A preparation method of D-psicose

Abstract

The present invention relates to the field of biotechnology, and more particularly to a method for preparing D-psicose, comprising the following steps: (1) using D-fructose as a substrate, adding a solvent and a first whole-cell catalyst, adjusting the pH of the reaction system to a weakly acidic state, controlling the temperature of the reaction system, and collecting the supernatant after the reaction is completed by centrifugation to obtain a D-allitol solution; and (2) taking the D-allitol solution in step (1), adjusting the pH to a neutral state, and adding a second whole-cell catalyst under ventilation and stirring to obtain a psicose solution after the reaction is completed. The above method greatly improves the conversion rate of D-fructose to D-psicose.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing D-psicose. Background Art

[0002] D-psicose is a rare and important ketohexose with 70% the sweetness of sucrose but almost zero energy. Its solvent properties and taste are similar to those of sucrose, and it can undergo the Maillard reaction. D-psicose is an ideal substitute for sucrose in the food and pharmaceutical industries. Furthermore, D-psicose has the potential to reduce obesity, lower blood sugar, and lower blood lipids, making it ideal for diabetics and obese individuals. Therefore, it has significant benefits in medical and healthcare settings.

[0003] Currently, there are four main pathways for the biosynthesis of D-psicose: (1) One-step isomerization of D-fructose: DPE is an isomerase that catalyzes the reversible reaction of D-fructose and D-psicose, but has a low conversion rate of about 30%. Therefore, large-scale production of D-psicose from D-fructose and product purification remain a challenge; (2) Two-step isomerization of D-glucose: Using glucose as a substrate, D-psicose is synthesized by glucose synthesis. Sugar isomerase converts it into fructose, which is then converted to D-psicose via DPE. (3) D-psicose is synthesized from starch via a multienzyme cascade reaction: 50 g / L starch (275 mm glucose equivalent) is synthesized using starch isomerase, α-glucan phosphatase, phosphoglucoamylase, phosphoglucoamylase, D-psicose 6-phosphate isomerase, and D-psicose 6-phosphate phosphatase. (4) Microbial conversion of allitol (D-allitol) produces D-psicose. Although the above methods are feasible, they suffer from low yields. Therefore, a method for preparing D-psicose was established to address this issue. Summary of the Invention

[0004] The technical problem to be solved by the present invention is: to address the deficiencies in the existing technology, to provide a method for preparing D-psicose, and to utilize the preparation method to improve the conversion rate of D-fructose and reduce production costs.

[0005] In order to solve the above technical problems, the technical solution of the present invention is:

[0006] A method for preparing D-psicose, comprising the following steps:

[0007] (1) Using D-fructose as a substrate, adding a solvent and a first whole-cell catalyst, adjusting the pH of the reaction system to a weakly acidic state, controlling the temperature of the reaction system, and centrifuging and collecting the supernatant after the reaction is completed to obtain the D-alliitol solution;

[0008] (2) Taking the D-alliitol solution in step (1), adjusting the pH to neutral, and adding the second whole-cell catalyst under ventilation and stirring, and obtaining a psicose solution after the reaction is completed.

[0009] As an improved technical solution, the first whole-cell catalyst is named AO1, and the first whole-cell catalyst is a whole-cell catalyst that can simultaneously express the allulose epimerase (DPE) gene, the allitol dehydrogenase (RDH) gene, and the formate dehydrogenase (FDH) gene; the second whole-cell catalyst is named PO3, and the second whole-cell catalyst is a whole-cell catalyst that can simultaneously express the allitol dehydrogenase (RDH) gene and the NADH oxidase (NOX) gene.

[0010] As an improved technical solution, the strain from which the allulose epimerase gene that can be expressed by the first whole-cell catalyst is derived is selected from Clostridiales, the strain from which the allitol dehydrogenase gene is derived is selected from Providencia alcalifaciens, and the strain from which the formate dehydrogenase gene is derived is selected from Starkeya; the strain from which the allitol dehydrogenase (RDH) gene that can be expressed by the second whole-cell catalyst is selected from Providencia alcalifaciens, and the strain from which the NADH oxidase (NOX) gene is derived is selected from Streptococcus pyogenes.

[0011] As an improved technical solution, the preparation of the first whole-cell catalyst includes the following operations: connecting the psicose epimerase gene (DPE), the allitol dehydrogenase gene (RDH), and the formate dehydrogenase gene (FDH) to the pYB1s plasmid, and transforming the resulting recombinant plasmid pYB1s-DPE-RDH-FDH into Escherichia coli BW25113 to obtain the first whole-cell catalyst;

[0012] The preparation of the second whole-cell catalyst includes the following operations: connecting the allitol dehydrogenase (RDH) gene and the NADH oxidase gene (NOX) to the pYB1s plasmid and transforming the obtained recombinant plasmid pYB1s-RDH-NOX into Escherichia coli BW25113 to obtain the second whole-cell catalyst.

[0013] As an improved technical solution, in step (1), D-fructose and the first whole-cell catalyst are added in a ratio of 50-200 g / L and a wet weight concentration of 5-30 g / L, respectively; the solvent is a sodium formate solution or a potassium formate solution, and the concentration of the sodium formate solution or the potassium formate solution is 0.5-2.0 mol / L; the pH of the reaction system is 5.8-6.8, the reaction temperature is 35-40°C, and the reaction time is 11-13 h.

[0014] As a preferred technical solution, in step (1), D-fructose and the first whole-cell catalyst are added in a ratio of 100 g / L and a wet weight concentration of 20 g / L, respectively, the pH of the reaction system is 6.5, the reaction temperature is 40°C, and the reaction time is 12 h.

[0015] As an improved technical solution, the D-alliitol solution and the second whole-cell catalyst in step (2) are added in a ratio of 50-200 g / L and a wet weight concentration of 5-30 g / L, respectively; air is introduced at a flow rate of 1.5-2.5 L / min, and the dissolved oxygen in the reaction system is maintained at 48-53% v / v, the stirring speed is gradually increased from the initial 500 rpm to 1000 rpm, the reaction time is controlled at 13-15 h, and the reaction temperature is controlled at 35-40°C.

[0016] As a preferred technical solution, the D-alliitol solution and the second whole-cell catalyst in step (2) are added at a ratio of 100 g / L and a wet weight concentration of 10 g / L, respectively, and air is introduced at a flow rate of 1.8 L / min to maintain the dissolved oxygen content in the reaction system at 50% v / v. The pH of the reaction system is 7.0, the reaction temperature is 40° C., and the reaction time is 14.5 h.

[0017] The reaction equations in step (1) and step (2) in the preparation method of the present invention are:

[0018] Reaction of step (1):

[0019]

[0020] Reaction of step (2):

[0021]

[0022] After adopting the above technical solution, the beneficial effects of the present invention are:

[0023] The present invention uses D-fructose as a substrate, adjusts the pH of the reaction system to a weakly acidic state, and employs a first whole-cell catalyst that simultaneously expresses the genes for psicose epimerase (DPE), allitol dehydrogenase (RDH), and formate dehydrogenase (FDH) to generate an NADH regeneration system, catalyzing D-fructose to D-alitol. A second whole-cell catalyst that simultaneously expresses the genes for allitol dehydrogenase (RDH) and NADH oxidase (NOX) is then employed to oxidize D-alitol to D-psicose via a NOX-driven NADH regeneration system. In this synthetic route, the two NADH regeneration systems are completed through FDH-driven reduction and NOX-driven oxidation, respectively. Using this two-step, multi-enzyme tandem bioconversion system, the maximum theoretical product yield of D-fructose to D-psicose is 100%. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the high-phase liquid phase detection spectrum of D-psicose in Example 4. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0026] Example 1

[0027] A method for preparing D-psicose comprises the following steps:

[0028] (1) Substrate D-fructose 200 g / L, sodium formate solution 2.0 mol / L, the first whole-cell catalyst added in an amount of 30 g (wet weight) / L, the first whole-cell catalyst (simultaneously expressing the psicose epimerase gene, the allitol dehydrogenase gene, and the formate dehydrogenase gene) was placed in a bioreactor, the pH of the reaction system was adjusted to 5.8, the temperature of the reaction system was controlled to 35°C, the reaction was terminated after 11 hours, and the supernatant collected by centrifugation was the D-alitol solution;

[0029] (2) 0.5 L of the D-alliitol solution in step (1) with a D-alliitol concentration of 200 g / L was placed in a 1.0 L bioreactor, and air was introduced at a flow rate of 2.5 L / min to maintain the dissolved oxygen content in the reaction system at 53% v / v. The pH was adjusted to neutral, and 30 g (wet weight) / L of the second whole-cell catalyst (simultaneously expressing the allitol dehydrogenase gene and the NADH oxidase gene) was added under stirring. The stirring speed of the reaction system was gradually increased from 500 rpm to 1000 rpm, and the temperature of the reaction system was controlled at 35° C. The reaction was terminated after 14 hours to obtain a D-psicose solution.

[0030] Example 2

[0031] A method for preparing D-psicose comprises the following steps:

[0032] (1) Substrate D-fructose 180 g / L, sodium formate solution 1.5 mol / L, the first whole-cell catalyst added in an amount of 25 g (wet weight) / L, the first whole-cell catalyst (simultaneously expressing the psicose epimerase gene, the allitol dehydrogenase gene, and the formate dehydrogenase gene) was placed in a bioreactor, the pH of the reaction system was adjusted to 6.0, the temperature of the reaction system was controlled to 37°C, the reaction was terminated after 11.5 hours, and the supernatant collected by centrifugation was the D-alitol solution;

[0033] (2) 0.5 L of the D-alliitol solution in step (1) with a concentration of 180 g / L was taken and placed in a bioreactor with a volume of 1 L. Air was introduced at a flow rate of 2.3 L / min to maintain the dissolved oxygen content in the reaction system at 52% v / v. 25 g (wet weight) / L of a second whole-cell catalyst (which can simultaneously express the allitol dehydrogenase gene and the NADH oxidase gene) was added. The pH was adjusted to neutral. The reaction system was stirred at a speed gradually increased from 500 rpm to 1000 rpm. The temperature of the reaction system was controlled at 37° C. The reaction was terminated after 14.5 hours of reaction to obtain a D-psicose solution.

[0034] Example 3

[0035] A method for preparing D-psicose comprises the following steps:

[0036] (1) The substrate D-fructose was 150 g / L, the sodium formate solution was 1.0 mol / L, and the first whole-cell catalyst was added in an amount of 20 g (wet weight) / L. The first whole-cell catalyst (which simultaneously expressed the psicose epimerase gene, the allitol dehydrogenase gene, and the formate dehydrogenase gene) was placed in a bioreactor. The pH of the reaction system was adjusted to 6.3, and the temperature of the reaction system was controlled to 38°C. The reaction was terminated after 12 hours, and the supernatant collected by centrifugation was the D-alitol solution.

[0037] (2) 0.5 L of the D-alliitol solution in step (1) with a concentration of 150 g / L was taken and placed in a bioreactor with a volume of 1 L. Air was introduced at a flow rate of 2 L / min to maintain the dissolved oxygen content in the reaction system at 51% v / v. 20 g (wet weight) / L of a second whole-cell catalyst (which can simultaneously express the allitol dehydrogenase gene and the NADH oxidase gene) was added. The pH was adjusted to neutral. The stirring speed of the reaction system was gradually increased from 500 rpm to 1000 rpm. The temperature of the reaction system was controlled at 38° C. The reaction was terminated after 15 hours of reaction to obtain a D-psicose solution.

[0038] Example 4

[0039] A method for preparing D-psicose mannose comprises the following steps:

[0040] (1) The substrate D-fructose was 100 g / L, the sodium formate solution was 1.5 mol / L, and the first whole-cell catalyst was added in an amount of 10 g (wet weight) / L. The first whole-cell catalyst (which simultaneously expressed the psicose epimerase gene, the allitol dehydrogenase gene, and the formate dehydrogenase gene) was placed in a bioreactor. The pH of the reaction system was adjusted to 6.5, and the temperature of the reaction system was controlled to 40°C. The reaction was terminated after 12 hours, and the supernatant collected by centrifugation was the D-alitol solution.

[0041] (2) 0.5 L of the D-alliitol solution in step (1) with a concentration of 100 g / L was placed in a bioreactor with a volume of 1 L, and air was introduced at a flow rate of 1.8 L / min to maintain the dissolved oxygen content in the reaction system at 50% v / v. 10 g (wet weight) / L of a second whole-cell catalyst (capable of simultaneously expressing the allitol dehydrogenase gene and the NADH oxidase gene) was added, the pH was adjusted to neutral, and the reaction system was stirred at a speed gradually increased from 500 rpm to 1000 rpm. The temperature of the reaction system was controlled at 40° C. The reaction was terminated after 14.5 hours to obtain a D-psicose solution.

[0042] Example 5

[0043] A method for preparing D-psicose comprises the following steps:

[0044] (1) Substrate D-fructose 50 g / L, sodium formate solution 1.0 mol / L, and the first whole-cell catalyst added in an amount of 5 g (wet weight) / L. The first whole-cell catalyst (simultaneously expressing the psicose epimerase gene, allitol dehydrogenase gene, and formate dehydrogenase gene) was placed in a bioreactor, the pH of the reaction system was adjusted to 6.8, the temperature of the reaction system was controlled at 40°C, and the reaction was terminated after 13 hours. The supernatant collected by centrifugation was the D-alitol solution;

[0045] (2) 0.5 L of the D-alliitol solution in step (1) with a concentration of 100 g / L was taken and placed in a bioreactor with a volume of 1 L. Air was introduced at a flow rate of 1.5 L / min to maintain the dissolved oxygen content in the reaction system at 49% v / v. 5 g (wet weight) / L of a second whole-cell catalyst (which can simultaneously express the allitol dehydrogenase gene and the NADH oxidase gene) was added. The pH was adjusted to neutral. The reaction system was stirred at a speed gradually increased from 500 rpm to 1000 rpm. The temperature of the reaction system was controlled at 39° C. The reaction was terminated after 13 hours to obtain a D-psicose solution.

[0046] Example 6

[0047] A method for preparing D-psicose comprises the following steps:

[0048] (1) The substrate D-fructose was 120 g / L, the potassium formate solution was 2.0 mol / L, and the first whole-cell catalyst was added in an amount of 15 g (wet weight) / L; the first whole-cell catalyst (which simultaneously expressed the psicose epimerase gene, the allitol dehydrogenase gene, and the formate dehydrogenase gene) was placed in a bioreactor, the pH of the reaction system was adjusted to 6.8, the temperature of the reaction system was controlled to 39°C, the reaction was terminated after 13 hours, and the supernatant collected by centrifugation was the D-alitol solution;

[0049] (2) 0.5 L of the D-alliitol solution in step (1) with a concentration of 120 g / L was taken and placed in a bioreactor with a volume of 1 L. Air was introduced at a flow rate of 1.6 L / min to maintain the dissolved oxygen content in the reaction system at 48% v / v. 15 g (wet weight) / L of a second whole-cell catalyst (which can simultaneously express the allitol dehydrogenase gene and the NADH oxidase gene) was added. The pH was adjusted to neutral. The reaction system was stirred at a speed gradually increased from 500 rpm to 1000 rpm. The temperature of the reaction system was controlled at 40° C. The reaction was terminated after 13 h to obtain a D-psicose solution.

[0050] Example 7

[0051] A method for preparing D-psicose comprises the following steps:

[0052] (1) The substrate D-fructose was 80 g / L, the potassium formate solution was 0.5 mol / L, and the first whole-cell catalyst was added in an amount of 5 g (wet weight) / L; the first whole-cell catalyst (which simultaneously expressed the psicose epimerase gene, the allitol dehydrogenase gene, and the formate dehydrogenase gene) was placed in a bioreactor, the pH of the reaction system was adjusted to 6.8, the temperature of the reaction system was controlled to 40°C, the reaction was terminated after 13 hours, and the supernatant collected by centrifugation was the D-alitol solution;

[0053] (2) 0.5 L of the D-alliitol solution in step (1) with a concentration of 50 g / L was taken and placed in a bioreactor with a volume of 1 L. Air was introduced at a flow rate of 2 L / min to maintain the dissolved oxygen content in the reaction system at 50% v / v. 5 g (wet weight) / L of a second whole-cell catalyst (which can simultaneously express the allitol dehydrogenase gene and the NADH oxidase gene) was added. The pH was adjusted to neutral. The stirring speed of the reaction system was gradually increased from 500 rpm to 1000 rpm. The temperature of the reaction system was controlled at 40° C. The reaction was terminated after 13 hours of reaction to obtain a D-psicose solution.

[0054] The first whole-cell catalyst preparation method in Examples 1-7 comprises the following steps:

[0055] (1) Construction of recombinant plasmid pYB1s-DPE-RDH-FDH: The pYB1s vector was double-digested with XhoI and SpeI, and the vector fragment was recovered. The artificially synthesized DPE gene fragment, RDH gene fragment, and FDH gene fragment (synthesized by Nanjing GenScript Biotechnology Co., Ltd.) were ligated with the recovered vector fragment using the Gibson method (Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, 3rd, Smith HO: Enzymatic assembly of DNA molecules upto several hundred kilobases. Nat Methods 2009, 6: 343-345.) to obtain a ligation product. The ligation product was transformed into DH5α competent cells and coated on a solid LB plate containing streptomycin. Incubate at 37°C overnight, pick a single clone to extract the plasmid, and perform sequencing verification. The vector with the correct sequencing result was named pYB1s-DPE-RDH-FDH.

[0056] (2) Inducible expression of recombinant proteins DPE, RDH and FDH: The vector pYB1s-DPE-RDH-FDH was transformed into Escherichia coli BW25113 by chemical transformation to obtain the first whole-cell catalyst. The recombinant bacteria (first whole-cell catalyst) were streaked onto LB plates containing agar (containing 50 μg / mL streptomycin) at a mass percentage concentration of 1.5 g / 100 mL and cultured at 37°C for 12 h. A single clone was picked and inoculated into liquid LB medium containing 50 μg / mL streptomycin, and cultured at 37°C for 10 h with shaking at 220 rpm; the culture was inoculated into 500 mL LB medium at a volume percentage of 1%, cultured at 37°C for 3 h with shaking, and arabinose was added at a final concentration of 2 g / L to induce expression, and cultured at 30°C for 12 h with shaking.

[0057] The preparation method of the second whole-cell catalyst in Examples 1-7 comprises the following steps:

[0058] (1) Construction of recombinant plasmid pYB1s-RDH-NOX: The pYB1s vector was double-digested with XhoI and SpeI, and the vector fragment was recovered. The artificially synthesized RDH gene fragment (from the strain selected from Providencia alcalifaciens) and NOX gene fragment (synthesized by Nanjing GenScript Biotechnology Co., Ltd.) were ligated with the recovered vector fragment using the Gibson method to obtain a ligation product. The ligation product was transformed into DH5α competent cells and coated on a streptomycin-containing LB solid plate. After incubation at 37°C overnight, a single clone was picked to extract the plasmid and sequenced for verification. The vector with the correct sequencing result was named pYB1s-RDH-NOX.

[0059] (2) Inducible expression of recombinant proteins RDH and NOX: The vector pYB1s-RDH-NOX was transformed into Escherichia coli BW25113 by chemical transformation to obtain the second whole-cell catalyst. The recombinant bacteria (second whole-cell catalyst) were streaked onto LB plates containing agar (containing 50 μg / mL streptomycin) at a mass percentage concentration of 1.5 g / 100 mL and cultured at 37°C for 12 h. A single clone was picked and inoculated into liquid LB medium containing 50 μg / mL streptomycin, and cultured at 37°C with shaking for 10 h at 220 rpm; the culture was inoculated into 500 mL LB medium at a volume percentage of 1%, cultured at 37°C with shaking for 3 h, and arabinose was added at a final concentration of 2 g / L to induce expression, and cultured at 30°C with shaking for 12 h.

[0060] In order to better demonstrate that the preparation process of the present invention has good technical effects, three comparative examples are given with reference to Example 4; the experimental results are shown in Table 1.

[0061] Comparative Example 1

[0062] The difference from Example 4 is that the recombinant plasmid constructed during the preparation of the first whole-cell catalyst is pYB1s-PRDH-SFDH-CBDPE, and the other operations are exactly the same.

[0063] Comparative Example 2

[0064] The difference from Example 4 is that the recombinant plasmid constructed during the preparation of the first whole-cell catalyst is pYB1s-CBDPE-SFDH-PRDH, and the other operations are exactly the same.

[0065] Comparative Example 3

[0066] The difference from Example 4 is that when preparing the second whole-cell catalyst, the RDH gene source strain is selected from Klebsiella oxytoca, and the other operations are exactly the same.

[0067] Comparative Example 4

[0068] The difference from Example 4 is that when preparing the second whole-cell catalyst, the RDH gene source strain is selected from Rubrivivax sp., and the other operations are exactly the same.

[0069] Comparative Example 5

[0070] The difference from Example 4 is that when preparing the second whole-cell catalyst, the RDH gene source strain is selected from Pantoea agglomerans, and the other operations are exactly the same.

[0071] Table 1

[0072]

[0073]

[0074] The data in Table 1 show that the preparation method of the present invention is suitable for industrial production, and improves the conversion rate of D-fructose and reduces production costs. The concentrations of D-fructose, allitol, and D-psicose were measured by high performance liquid chromatography (Aminex HPX-87C column (7.8*300mm), detector (RID), mobile phase: double distilled water, column temperature 80°C, flow rate: 0.6ml / min). Detailed detection is shown in the attached Figure 1 The protein sequences of allulose epimerase, allitol dehydrogenase, formate dehydrogenase, allitol dehydrogenase, and NADH oxidase are detailed in the sequence listing.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing D-psicose, characterized in that The preparation method comprises the following steps: (1) D-fructose is used as a substrate, a solvent and a first whole-cell catalyst are added, the pH of the reaction system is adjusted to be weakly acidic, the temperature of the reaction system is controlled, and the supernatant collected by centrifugation after the reaction is completed is a D-alliitol solution; the first whole-cell catalyst is named AO1, and the first whole-cell catalyst is a whole-cell catalyst that can simultaneously express a psicose epimerase gene, an allitol dehydrogenase gene, and a formate dehydrogenase gene; the strain from which the psicose epimerase gene that can be expressed by the first whole-cell catalyst is selected from Clostridiales, the strain from which the allitol dehydrogenase gene is selected from Providencia alcalifaciens, and the strain from which the formate dehydrogenase gene is selected from Starkeya; (2) Taking the D-alliitol solution in step (1), adjusting the pH to neutral, and adding a second whole-cell catalyst under ventilation and stirring, and obtaining a psicose solution after the reaction is completed; the second whole-cell catalyst is named PO3, and the second whole-cell catalyst is a whole-cell catalyst that can simultaneously express the allitol dehydrogenase gene and the NADH oxidase gene; the allitol dehydrogenase (RDH) gene that can be expressed by the second whole-cell catalyst is derived from a strain selected from Providencia alcalifaciens, and the NADH oxidase gene is derived from a strain selected from Streptococcus pyogenes.

2. The method for preparing D-psicose according to claim 1, wherein: The preparation of the first whole-cell catalyst comprises the following steps: ligating a psicose epimerase gene, an allitol dehydrogenase gene, and a formate dehydrogenase gene to a pYB1s plasmid, and transforming the resulting recombinant plasmid pYB1s-DPE-RDH-FDH into Escherichia coli BW25113 to obtain a first whole-cell catalyst; The preparation of the second whole-cell catalyst includes the following operations: connecting the allitol dehydrogenase gene and the NADH oxidase gene to the pYB1s plasmid and transforming the obtained recombinant plasmid pYB1s-RDH-NOX into Escherichia coli BW25113 to obtain the second whole-cell catalyst.

3. The method for preparing D-psicose according to claim 1, wherein: In step (1), D-fructose and the first whole-cell catalyst are added in a ratio of 50-200 g / L and a wet weight concentration of 5-30 g / L, respectively; the solvent is a sodium formate solution or a potassium formate solution; the concentration of the sodium formate solution or the potassium formate solution is 0.5-2.0 mol / L; the pH of the reaction system is 5.8-6.8, the reaction temperature is 35-40° C., and the reaction time is 11-13 h.

4. The method for preparing D-psicose according to claim 3, wherein: In step (1), D-fructose and the first whole-cell catalyst were added at a ratio of 100 g / L and a wet weight concentration of 10 g / L, respectively. The pH of the reaction system was 6.5, the reaction temperature was 40° C., and the reaction time was 12 h.

5. The method for preparing D-psicose according to claim 1, wherein: The D-alliitol solution and the second whole-cell catalyst in step (2) are added in a ratio of 50-200 g / L and a wet weight concentration of 5-30 g / L, respectively; air is introduced at a flow rate of 1.5-2.5 L / min, and the dissolved oxygen in the reaction system is maintained at 48-53% v / v. The stirring speed is gradually increased from 500 rpm at the beginning to 1000 rpm. The reaction time is controlled at 13-15 h, and the reaction temperature is controlled at 35-40°C.

6. The method for preparing D-psicose according to claim 5, wherein: The D-alliitol solution and the second whole-cell catalyst in step (2) were added at a ratio of 100 g / L and a wet weight concentration of 10 g / L, respectively. Air was introduced at a flow rate of 1.8 L / min to maintain the dissolved oxygen content in the reaction system at 50% v / v. The pH of the reaction system was 7.0, the reaction temperature was 40° C., and the reaction time was 14.5 h.

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