A method for preparing d-ribose

Abstract

The present application relates to the technical field of biological chemical industry, and particularly relates to a preparation method of D-ribose. In the method, the fermentation liquor of D-ribose is flocculated by a flocculating agent and then rapidly filtered to obtain a filtration mother liquor. The filtration mother liquor is subjected to decolorization, desalination, purification and concentration to obtain a concentrated liquor. The concentrated liquor is mixed with ether and then sent to a crystallization tank. Further concentration and crystallization are carried out under azeotropic conditions. The obtained crystals are vacuum dried to obtain the product. The ether-water mixed steam generated in the crystallization process is condensed and separated. The obtained ether is heated by heat exchange and then recycled. The method can realize continuous crystallization process, effectively improve the production efficiency, and effectively ensure the purity of the product. In addition, the ether-water steam generated in the azeotropic concentration is easy to separate, which can effectively reduce the energy consumption of separation and facilitate the recycling of ether.

Description

Technical Field

[0001] This invention relates to the field of biochemical technology, specifically to a method for preparing D-ribose. Background Technology

[0002] D-ribose is a crucial component of nucleic acids, the genetic material in living organisms. It plays a pivotal role in the metabolism of nucleosides, proteins, and lipids, possessing vital physiological functions and broad application prospects. As a naturally occurring component in all cells, D-ribose is closely related to the formation of adenosine nucleotides and the regeneration of ATP, making it one of the most fundamental energy sources for life metabolism. It plays a key role in the metabolism of the heart and skeletal muscles, promoting the recovery of ischemic and hypoxic tissues. Nucleic acid drugs are an important means of treating viruses, tumors, and AIDS. D-ribose is an important intermediate in many nucleic acid drugs and can be used in the production of many drugs, including triazole nucleosides, adenosine, thymidine, cytidine, fluoroadenosine nucleosides, 2-methyladenosine, vitatoxin, pyrazole toxin, and adenosyldanine. D-ribose has a wide range of applications and is closely related to people's lives. Researching its different synthetic pathways is of great significance for medicine, food, agriculture, and other fields.

[0003] Currently, there are three methods for producing D-ribose: one is extraction from yeast nucleic acid; the second is chemical synthesis using glucose and arabinose as raw materials; and the third is fermentation with glucose using transketolase-deficient strains, with the third being the most common. However, the separation and purification of D-ribose is extremely difficult due to the presence of various ions, pigments, pentoses, hexoses and their phosphate esters, as well as proteins and other macromolecular impurities in the fermentation broth. Existing preparation and purification methods generally involve the following steps: centrifugation to remove bacterial cells from the fermentation broth, decolorization with activated carbon, removal of anions and cations from the fermentation broth through ion exchange, concentration under reduced pressure, and precipitation and crystallization with four times the volume of ethanol to obtain the product.

[0004] However, in the above-mentioned process, the crystallization of D-ribose using ethanol is carried out in batches, which is not conducive to improving production efficiency. Single-batch operation results in product residue in the crystallization mother liquor, leading to a significant decrease in product yield. Furthermore, ethanol and water are miscible during crystallization, making the treatment of the crystallization mother liquor complex and ethanol recovery difficult. Therefore, the entire preparation and purification process has low production efficiency, hindering the improvement of enterprise production efficiency and the reduction of production costs. Thus, a more efficient method for preparing D-ribose that can effectively reduce enterprise production costs is an urgent problem to be solved. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing D-ribose, in order to solve the problems of low production efficiency, difficulty in separating the crystallization mother liquor, and reduced product yield due to partial loss of D-ribose in existing preparation and purification methods in practical applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a method for preparing D-ribose, characterized by comprising the following steps:

[0007] S1. Add flocculant to the fermentation broth of D-ribose and filter to obtain the mother liquor;

[0008] S2. The mother liquor obtained from S1 is decolorized, desalted, purified and concentrated to obtain a concentrated solution.

[0009] S3. The concentrated solution obtained in S2 and diethyl ether are introduced into the crystallization tank in a certain proportion;

[0010] S4. Maintain a stable amount of liquid in the crystallizer and keep it in an azeotropic state. Continue to concentrate and crystallize in the azeotropic state to obtain ribose crystals.

[0011] S5. Vacuum drying of the ribose crystals obtained in S4 yields the D-ribose product.

[0012] Furthermore, in S1, the fermentation broth for D-ribose is produced by fermenting glucose as raw material and using a transketolase-deficient mutant strain of Bacillus subtilis as the production strain.

[0013] Furthermore, in S1, the flocculant is one of polyacrylamide, chitosan, or chitosan-polyacrylamide copolymer.

[0014] Furthermore, in S2, decolorization is performed using activated carbon or macroporous adsorption resin; desalination is performed using anion and cation exchange resins; and purification is performed using nanofiltration membranes.

[0015] Furthermore, the nanofiltration membrane has a molecular weight cutoff of 400 to 600.

[0016] Furthermore, a portion of the residual liquid obtained after nanofiltration is refluxed back to the front end of the nanofiltration membrane, with a reflux-to-discharge ratio of 3 to 5:1.

[0017] Furthermore, in S3, the volume ratio of the concentrate to the diethyl ether is 1:99-100, and the temperature of the diethyl ether is controlled at 25-30°C.

[0018] Furthermore, in S4, the ether-water mixture gas distilled in the azeotropic state is condensed to separate ether and water, and the separated ether is returned to S3 for recycling after heat exchange.

[0019] Furthermore, in S5, the vacuum drying temperature is 40–60°C.

[0020] Furthermore, in step S5, the mixed gas obtained by vacuum drying is mixed with the ether-water mixed gas, and then condensed and separated.

[0021] The beneficial effects of this invention are:

[0022] 1. Adding flocculants to the fermentation broth can cause the suspended solids in the broth to settle quickly, thus obtaining a relatively pure mother liquor through simple filtration. This process is more efficient and less costly.

[0023] 2. The mother liquor is decolorized, desalted, purified and concentrated to obtain a concentrated solution. Decolorization can be performed using macroporous adsorption resin, desalting can be performed using anion and cation exchange resin, and purification can be performed using a nanofiltration membrane with a molecular weight cutoff of 400-600, which makes the purified solution purer and effectively ensures the purity of the product.

[0024] 3. The residual liquid from the nanofiltration membrane is refluxed at a certain reflux ratio to reduce product loss and ensure product yield;

[0025] 4. An azeotropic system of ether-water is used in the crystallization tank to concentrate and crystallize the feed solution. During the process, the amount of feed solution added is consistent with the evaporation rate of the system, so as to achieve a continuous concentration and crystallization process, thereby effectively improving production efficiency.

[0026] 5. Due to the incompatibility between diethyl ether and water, the vapor obtained during azeotropic concentration and subsequent vacuum drying will separate into layers after condensation. This allows for the separation of diethyl ether and water, resulting in higher efficiency, lower cost, and easier recycling of the recovered diethyl ether. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the process flow of the preparation method of the present invention. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0029] like Figure 1 As shown, the principle involved in the method and process of this invention is as follows:

[0030] The process of this invention is based on the existing relatively mature D-ribose fermentation broth, which uses glucose as raw material and a transketolase-deficient mutant strain of Bacillus subtilis as the production strain. Since the fermentation process is relatively mature, it will not be described in detail here.

[0031] The D-ribose fermentation broth contains bacterial cells, large particulate impurities, culture medium ions, and proteins, thus requiring purification. Adding flocculants can cause bacterial cells, large particulate impurities, and proteins to flocculate, enabling their rapid and efficient removal. Organic flocculants and natural polymeric flocculants can be used, with polyacrylamide, chitosan, and chitosan-polyacrylamide copolymers being preferred. This ensures effective flocculation and sedimentation without introducing large amounts of ions, thus reducing the load on subsequent desalination processes.

[0032] The mother liquor obtained after flocculation filtration removes large particulate impurities and most bacterial cells, but molecular groups, such as pigment groups, are not removed, thus requiring further decolorization. Traditional decolorization often uses activated carbon, but this generates a large amount of solid waste. Existing methods often use macroporous adsorption resins for adsorption and decolorization, which can be regenerated afterward, making it economical and environmentally friendly. Among them, medium-polarity and polar macroporous adsorption resins can be selected. After a period of use, 80% ethanol is used for elution and regeneration, and the pH of the ethanol eluent should be controlled at 9-11.

[0033] After decolorization, the pigment groups in the filtrate are removed, but the anions and cations remain, necessitating desalination. Desalination is performed using cation and anion exchange resins. In industrial production, the decolorized solution flows sequentially through a cation exchange tower and an anion exchange tower, with the cation exchange tower filled with H₂O. + Strong acid cation exchange resin, anion exchange tower filled with OH... - The weakly basic anion exchange resin can achieve desalination. After a period of use, the cation exchange tower is backwashed and regenerated with hydrochloric acid solution, and then washed with water until neutral. The anion exchange tower is backwashed and regenerated with sodium hydroxide solution, and then washed with water until neutral.

[0034] The desalinated feed solution needs further purification to remove small molecule impurities such as nucleotides, amino acids, and oligosaccharides. The purification is carried out using a nanofiltration membrane with a molecular weight cutoff of 400-600, an operating pressure of 1.5-2 MPa, and a dialysis water volume of 50-60% of the feed volume. The remaining liquid is partially refluxed to the front end of the nanofiltration membrane at a reflux ratio of 3-5:1.

[0035] After the above purification operation, a pure purified liquid is obtained. The purified liquid is then concentrated under reduced pressure, with the temperature controlled at 50-60℃ and the vacuum degree at -90 to -80 kPa. The purified liquid is concentrated to a viscous state and then quantitatively delivered to a crystallization tank, such as a peristaltic pump or metering pump. At the same time, diethyl ether is pumped into the crystallization tank, with the volume ratio of the concentrated liquid to diethyl ether being 1:99-100, and the temperature of the diethyl ether being controlled at 25-30℃.

[0036] The mixed liquid is heated in the crystallization tank to 34°C. At this temperature, ether and water undergo azeotropic reaction (ether is in excess in the system; initially, the material is heated to 35°C, which is the boiling point of ether, and the excess ether will evaporate rapidly; when the ratio of ether to water reaches the azeotropic ratio, azeotropy occurs naturally), and is continuously evaporated. Due to the carryover of water, ribose crystals precipitate. By controlling the evaporation rate to be consistent with the amount of liquid added, a continuous azeotropic concentration and crystallization process can be achieved, thereby greatly improving production efficiency.

[0037] During the azeotropic concentration and crystallization process, the generated ether-water mixed vapor is collected in the condenser after condensation, and the ether and water separate into layers, thus facilitating the separation of ether and water (the specific gravity of ether is less than that of water, and the ether can be easily recovered by overflow in the condenser). The recovered ether is at a low temperature, and is heated to 25-30°C through a heat exchanger during the process, and then recycled.

[0038] As for the crystals produced in the crystallization tank, a continuous discharge device can be used to discharge them. There are many existing continuous discharge devices for crystallization tanks. The material discharged from the crystallization tank is wet material, which can be dried under vacuum at 40-60℃. The mixed gas of ether and water generated by vacuuming can be condensed in a condenser.

[0039] Example 1

[0040] A method for preparing D-ribose includes the following steps:

[0041] S1. Preparation of fermentation broth

[0042] Bacillus subtilis was inoculated into a fermentation medium containing glucose substrate, with 200 kg of glucose added (180-200 g / L). Air was introduced to carry out aerobic fermentation and obtain fermentation broth.

[0043] Fermentation conditions were as follows: inoculum size 10.0%; tank temperature 36.0℃; aeration rate 0.5 vvm; tank pressure 0.05 MPa; culture time 40–50 h.

[0044] Add 0.001% polyacrylamide to the above fermentation broth. After sufficient flocculation, perform plate and frame filtration. After filtration stabilization, collect the filtrate. Circulate the initial filtrate for filtration to obtain the mother liquor.

[0045] S2. The mother liquor obtained from S1 is subjected to decolorization with macroporous adsorption resin (BJ7514, Jiangsu Jinshan New Material Co., Ltd.), desalination with anion and cation exchange resins, purification with nanofiltration membrane, and vacuum concentration. The nanofiltration membrane has a molecular weight cutoff of 500 and an operating pressure of 2 MPa. The vacuum concentration temperature is 60℃ and the vacuum degree is -80 kPa. The purified liquid is concentrated to a viscous state.

[0046] S3. The concentrated solution obtained in S2 and diethyl ether are introduced into the crystallization tank at a ratio of 1:100, and the temperature of the diethyl ether is controlled at 25-30℃.

[0047] S4. Maintain the temperature in the crystallizer at 34°C and keep the amount of liquid stable and in an azeotropic state. In the azeotropic state, continuous concentration and crystallization are carried out. During the concentration process, mixed vapor of ether-water and ribose crystals are obtained.

[0048] S5. The ribose crystals obtained in S4 were vacuum dried at 60°C for 8–10 h to obtain 139.3 kg of D-ribose product.

[0049] The D-ribose product prepared above had a yield of 83.6% relative to the raw glucose, and its purity was 99.3% as determined by liquid chromatography.

[0050] Comparative Example 1

[0051] The preparation method of Example 1 is compared with existing mature methods.

[0052] S1. Take the fermentation broth under the same conditions as in Example 1, centrifuge and filter it, and then decolorize it through macroporous adsorption resin (BJ7514, Jiangsu Jinshan New Material Co., Ltd.).

[0053] S2. Take the decolorized solution from S1 and desalinate it through anion and cation exchange resins.

[0054] S3, at a temperature of 60℃ and a vacuum of -80KPa, the desalted S2 solution is concentrated to a viscous state;

[0055] S4. Add four times the volume of anhydrous ethanol to the concentrated solution of S3, and crystallize overnight at 4°C to obtain ribose crystals.

[0056] S5. The ribose crystals obtained in S4 were vacuum dried at 60°C for 8–10 h to obtain 108.7 kg of D-ribose product.

[0057] The D-ribose product prepared above had a yield of 65.2% relative to the raw glucose, and its purity was 98.6% as determined by liquid chromatography.

[0058] Comparison reveals that the product obtained by the preparation method of this invention is superior to the product obtained by conventional methods in terms of both yield and purity, and has a positive effect on improving product quality.

[0059] In addition, the preparation method of Example 1 of the present invention is compared with the preparation method of Comparative Example 1 in terms of preparation efficiency and treatment of crystallization mother liquor.

[0060] Regarding preparation efficiency, the method of the present invention is a continuous azeotropic concentration and crystallization process. In Example 1, the processing time of one fermentation tank material is about 6 to 8 hours. However, using the method of Comparative Example 1, even without considering the time for cleaning and idle time of the crystallization tank, the crystallization step still requires 8 to 12 hours. It can be seen that the method of the present invention significantly improves the preparation efficiency.

[0061] Furthermore, regarding the treatment of the crystallization mother liquor, the method of this invention does not generate any crystallization mother liquor. The ether and water in the feed solution are simultaneously distilled off under azeotropic conditions. The mixed vapor of the distilled ethanol and water is condensed and separated in a condenser, making the entire process simpler. In contrast, the method of Comparative Example 1 uses a mixture of ethanol and water as the crystallization mother liquor. Since ethanol and water are miscible and also contain dissolved products, subsequent separation requires distillation, making the entire process complex. In comparison, the method of this invention is much simpler to operate and is significantly superior to existing mature methods.

[0062] This invention is not limited to the preferred embodiments described above. Anyone can derive other forms of products under the guidance of this invention. However, regardless of any changes made in their shape or structure, any technical solution that is the same as or similar to this application falls within the protection scope of this invention.

Claims

1. A method for preparing D-ribose, characterized in that, Includes the following steps: S1. Add flocculant to the fermentation broth of D-ribose and filter to obtain the mother liquor; S2. The mother liquor obtained from S1 is decolorized, desalted, purified and concentrated to obtain a concentrated solution. Decolorization is performed using macroporous adsorption resin; desalination is performed using anion and cation exchange resins; purification is performed using nanofiltration membrane; the molecular weight cutoff of the nanofiltration membrane is 400-600; part of the net liquid obtained after filtration by the nanofiltration membrane is refluxed back to the front end of the nanofiltration membrane, and the ratio of reflux to discharge is 3-5:

1. S3. The concentrated solution obtained in S2 and diethyl ether are introduced into the crystallization tank in a certain proportion; The volume ratio of the concentrate to diethyl ether is 1:99~100, and the temperature of the diethyl ether is controlled at 25~30℃; S4. Maintain a stable amount of liquid in the crystallizer and keep it in an azeotropic state. Continue to concentrate and crystallize in the azeotropic state to obtain ribose crystals. The ether-water mixture gas distilled out under azeotropic conditions is condensed to separate ether and water. The separated ether is returned to S3 for recycling after heat exchange. S5. Vacuum drying of the ribose crystals obtained in S4 yields the D-ribose product.

2. The method for preparing D-ribose according to claim 1, characterized in that: In S1, the fermentation broth for D-ribose is produced by fermenting glucose as raw material and using a transketolase-deficient mutant strain of Bacillus subtilis as the production strain.

3. The method for preparing D-ribose according to claim 1, characterized in that: In S1, the flocculant is one of polyacrylamide, chitosan, or chitosan-polyacrylamide copolymer.

4. The method for preparing D-ribose according to claim 1, characterized in that: In S5, the vacuum drying temperature is 40~60℃.

5. The method for preparing D-ribose according to claim 1, characterized in that: In step S5, the mixed gas obtained by vacuum drying is mixed with the ether-water mixed gas, and then condensed and separated.

Images