis of fructose 1,6-bisphosphate: 6L of culture medium was added to a 10L stirred tank, and 200g / L of Saccharomyces cerevisiae was inoculated for catalytic synthesis culture. After 5 hours of catalytic synthesis at 30℃ in the catalytic synthesis tank, the yield stabilized at 68g / L, and the catalytic synthesis was stopped.
[0040] Example 7
[0041] Preparation of catalytic synthesis medium: glucose 120 g / L, sodium dihydrogen phosphate 60 g / L, magnesium chloride 8 g / L, ammonium chloride 5 g / L, potassium chloride 2 g / L, malondialdehyde 0.2 g / L, solvent is water, pH = 6.0.
[0042] Catalytic synthesis of fructose 1,6-bisphosphate: 6L of culture medium was added to a 10L stirred tank, and 200g / L of Saccharomyces cerevisiae was inoculated for catalytic synthesis culture. After 5 hours of catalytic synthesis at 30℃ in the catalytic synthesis tank, the yield stabilized at 62g / L, and the catalytic synthesis was stopped.
[0043] Example 8
[0044] Optimization experiment of formaldehyde addition. Different amounts of formaldehyde were added to the catalytic synthesis medium, while other components were the same as the catalytic synthesis medium in Example 12. Saccharomyces cerevisiae were inoculated into the catalytic synthesis medium for catalytic synthesis culture, and the catalytic synthesis method was also the same as in Example 12. The optimal amount of formaldehyde was investigated using FDP yield as the evaluation index. The formaldehyde addition amount in the catalytic synthesis medium and the corresponding FDP yield are shown in Table 1.
[0045] Table 1. FDP production under different formaldehyde addition levels
[0046] Serial Number Formaldehyde content (g / L) FDP production (g / L) 1 0.1 69 2 0.2 85 3 0.3 80 4 0.4 74 5 0.5 68
[0047] Example 9
[0048] The reaction solutions obtained in Examples 1-8 were centrifuged at 8000 rpm for 20 min to separate solids and liquids, and the supernatant containing FDP was collected. The supernatant was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 6000 Da to remove proteins, and the permeate was collected. The permeate was desalted using a nanofiltration membrane with a molecular weight cutoff of 150 Da, and the retentate was collected. This retentate was spray-dried using a spray dryer (inlet temperature 180°C, outlet temperature 100°C) to obtain FDP powder.
[0049] This invention provides a method and approach for preparing fructose-1,6-bisphosphate. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.
Claims
1. A method for preparing 1,6-fructose diphosphate, characterized by, The yeast is inoculated into a culture medium to react with glucose as a substrate, the obtained reaction liquid is subjected to solid-liquid separation, the supernatant is collected, ultrafiltration is performed, the permeate is collected, nanofiltration is performed, the retentate is collected, and drying is performed, to obtain 1,6-fructose diphosphate. The culture medium contains an aldehyde compound.
2. The method of claim 1, wherein, The aldehyde compound is any one or a combination of formaldehyde, acetaldehyde, propyl aldehyde, butyl aldehyde, glyoxal and malondialdehyde.
3. The method of claim 1, wherein, The concentration of the aldehyde compound in the culture medium is 0.1-1 g / L.
4. The method of claim 1, wherein, The yeast is Saccharomyces cerevisiae, and the yeast is wet yeast.
5. The method of claim 4, wherein, The inoculation amount of the yeast is 50-500 g / L.
6. The method of claim 1, wherein, The reaction conditions are as follows: temperature is 20-40 DEG C, and time is 1-10 h.
7. The method of claim 1, wherein, The culture medium comprises the following components: glucose 20-200 g / L, sodium phosphate dibasic 10-100 g / L, magnesium chloride 2-20 g / L, ammonium chloride 1-10 g / L, and potassium chloride 1-10 g / L; and the pH of the culture medium is 4.0-8.
0.
8. The method of claim 1, wherein, The solid-liquid separation is centrifugation at 5000-10000 rpm for 10-30 min.
9. The method of claim 1, wherein, The ultrafiltration membrane used in the ultrafiltration has a molecular weight cut-off of 4000-8000 Da.
10. The method of claim 1, wherein, The nanofiltration membrane used in the nanofiltration has a molecular weight cut-off of 100-300 Da; and the drying is spray drying.