A method for preparing D-allose

Abstract

This invention discloses a method for preparing D-allose, relating to the field of biotransformation technology. Using D-allulose as a substrate, whole cells containing recombinant ribose-5-phosphate isomerase are added to a phosphate buffer system for biotransformation, yielding a transformation solution containing D-allose. The transformation solution is then filtered, and the supernatant is collected. Glucose isomerase and glucose oxidase are added to the supernatant, along with calcium carbonate, to convert unconverted D-allulose into calcium gluconate. The resulting mixture is then purified to obtain D-allose and calcium gluconate, respectively. This process is simple, achieves simultaneous high-value separation of the main product and byproduct, and improves the economic benefits of the product.

Description

Technical Field

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

[0002] D-Allose is a rare aldose, belonging to the category of all-natural, low-calorie sweeteners. Due to its unique physiological functions, such as anti-inflammatory, anti-tumor, immunosuppressive, and food texture-maintaining properties at low temperatures, D-Allose has broad application prospects in functional foods, pharmaceuticals, and health products. However, because D-Allose is present in extremely small amounts in nature, it is difficult to obtain through direct extraction. Therefore, developing efficient and low-cost biosynthetic preparation methods has become a current research hotspot.

[0003] Currently, the main route for preparing D-allose is to convert D-allulose to D-allose using isomerases. Although this technical route has made some progress in laboratory research, it still faces many challenges in industrial-scale applications:

[0004] 1. Low conversion rate and substrate residue: Due to the thermodynamic equilibrium of the enzymatic reaction, the conversion rate of D-allulose to D-allose catalyzed by ribose-5-phosphate isomerase is usually low (generally around 30%-40%). This means that after the reaction, a large amount of unconverted D-allulose remains in the system.

[0005] 2. Difficulty and high cost in separation and purification: Due to the extreme similarity in molecular structure, polarity, and physicochemical properties between D-allose and unreacted D-allulose, traditional chromatographic separation or crystallization methods are difficult to achieve efficient separation. To obtain high-purity D-allose, multiple repeated chromatography or complex derivatization steps are often required, resulting in reduced yield, huge solvent consumption, and high production costs.

[0006] 3. Byproduct treatment and resource waste: Existing technologies typically treat unreacted D-allulose as an impurity for removal or recycling, but the recycling process can easily lead to the accumulation of side reactions or a decrease in enzyme activity. Furthermore, the lack of value-added treatment methods for residual substrates results in the waste of raw materials. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a method for preparing D-allose that addresses the shortcomings of existing technologies. This method is simple, achieves simultaneous high-value separation of main products and by-products, and improves the economic benefits of the products.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0009] A method for preparing D-allose includes the following steps:

[0010] A: Using D-allulose as a substrate, whole cells containing recombinant ribose-5-phosphate isomerase were added to a phosphate buffer system for biotransformation to obtain a transformation solution containing D-allulose.

[0011] B: Filter and separate the conversion liquid obtained in step A, and collect the clear liquid;

[0012] C: Add glucose isomerase and glucose oxidase to the clear liquid obtained in step B, and add calcium carbonate to convert the unconverted D-allulose into calcium gluconate.

[0013] D: The mixture obtained in step C is separated and purified to obtain D-allose and calcium gluconate, respectively.

[0014] Preferably, the specific process of biotransformation in step A is as follows: prepare a D-allulose solution with a concentration of 600-700 g / L, add 50-100 g / L of whole-cell wet bacterial cells containing recombinant ribose-5-phosphate isomerase, add 0.5-1 mM MnCl2, and react in a phosphate buffer solution with a concentration of 20-50 mM and a pH of 7-8 at 60-80°C for 4-6 h.

[0015] Preferably, the filtration method in step B is ceramic membrane filtration. The collected clear liquid from the ceramic membrane enters the next step, while the retained concentrated liquid from the ceramic membrane is a bacterial concentrate containing enzymes, which is returned to step A for recycling and biotransformation.

[0016] Preferably, the glucose isomerase and glucose oxidase mentioned in step C are immobilized dual enzymes, and the amount added accounts for 5-10 g / L of the supernatant.

[0017] Preferably, in step C, calcium carbonate is added in the form of a slurry via a feed method, wherein the amount of calcium carbonate added is 0.3-0.4 times the weight of the substrate D-allulose, the reaction process is controlled with pH value of 6.0-7.0, temperature of 30-35℃, dissolved oxygen ≥20%, and reaction time of 6-8h.

[0018] Preferably, the separation and purification in step D includes: first filtering and washing the reaction solution, and collecting the filtrate; filtering the filtrate through an ultrafiltration membrane and collecting the ultrafiltration membrane supernatant; adding activated carbon to the ultrafiltration membrane supernatant for decolorization, concentrating it, adding calcium gluconate seed crystals to induce crystallization, filtering to separate the calcium gluconate crystals, and retaining the filtrate.

[0019] Preferably, the activated carbon decolorization conditions are: the amount of activated carbon added is 1-2% v / v of the liquid volume, and the decolorization temperature is 50-60℃;

[0020] The ultrafiltration membrane supernatant was decolorized with activated carbon and then concentrated to a solid content of 40-50% w / w.

[0021] The amount of calcium gluconate seed crystals added is 0.5-1% v / v of the liquid volume. After holding at this temperature for 0.5-1h, the temperature is lowered to 5-15℃ for crystallization.

[0022] Preferably, the filtrate after separating calcium gluconate is passed sequentially through a cation exchange resin and an anion exchange resin at a flow rate of 1-2 BV. The column chromatography solution is collected, concentrated, and then 95% ethanol is added for crystallization. After filtration and drying, D-allose product is obtained.

[0023] Preferably, the column chromatography solution is concentrated to a solid content >80% w / w, and the amount of 95% ethanol added is 1-2 times the volume of the concentrate.

[0024] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0025] 1. After the first isomerization reaction, instead of directly performing the challenging allosugar / allulose separation, an immobilized glucose isomerase and glucose oxidase dual-enzyme system is introduced to directionally convert approximately 65% ​​of the remaining unreacted D-allulose into gluconic acid, which further reacts with calcium carbonate to form calcium gluconate. D-allose and calcium gluconate differ significantly in solubility, charge properties, and crystallization behavior. This strategy transforms a "difficult-to-separate mixture of isomers" into an "easily separable mixture of sugars and salts," greatly reducing the difficulty and cost of subsequent separation and purification. Simultaneously, it transforms the byproduct calcium gluconate into an independent product with market value, achieving full high-value utilization of the raw materials.

[0026] 2. By utilizing ceramic membrane filtration technology, enzyme-containing bacteria are retained and returned to the reaction system for continued catalysis, while the supernatant permeates through the membrane to the next process. This "reaction-separation" coupling mode not only improves equipment utilization but also significantly reduces the amount of fresh bacteria required, thereby substantially lowering production costs.

[0027] 3. In the second conversion step, immobilized glucose isomerase and glucose oxidase were used, and the pH value (6.0-7.0) was precisely controlled by adding calcium carbonate. The immobilized enzyme is easy to recover and reuse through filtration, reducing enzyme consumption; the addition of calcium carbonate not only neutralized the generated acid but also maintained the optimal pH environment required for enzyme activity, ensuring that both enzymes remained at their highest activity level throughout the reaction, thus guaranteeing that the 65% conversion rate target was achieved.

[0028] 4. Utilizing the characteristic that calcium gluconate's solubility decreases at low temperatures, most of the calcium gluconate is preferentially removed in crystalline form through steps such as adding seed crystals and cooling crystallization, significantly reducing the load on subsequent resin columns. For remaining trace impurities, specific cation exchange resins and anion exchange resins are used in series for column chromatography. This combination effectively removes residual metal ions, pigments, and trace organic acids, greatly improving the purity of the product D-allose solution.

[0029] 5. Throughout the entire process, the bacterial cells are recycled, the immobilized enzymes are reused, and the byproduct calcium gluconate is recovered and reused, resulting in no large-scale waste substrate discharge. The ultrafiltration concentrate and a small amount of waste liquid are treated in an environmentally friendly manner, and the activated carbon decolorization and resin regeneration processes are controllable. Compared with traditional multiple chromatography processes, this significantly reduces the consumption of organic solvents and chemical reagents, meeting the requirements of green manufacturing. Attached Figure Description

[0030] Figure 1 This is the liquid chromatogram of D-allose in Example 1 of the present invention. Detailed Implementation

[0031] The present invention will be further illustrated below with reference to the embodiments. Example 1

[0032] 1. Prepare 50 L of 600 g / L D-allulose solution, add whole-cell wet bacterial cells containing recombinant ribose-phosphoisomerase at a concentration of 50 g / L, add 0.5 mM MnCl2 solution, and incubate at 60 °C for 6 h in 20 mM phosphate buffer (pH 7) to obtain 52 L of conversion solution containing D-allulose, with a conversion rate of 32.3%.

[0033] 2. The conversion solution is filtered through a ceramic membrane, and 47L of the clear solution and 5L of the concentrated solution containing enzymes are collected. The solution is then returned to step 1 for further conversion.

[0034] 3. Immobilized glucose isomerase and glucose oxidase were added to the ceramic membrane supernatant at a concentration of 5 g / L. Then, calcium carbonate slurry was added by feeding, with the amount of calcium carbonate being 0.3 times the weight of the substrate D-allulose. The conversion was controlled at pH 6.0, temperature 30℃, dissolved oxygen ≥20%, and the conversion time was 8 h.

[0035] 4. Filter the conversion solution, rinse the filter residue with water, collect the filtrate for later use, and use the immobilized enzyme for conversion again.

[0036] 5. The filtrate is filtered through an ultrafiltration membrane, and 116L of the ultrafiltration membrane supernatant is collected. The concentrated liquid is then treated in an environmentally friendly manner.

[0037] 6. Add 1% v / v activated carbon to the ultrafiltration membrane supernatant for decolorization at 50℃. Concentrate the decolorized solution to a solid content of 40% w / w, yielding 75L of concentrate. Add 0.5% v / v calcium gluconate seed crystals to the concentrate, keep warm for 0.5h, cool to 15℃, filter, wash with water, and the filter cake is calcium gluconate crystals. After drying, the amount is 21615g, yield 89.1%, purity 99.2%, and 52L of filtrate is collected for later use.

[0038] 7. The filtrate is first passed through a cation exchange resin column, then through an anion exchange resin column, and the column pass solution is collected. The column flow rate is 1 BV. The cation exchange resin used is D-67, and the anion exchange resin used is SQD-936. The column pass solution is D-aloose solution with a purity of 95.7%.

[0039] 8. The D-allose solution was concentrated by column chromatography to a solid content of 81% w / w, yielding 12 L of concentrate. 95% ethanol was added to the concentrate to crystallize, and the solution was filtered and dried to obtain 7955 g of D-allose product, with a yield of 82.1% and a purity of 99.5%. The amount of 95% ethanol added was 1 times the volume of the concentrate. Example 2

[0040] 1. Prepare 50 L of 650 g / L D-allulose solution, add whole-cell wet bacterial cells containing recombinant ribose-phosphoisomerase at a concentration of 75 g / L, add 0.75 mM MnCl2 solution, and incubate at 70 °C for 5 h in 35 mM phosphate buffer (pH 7.5) to obtain a conversion solution containing D-allulose with a conversion rate of 33.5%.

[0041] 2. The conversion solution is filtered through a ceramic membrane, and 48L of the clear solution and 6L of the concentrated solution containing enzymes are collected. The conversion solution is then returned to step 1 for further conversion.

[0042] 3. Immobilized glucose isomerase and glucose oxidase were added to the ceramic membrane supernatant at a concentration of 8 g / L. Then, calcium carbonate slurry was added by feeding, with the amount of calcium carbonate being 0.35 times the weight of the substrate D-allulose. The conversion was controlled at pH 7.0, temperature 32℃, dissolved oxygen ≥20%, and the conversion time was 7 h.

[0043] 4. Filter the conversion solution, rinse the filter residue with water, collect the filtrate for later use, and use the immobilized enzyme for conversion again.

[0044] 5. The filtrate is filtered through an ultrafiltration membrane, and 125L of the ultrafiltration membrane supernatant is collected. The concentrated liquid is then treated in an environmentally friendly manner.

[0045] 6. Add 1.5% v / v activated carbon to the ultrafiltration membrane supernatant for decolorization at 55℃. Concentrate the decolorized solution to a solid content of 45% w / w, yielding 73L of concentrate. Add 0.8% v / v calcium gluconate seed crystals to the concentrate, keep warm for 0.8h, cool to 10℃, filter, wash with water, and the filter cake is calcium gluconate crystals. After drying, the amount is 22898g, with a yield of 88.7% and a purity of 99.3%. Collect 51L of filtrate for later use.

[0046] 7. The filtrate is first passed through a cation exchange resin column, then through an anion exchange resin column, and the column pass solution is collected. The column flow rate is 1.5 BV. The cation exchange resin used is D-67, and the anion exchange resin used is SQD-936. The column pass solution is a D-aloose solution with a purity of 95.5%.

[0047] 8. The D-allose solution was concentrated by column chromatography to a solid content of 82% w / w, yielding 13.3 L of concentrate. 95% ethanol was added to the concentrate to crystallize, and the solution was filtered and dried to obtain 9112 g of D-allose product, with a yield of 83.7% and a purity of 99.4%. The amount of 95% ethanol added was 1.5 times the volume of the concentrate. Example 3

[0048] 1. Prepare 50 L of 700 g / L D-allulose solution, add whole-cell wet bacterial cells containing recombinant ribose-phosphoisomerase at a concentration of 100 g / L, add 1 mM MnCl2 solution, and incubate at 80 °C for 4 h in 50 mM phosphate buffer (pH 8) to obtain a conversion solution containing D-allulose with a conversion rate of 35.0%.

[0049] 2. The conversion solution is filtered through a ceramic membrane, and 49L of the clear solution and 8L of the concentrated solution containing enzymes are collected. The conversion solution is then returned to step 1 for further conversion.

[0050] 3. Immobilized glucose isomerase and glucose oxidase were added to the ceramic membrane supernatant at a concentration of 10 g / L. Then, calcium carbonate slurry was added by feeding, with the amount of calcium carbonate being 0.4 times the weight of the substrate D-allulose. The conversion was controlled at pH 7.0, temperature 35℃, dissolved oxygen ≥20%, and the conversion time was 6 h.

[0051] 4. Filter the conversion solution, rinse the filter residue with water, collect the filtrate for later use, and use the immobilized enzyme for conversion again.

[0052] 5. The filtrate is filtered through an ultrafiltration membrane, and 134L of the ultrafiltration membrane supernatant is collected. The concentrated liquid is then treated in an environmentally friendly manner.

[0053] 6. Add 2% v / v activated carbon to the ultrafiltration membrane supernatant for decolorization at 60℃. Concentrate the decolorized solution to a solid content of 50% w / w, yielding 70L of concentrate. Add 1% v / v calcium gluconate seed crystals to the concentrate, keep warm for 1 hour, cool to 5℃, filter, wash with water, and the filter cake is calcium gluconate crystals. After drying, the amount is 24266g, with a yield of 89.3% and a purity of 99.2%. Collect 46L of filtrate for later use.

[0054] 7. The filtrate is first passed through a cation exchange resin column, then through an anion exchange resin column, and the column pass solution is collected. The column flow rate is 2 BV. The cation exchange resin used is D-67, and the anion exchange resin used is SQD-936. The column pass solution is D-aloose solution with a purity of 95.3%.

[0055] 8. The D-allose solution was concentrated by column chromatography to a solid content of 85% w / w, yielding 14.4 L of concentrate. 95% ethanol was added to the concentrate to crystallize, and the solution was filtered and dried to obtain 10363 g of D-allose product, with a yield of 84.6% and a purity of 99.5%. The amount of 95% ethanol added was twice the volume of the concentrate.

[0056] 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 preparing D-allose, characterized in that... Includes the following steps: A: Using D-allulose as a substrate, whole cells containing recombinant ribose-5-phosphate isomerase were added to a phosphate buffer system for biotransformation to obtain a transformation solution containing D-allulose. B: Filter and separate the conversion liquid obtained in step A, and collect the clear liquid; C: Add glucose isomerase and glucose oxidase to the clear liquid obtained in step B, and add calcium carbonate to convert the unconverted D-allulose into calcium gluconate. D: The mixture obtained in step C is separated and purified to obtain D-allose and calcium gluconate, respectively.

2. The method for preparing D-allose as described in claim 1, characterized in that... The specific process of biotransformation in step A is as follows: prepare a D-allulose solution with a concentration of 600-700 g / L, add 50-100 g / L of whole-cell wet bacterial cells containing recombinant ribose-5-phosphate isomerase, add 0.5-1 mM MnCl2 solution, and react at 60-80℃ for 4-6 h in a phosphate buffer solution with a concentration of 20-50 mM and a pH of 7-8.

3. The method for preparing D-allose as described in claim 1, characterized in that: In step B, the filtration method is ceramic membrane filtration. The collected clear liquid from the ceramic membrane enters the next step, while the retained concentrated liquid from the ceramic membrane is the enzyme-containing bacterial concentrate, which is returned to step A for recycling and biotransformation.

4. The method for preparing D-allose as described in claim 1, characterized in that: The glucose isomerase and glucose oxidase mentioned in step C are immobilized dual enzymes, and the amount added accounts for 5-10 g / L of the supernatant volume.

5. The method for preparing D-allose as described in claim 4, characterized in that: In step C, calcium carbonate is added in the form of a slurry via a feed-feed method. The amount of calcium carbonate added is 0.3-0.4 times the weight of the substrate D-allulose. The reaction process is controlled with a pH of 6.0-7.0, a temperature of 30-35℃, dissolved oxygen ≥20%, and a reaction time of 6-8 hours.

6. The method for preparing D-allose as described in claim 1, characterized in that, Step D, the separation and purification process, includes: first, filtering and washing the reaction solution, and collecting the filtrate; filtering the filtrate through an ultrafiltration membrane and collecting the supernatant; adding activated carbon to the supernatant for decolorization, concentrating it, adding calcium gluconate seed crystals to induce crystallization, filtering to separate the calcium gluconate crystals, and retaining the filtrate.

7. The method for preparing D-allose as described in claim 6, characterized in that, The conditions for activated carbon decolorization are as follows: the amount of activated carbon added is 1-2% v / v of the liquid volume, and the decolorization temperature is 50-60℃. The ultrafiltration membrane supernatant was decolorized with activated carbon and then concentrated to a solid content of 40-50% w / w. The amount of calcium gluconate seed crystals added is 0.5-1% v / v of the liquid volume. After holding at this temperature for 0.5-1h, the temperature is lowered to 5-15℃ for crystallization.

8. The method for preparing D-allose as described in claim 6, characterized in that, The filtrate after separating calcium gluconate was passed sequentially through a cation exchange resin and an anion exchange resin at a flow rate of 1-2 BV. The column chromatography solution was collected, concentrated, and then 95% ethanol was added for crystallization. After filtration and drying, D-aloose was obtained as the final product.

9. The method for preparing D-allose as described in claim 8, characterized in that: Concentrate the column chromatography solution to a solid content >80% w / w, and add 95% ethanol at a volume of 1-2 times that of the concentrate.

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