Separation and preparation method of D-allose
By combining whole-cell wet cell transformation with recombinant ribose-5-phosphate isomerase, membrane filtration, and resin treatment with selective crystallization using an ethanol-water system, the high-cost separation problem in D-allose production was solved, achieving low-cost and high-efficiency separation and purification of D-allose.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHUCHENG HAOTIAN PHARMA CO LTD
- Filing Date
- 2026-01-27
- Publication Date
- 2026-05-12
AI Technical Summary
Existing D-allose production processes involve high separation and purification costs. Traditional chromatographic separation techniques require expensive equipment, are complex to operate, and consume large amounts of solvents, making it difficult to achieve continuous and large-scale production.
The whole-cell wet bacterial transformation using recombinant ribose-5-phosphate isomerase was combined with treatment using ceramic membranes, ultrafiltration membranes, cation exchange resin columns, and anion exchange resin columns. D-aloose was then selectively separated by crystallization using an ethanol-water system, reducing the number of chromatographic separation steps.
It reduces equipment investment and operating costs, improves the purity and yield of D-allose, achieves low-cost and efficient separation, conforms to green chemistry principles, and reduces solvent and wastewater emissions.
Smart Images
Figure CN122011056A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of D-allose production technology, and specifically to a method for separating and preparing D-allose. Background Technology
[0002] D-Allose is a rare sugar with potential bioactivity and applications, attracting widespread attention, particularly in the pharmaceutical, food, and functional health product fields. Currently, the preparation of D-Allose mainly relies on biocatalysis, using allulose as a substrate and converting it to D-Allose via ribose-5-phosphate isomerase catalysis. However, existing production processes face the problem of high separation and purification costs. Traditional methods generally rely on chromatographic separation techniques (such as simulated moving bed chromatography), which, while providing good separation results, involve expensive equipment investment, complex operation, high solvent consumption, and are difficult to scale up continuously, resulting in persistently high production costs for D-Allose. Summary of the Invention
[0003] The technical problem to be solved by the present invention is to provide a method for separating and preparing D-allose, which has good separation effect and low production cost, in order to address the shortcomings of the existing technology.
[0004] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:
[0005] A method for isolating and preparing D-allose includes the following steps:
[0006] A: D-allulose solution was converted into whole-cell wet bacteria containing recombinant ribose-5-phosphate isomerase, and the conversion solution was collected.
[0007] B: The conversion solution is sequentially treated through a ceramic membrane, an ultrafiltration membrane, a cation exchange resin column, and an anion exchange resin column, and the desalination solution is collected.
[0008] C: Concentrate the desalting solution to a solid content of 70-90% w / w and collect the concentrate;
[0009] D: Add ethanol to the concentrate, keep warm at 50-60℃ for 1-2 hours, then cool down to 22-28℃, and filter the cake to obtain crude D-aloose.
[0010] Preferably, the conversion system in step A further includes manganese chloride solution and phosphate buffer, the conversion temperature is 60-80℃, and the conversion time is 4-6h;
[0011] The concentration of D-allulose solution is 600-700 g / L, the amount of whole-cell wet bacteria containing recombinant ribose-5-phosphate isomerase is 50-100 g / L, the amount of manganese chloride solution is 0.5-1 mM, the amount of phosphate buffer is 20-50 mM, and the pH of phosphate buffer is 7-8.
[0012] Preferably, the ceramic membrane in step B has a pore size of 20-100 nm and the ultrafiltration membrane has a molecular weight cutoff of 2000-10000 Da.
[0013] Preferably, in step B, the flow rate of both the cation exchange resin column and the anion exchange resin column is 1-3 BV, and the conductivity of the desalination solution is <50 μs / cm.
[0014] Before use, cation exchange resin columns should be soaked in 4-5% wt hydrochloric acid solution for 1-2 hours, and then washed with deionized water until neutral. Before use, anion exchange resin columns should be soaked in 4-5% wt sodium hydroxide solution for 1-2 hours, and then washed with deionized water until neutral.
[0015] Preferably, the concentration temperature in step C is 50-70℃ and the vacuum degree is <-0.09MPa.
[0016] Preferably, the amount of ethanol added in step D is 1-1.5 times the volume of the concentrated liquid.
[0017] Preferably, the crude D-allose product of step D contains >95% D-allose.
[0018] Preferably, in step D, the crude D-allose is dissolved in purified water and then decolorized. Ethanol is added to the decolorized solution, the temperature is lowered to 10-15°C, filtered, washed with ethanol, and dried to obtain the finished D-allose product.
[0019] Preferably, the amount of purified water added is 0.5-1 times the weight of crude D-allose, the dissolution temperature is 60-70℃, the amount of activated carbon added during decolorization is 1-3% of the weight of crude D-allose, and the decolorization time is 30-60 min.
[0020] Preferably, the amount of ethanol added is 1-1.5 times the volume of the decolorizing liquid, and the D-allose content in the obtained D-allose product is >99%.
[0021] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:
[0022] 1. The solubility of D-allose in the ethanol-water system is lower than that of D-allulose, with the greatest difference observed at 25°C. This invention is the first to employ "selective crystallization separation in an ethanol-water system" as an alternative to the chromatographic separation method commonly relied upon in the industry. Chromatographic equipment is expensive, energy-intensive, and requires frequent regeneration due to resin depletion. This method uses only conventional crystallization vessels and filtration equipment, significantly reducing equipment investment and lowering the barrier to industrialization. Furthermore, by precisely controlling the ethanol-water ratio and crystallization conditions, highly efficient separation of D-allose and D-allulose is achieved. Ethanol can be efficiently recovered and reused, greatly reducing solvent consumption and avoiding the use of large amounts of acid and alkali regeneration solutions and rinsing water in chromatographic separation, thus significantly reducing operating costs.
[0023] 2. Through a "ceramic membrane filtration" step, whole-cell wet bacterial cells containing active enzymes are efficiently retained and directly returned to the conversion reactor for reuse. This not only avoids the cost of each enzyme feed but also, because the enzyme activity remains good under suitable conditions, the catalyst utilization rate can be increased several times, fundamentally reducing the unit cost of biocatalysts. Furthermore, the mother liquor after separating crude D-allose is rich in unconverted D-allulose, which, after dealcoholization, can be directly returned to the first conversion step as substrate for reuse. This achieves a closed-loop substrate cycle, increasing the D-allulose conversion rate from approximately 35% per cycle to close to the theoretical total yield, maximizing raw material utilization, and significantly reducing production costs.
[0024] 3. A pretreatment combination of "ceramic membrane → ultrafiltration membrane" is employed. The ceramic membrane ensures complete catalyst recovery, while the ultrafiltration membrane provides effective protection for the subsequent ion exchange resin, preventing resin contamination and clogging, extending resin lifespan, and ensuring long-term stable operation of the ion exchange process. The ion exchange desalination process effectively removes inorganic salt ions and charged impurities, keeping the feed solution conductivity consistently below 50 μS / cm, creating a high-purity material environment for subsequent crystallization. After concentration, selective crystallization with ethanol is employed; the steps are clear, the conditions are mild and controllable, and the final product has a stable purity >99%, good crystal form, and is easy to process.
[0025] 4. Throughout the process, water is primarily used for preparing buffer solutions, resin cleaning, and product purification and dissolution, with a significant amount of process water (such as membrane filtration clarified liquid and washing water) being recycled within the system. Compared to traditional chromatographic separation and extensive rinsing processes, overall process water consumption is greatly reduced. Simultaneously, the recycling of microbial cells reduces the generation of biological solid waste; substrate reuse lowers the organic load in wastewater; and the recovery and reuse of ethanol solvent reduces VOC emissions. The amount of waste acid and alkali generated during ion exchange resin regeneration is also reduced due to effective pretreatment at the front end. The entire process embodies the green chemistry principles of "reduction, reuse, and recycling." Attached Figure Description
[0026] Figure 1This is the liquid chromatogram of D-allose in Example 1 of the present invention. Detailed Implementation
[0027] The present invention will be further illustrated below with reference to the embodiments. Example 1
[0028] 1. Prepare 20 L of 600 g / L D-allulose solution, add whole-cell wet bacterial cells containing recombinant ribose-5-phosphate isomerase at a concentration of 50 g / L, add 0.5 mM MnCl2, add 20 mM phosphate buffer (pH 7), and incubate at 60 °C for 6 h to obtain 21 L of incubation solution containing D-allulose.
[0029] 2. The conversion solution was filtered through a ceramic membrane, and 28L of the clear solution was collected. The concentrated solution was the bacterial solution, which was collected and reused for conversion. The pore size of the ceramic membrane was 20nm.
[0030] 3. Pass the ceramic membrane supernatant through the ultrafiltration membrane and collect 35L of the supernatant. The molecular weight cutoff of the ultrafiltration membrane is 2000 Da.
[0031] 4. The ultrafiltration membrane supernatant is passed through a cation exchange resin column at a flow rate of 1 BV, and then through an anion exchange resin column at a flow rate of 1 BV for desalination, yielding 42 L of desalted solution. The conductivity of the desalted solution is <50 μS / cm. The cation exchange resin used is SQ-605, and the anion exchange resin is SQD-936. Before use, the cation exchange resin is soaked in 4% wt hydrochloric acid solution for 2 h, and then washed with deionized water until neutral. Before use, the anion exchange resin is soaked in 4% wt sodium hydroxide solution for 2 h, and then washed with deionized water until neutral.
[0032] 5. The desalting solution was concentrated at 50℃ and a vacuum of <-0.09MPa to a solid content of 70% w / w, yielding 17L of concentrate.
[0033] 6. When the temperature of the concentrate is 50℃, add 95% ethanol with a volume equal to that of the concentrate, keep warm for 2 hours, then control the stirring speed to 30 rpm, cool the liquid to 28℃, filter and collect 29L of filter cake and filtrate, wherein the filter cake is D-alokose wet crude product, 4500g, with a content of 96.4%, and the filtrate is de-ethanolified and returned to step 1 for further conversion.
[0034] 7. Dissolve crude D-allose in 0.5 times its weight of water at 60°C. Add 1% of the crude product weight of activated carbon for decolorization for 60 minutes. After filtration, collect 5.1 L of the decolorized solution.
[0035] 8. Slowly add ethanol of 1 volume to the decolorizing solution to crystallize, cool to 15°C, filter, wash with ethanol, and dry to obtain D-allose product, 2650g, total yield 63.1%, purity 99.6%. Example 2
[0036] 1. Prepare 20 L of 650 g / L D-allulose solution, add whole-cell wet bacterial cells containing recombinant ribose-5-phosphate isomerase at a concentration of 80 g / L, add 0.8 mM MnCl2, add 30 mM phosphate buffer (pH 8), and invert at 70 °C for 5 h to obtain 22 L of inversion solution containing D-allulose.
[0037] 2. The conversion solution was filtered through a ceramic membrane, and 29 L of the clear solution was collected. The concentrated solution was the bacterial solution, which was collected and reused for conversion. The pore size of the ceramic membrane was 50 nm.
[0038] 3. Pass the ceramic membrane supernatant through the ultrafiltration membrane and collect 37 L of the supernatant. The molecular weight cutoff of the ultrafiltration membrane is 8000 Da.
[0039] 4. The ultrafiltration membrane supernatant was passed through a cation exchange resin column at a flow rate of 2 BV, and then through an anion exchange resin column at a flow rate of 2 BV for desalination, yielding 44 L of desalted solution. The conductivity of the desalted solution was made <50 μS / cm. The cation exchange resin used was SQ-605, and the anion exchange resin used was SQD-936. Before use, the cation exchange resin was soaked in 4.5% wt hydrochloric acid solution for 1.5 h, and then washed with deionized water until neutral. Before use, the anion exchange resin was soaked in 4.5% wt sodium hydroxide solution for 1.5 h, and then washed with deionized water until neutral.
[0040] 5. The desalting solution was concentrated at 60℃ and a vacuum of <-0.09MPa to a solid content of 80% w / w, yielding 16L of concentrated solution.
[0041] 6. When the temperature of the concentrate is 55℃, add 1.2 times the volume of 95% ethanol and keep warm for 1.5h. Then control the stirring speed to 80rpm and cool the liquid to 25℃. Filter and collect 30L of filter cake and filtrate. The filter cake is 4900g of D-allose wet crude product with a content of 96.5%. After the filtrate is de-ethanolified, return it to step 1 for further conversion.
[0042] 7. D-Allose crude product was dissolved in 0.8 times its weight of water at 65℃. Activated carbon at 2% of the crude product weight was added for decolorization for 45 minutes. After filtration, 7.3L of the decolorized solution was collected.
[0043] 8. Slowly add 1.2 times the volume of ethanol to the decolorizing solution to crystallize, cool to 12°C, filter, wash with ethanol, and dry to obtain D-allose product, 2920g, yield 64.2%, purity 99.5%. Example 3
[0044] 1. Prepare 20 L of 700 g / L D-allulose solution, add whole-cell wet bacterial cells containing recombinant ribose-5-phosphate isomerase at a concentration of 100 g / L, add 1 mM MnCl2, add 50 mM phosphate buffer (pH 7), and incubate at 80 °C for 4 h to obtain 23 L of incubation solution containing D-allulose.
[0045] 2. The conversion solution was filtered through a ceramic membrane, and 31 L of the clear solution was collected. The concentrated solution was the bacterial solution, which was collected and reused for conversion. The pore size of the ceramic membrane was 100 nm.
[0046] 3. Pass the ceramic membrane supernatant through the ultrafiltration membrane and collect 40L of the supernatant. The molecular weight cutoff of the ultrafiltration membrane is 10000Da.
[0047] 4. The ultrafiltration membrane supernatant is passed through a cation exchange resin column at a flow rate of 3 BV, and then through an anion exchange resin column at a flow rate of 3 BV for desalination, yielding 51 L of desalted solution. The conductivity of the desalted solution is <50 μS / cm. The cation exchange resin used is SQ-605, and the anion exchange resin used is SQD-936. Before use, the cation exchange resin is soaked in 5% wt hydrochloric acid solution for 1 h, and then washed with deionized water until neutral. Before use, the anion exchange resin is soaked in 5% wt sodium hydroxide solution for 1 h, and then washed with deionized water until neutral.
[0048] 5. The desalting solution was concentrated at 70℃ and a vacuum of <-0.09MPa to a solid content of 90% w / w, yielding 15L of concentrated solution.
[0049] 6. When the temperature of the concentrate is 60℃, add 1.5 times the volume of 95% ethanol and keep warm for 1 hour. Then control the stirring speed to 100 rpm and cool the liquid to 22℃. Filter and collect 32L of filter cake and filtrate. The filter cake is 5400g of D-allose wet crude product with a content of 95.7%. After the filtrate is de-ethanolified, it is returned to step 1 for further conversion.
[0050] 7. Dissolve crude D-allose in water at 1 times its weight at 70°C. Add 3% of the crude product weight of activated carbon for decolorization for 30 minutes. After filtration, collect 9L of the decolorized solution.
[0051] 8. Slowly add 1.5 times the volume of ethanol to the decolorizing solution to crystallize, cool to 10°C, filter, wash with ethanol, and dry to obtain D-allose product, 3180g, yield 64.9%, purity 99.6%.
[0052] The solubility of D-allose and D-allulose in the ethanol-water system at different temperatures was detected, and the results are shown in the table below:
[0053] Table 1
[0054]
[0055] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.
Claims
1. A method for isolating and preparing D-allose, characterized in that... Includes the following steps: A: D-allulose solution was converted into whole-cell wet bacteria containing recombinant ribose-5-phosphate isomerase, and the conversion solution was collected. B: The conversion solution is sequentially treated through a ceramic membrane, an ultrafiltration membrane, a cation exchange resin column, and an anion exchange resin column, and the desalination solution is collected. C: Concentrate the desalting solution to a solid content of 70-90% w / w and collect the concentrate; D: Add ethanol to the concentrate, keep warm at 50-60℃ for 1-2 hours, then cool down to 22-28℃, and filter the cake to obtain crude D-allose.
2. The method for isolating and preparing D-allose as described in claim 1, characterized in that: The conversion system in step A also includes manganese chloride solution and phosphate buffer, with a conversion temperature of 60-80℃ and a conversion time of 4-6 hours; The concentration of D-allulose solution is 600-700 g / L, the amount of whole-cell wet bacteria containing recombinant ribose-5-phosphate isomerase is 50-100 g / L, the amount of manganese chloride solution is 0.5-1 mM, the amount of phosphate buffer is 20-50 mM, and the pH of phosphate buffer is 7-8.
3. The method for separating and preparing D-allose as described in claim 1, characterized in that: The ceramic membrane in step B has a pore size of 20-100 nm and the ultrafiltration membrane has a molecular weight cutoff of 2000-10000 Da.
4. The method for separating and preparing D-allose as described in claim 1, characterized in that: In step B, the flow rate of both the cation exchange resin column and the anion exchange resin column is 1-3 BV, and the conductivity of the desalting solution is <50 μs / cm. Before use, cation exchange resin columns should be soaked in 4-5% wt hydrochloric acid solution for 1-2 hours, and then washed with deionized water until neutral. Before use, anion exchange resin columns should be soaked in 4-5% wt sodium hydroxide solution for 1-2 hours, and then washed with deionized water until neutral.
5. The method for isolating and preparing D-allose as described in claim 1, characterized in that: In step C, the concentration temperature is 50-70℃ and the vacuum degree is <-0.09MPa.
6. The method for isolating and preparing D-allose as described in claim 1, characterized in that: In step D, the amount of ethanol added is 1-1.5 times the volume of the concentrated liquid.
7. The method for isolating and preparing D-allose as described in claim 1, characterized in that: The crude D-allose product from step D contains >95% D-allose.
8. The method for isolating and preparing D-allose as described in claim 1, characterized in that: In step D, the crude D-allose is dissolved in purified water and then decolorized. Ethanol is added to the decolorized solution, the temperature is lowered to 10-15℃, filtered, washed with ethanol, and dried to obtain the finished D-allose product.
9. The method for isolating and preparing D-allose as described in claim 8, characterized in that: The amount of purified water added is 0.5-1 times the weight of crude D-allose, the dissolution temperature is 60-70℃, the amount of activated carbon added during decolorization is 1-3% of the weight of crude D-allose, and the decolorization time is 30-60 minutes.
10. The method for isolating and preparing D-allose as described in claim 8, characterized in that: The amount of ethanol added is 1-1.5 times the volume of the decolorizing liquid, and the D-allose content in the obtained D-allose product is >99%.