A type of Co-added 2+ Methods to improve the yield of D-allulose produced by multi-enzyme catalysis

Abstract

The application discloses a kind of added Co 2+ The application discloses a method for improving the yield of D-allulose produced by multi-enzyme catalysis, belonging to the field of biotechnology. The application provides a method for producing D-allulose by multi-enzyme catalysis, which comprises adding sucrose phosphorylase from Bifidobacterium adolescentis, fructokinase from Clostridium acetobutylicum, D-allulose-6-phosphate phosphatase from Clostridium thermocellum, and D-allulose-6-phosphate-3-epimerase from Pantoea sp to a reaction system containing sucrose and ATP, and then adding a metal ion solution to the reaction system to produce D-allulose. The application synthesizes an important low-calorie functional rare sugar D-allulose using inexpensive sucrose as a substrate, and experiments show that the addition of cobalt ions can increase the yield of D-allulose by five times.

Description

Technical Field

[0001] This invention relates to a method for adding Co 2+ Methods to improve the yield of D-allulose produced by multi-enzyme catalysis fall under the field of biotechnology. Background Technology

[0002] Rare sugars are monosaccharides and sugar alcohols that are rarely found in nature. D-allulose, the C-3 epimer of D-fructose, is a rare sugar found only in a few species in nature. Numerous studies have shown that D-allulose can regulate various physiological functions, thereby inhibiting the rise in blood sugar, improving lipid metabolism, and reducing the accumulation of body fat; it can also be used as a food ingredient and dietary additive.

[0003] Currently, the industrial production of D-allulose mainly relies on the epimerization reaction of D-allulose 3 epimerase. However, this method suffers from low conversion rates and high costs. Therefore, obtaining a high-conversion, low-cost method for preparing D-allulose is of great significance for industrial production. Summary of the Invention

[0004] This invention provides a method for producing D-allulose from sucrose, and involves adding Co... 2+ This invention significantly increases the product yield. Starting from inexpensive sucrose, D-allulose is synthesized via phosphorylation and dephosphorylation. Because the final step is dephosphorylation, the theoretical yield of allulose can reach 100%. To improve the yield of D-allulose synthesized by this method, the authors experimentally investigated the effect of metal ions on D-allulose production.

[0005] This invention provides a method for the multi-enzyme catalytic production of D-allulose. The method involves adding sucrose phosphorylase from Bifidobacterium adolescentis, fructokinase from Clostridium acetobutylicum, D-allulose-6-phosphate phosphatase from Clostridium thermocellum, and D-allulose-6-phosphate-3-epimerase from Pantoea sp. to a reaction system containing sucrose and ATP. Simultaneously, a metal ion solution is added to the reaction system before proceeding with the reaction to produce D-allulose.

[0006] In one embodiment of the present invention, the sucrose phosphorylase, fructose kinase, D-allulose-6-phosphate phosphatase, and D-allulose-6-phosphate-3-epimerase are all recombinant enzymes expressed using recombinant Escherichia coli.

[0007] In one embodiment of the present invention, the recombinant Escherichia coli uses E. coli BL21(DE3) as the expression host.

[0008] In one embodiment of the present invention, the amino acid sequence of the sucrose phosphorylase is shown in SEQ ID NO.1; the amino acid sequence of the fructose kinase is shown in SEQ ID NO.2; the amino acid sequence of the D-allulose-6-phosphate phosphatase is shown in SEQ ID NO.3; and the amino acid sequence of the D-allulose-6-phosphate-3-epimerase is shown in SEQ ID NO.4.

[0009] In one embodiment of the present invention, the nucleotide sequence encoding the sucrose phosphorylase is shown in SEQ ID NO. 5; the nucleotide sequence encoding the fructose kinase is shown in SEQ ID NO. 6; the nucleotide sequence encoding the D-allulose-6-phosphate phosphatase is shown in SEQ ID NO. 7; and the nucleotide sequence encoding the D-allulose-6-phosphate-3-epimerase is shown in SEQ ID NO. 8.

[0010] In one embodiment of the present invention, the metal ion is Co. 2+ .

[0011] In one embodiment of the present invention, the metal ion Co 2+ The addition amount is 0.1–10.0 mmol / L.

[0012] In one embodiment of the present invention, the metal ion Co 2+ The addition amount is 0.5–10.0 mmol / L.

[0013] In one embodiment of the present invention, the reaction conditions in the reaction system are: 40-50°C, pH 7.5-8.5, and reaction time of 6-48 h.

[0014] In one embodiment of the present invention, the amount of sucrose added is 5-20 mM.

[0015] In one embodiment of the present invention, the amount of substrate ATP added is 5-20 mM.

[0016] In one embodiment of the present invention, the reaction system further contains a buffer solution.

[0017] In one embodiment of the present invention, the buffer solution is Tris-HCl (50 mM, pH 8.0).

[0018] This invention also provides a method for improving the yield of D-allulose, comprising adding the metal ion Co to a reaction system containing sucrose, ATP, sucrose phosphorylase from Bifidobacterium adolescentis, fructokinase from Clostridium acetobutylicum, D-allulose-6-phosphate phosphatase from Clostridium thermocellum, and D-allulose-6-phosphate-3-epimerase from Pantoea sp. 2+ The solution was reacted to prepare D-allulose.

[0019] In one embodiment of the present invention, the Co 2+ The concentration of the solution is 0.1–10.0 mmol / L.

[0020] Beneficial effects

[0021] (1) This invention provides a method for producing D-allulose from sucrose, and by adding Co 2+ This invention significantly increases the product yield by synthesizing D-allulose from inexpensive sucrose through phosphorylation and dephosphorylation. Since the final step is a dephosphorylation reaction, the theoretical yield of allulose can reach 100%. To improve the yield of D-allulose by this method, the authors investigated the effect of metal ions on the production of D-allulose through experiments.

[0022] (2) This invention provides a method for synthesizing D-allulose, which uses inexpensive sucrose as a substrate to synthesize an important low-calorie functional rare sugar, D-allulose. Experiments have shown that adding cobalt ions can increase the yield of D-allulose to five times the original yield. Attached Figure Description

[0023] Figure 1 SDS-PAGE analysis of cell lysate supernatant and precipitate containing sucrose phosphorylase (SP), fructokinase (FRK), D-allulose-6-phosphate 3-epimerase (A6PE), and D-allulose-6-phosphate phosphatase (A6PP). Wherein, M: Blue Plus II Protein Marker; 1: SP supernatant; 2: SP precipitate; 3: FRK supernatant; 4: FRK precipitate; 5: A6PP supernatant; 6: A6PP precipitate; 7: A6PE supernatant; 8: A6PE precipitate.

[0024] Figure 2 The effect of different metal ions on the reaction.

[0025] Figure 3 Different Co 2+ The effect of concentration on the reaction. Detailed Implementation

[0026] The E. coli BL21(DE3), pET-28a(+), and pET-42b(+) plasmids used in the following examples were purchased from Takara.

[0027] The sucrose phosphorylase (SP) mentioned in the examples is derived from Bifidobacterium adolescentis, the fructokinase (FRK) is derived from Clostridium acetobutylicum, the D-alulose-6-phosphate 3-epimerase (A6PE) is derived from Pantoea sp, and the D-alulose-6-phosphate phosphatase (A6PP) is derived from Clostridium thermocellum.

[0028] The culture media involved in the following examples are as follows:

[0029] LB liquid medium: yeast extract 5.0 g·L -1 10.0 g / L of peptone -1 NaCl 10.0 g·L -1 Kanamycin 50 mg / L -1 .

[0030] LB solid medium: yeast extract 5.0 g·L -1 10.0 g / L of peptone -1 NaCl 10.0 g·L -1 15g / L of agar powder -1 Kanamycin 50 mg / L -1 .

[0031] Example 1: Construction of recombinant plasmids and recombinant bacterial strains

[0032] The specific steps are as follows:

[0033] (1) The following genes were obtained from NCBI: sucrose phosphorylase SP gene (Reference Sequence No.: WP_011679246.1), fructose kinase FRK gene (Reference Sequence No.: KHD36265.1), D-allulose-6-phosphate 3-epimerase A6PE gene (Reference Sequence No.: WP_039379501.1), and D-allulose-6-phosphate phosphatase A6PP gene (Reference Sequence No.: WP_003512401.1). Based on the codon preference of E. coli, the genes were optimized and sent to the company for synthesis. The following genes were obtained: sucrose phosphorylase (SP) gene (nucleotide sequence as shown in SEQ ID NO.5), fructokinase (FRK) gene (nucleotide sequence as shown in SEQ ID NO.6), D-allulose-6-phosphate phosphatase (A6PP) gene (nucleotide sequence as shown in SEQ ID NO.7), and D-allulose-6-phosphate-3-epimerase (A6PE) gene (nucleotide sequence as shown in SEQ ID NO.8);

[0034] (2) The above genes: sucrose phosphorylase (SP) gene, fructokinase (FRK) gene, D-alulose-6-phosphate phosphatase (A6PP) gene, and D-alulose-6-phosphate-3-epimerase (A6PE) were respectively ligated to pET28a plasmid (synthesized by the company) to prepare pET28a-SP, pET28a-FRK, pET28a-A6PP, and pET28a-A6PE.

[0035] (3) The recombinant plasmids pET28a-SP, pET28a-FRK, and pET28a-A6PP synthesized by the company were transformed into E. coli BL21(DE3). The transformation products were spread on LB solid medium and cultured at 37℃ for 12-14 h. The transformants were picked from the LB solid medium to obtain recombinant E. coli E. coli BL21(DE3) / pET28a-FRK, E. coli BL21(DE3) / pET28a-SP, and E. coli BL21(DE3) / pET28a-A6PP containing the recombinant plasmids.

[0036] (4) The target gene of the recombinant plasmid pET28a-A6PE synthesized by the company was extracted and ligated with the product of double digestion (EcoRI and XhoI) of pET-42b(+) plasmid. The ligation product was transformed into E. coli BL21(DE3). The transformation product was plated on LB solid medium and cultured at 37℃ for 12-14 h. Transformants were picked from LB solid medium. If the sequencing was correct, recombinant E. coli BL21(DE3) / pET42b-A6PE containing the recombinant plasmid was obtained.

[0037] Example 2: Enzyme Expression and Purification

[0038] The specific steps are as follows:

[0039] 1. Expression and purification of sucrose phosphorylase (SP), fructokinase (FRK), and D-allulose-6-phosphate phosphatase (A6PP)

[0040] (1) Enzyme expression

[0041] The recombinant Escherichia coli E. coli BL21(DE3) / pET28a-FRK, E. coli BL21(DE3) / pET28a-SP, and E. coli BL21(DE3) / pET28a-A6PP obtained in Example 1 were inoculated into LB liquid medium and cultured at 37°C for 10–12 h to obtain seed culture.

[0042] The above seed culture was inoculated into LB liquid medium at an inoculation rate of 1% (v / v) and cultured at 37°C and 180 rpm until OD reached. 600 After the concentration was increased to 0.8–0.9, IPTG was added to the culture medium to a final concentration of 0.2 mmol / L, and the culture was continued at 25 °C and 180 rpm for 12 h to obtain the fermentation broth.

[0043] The fermentation broth was centrifuged at 4℃ and 8000 rpm for 10 min, and the bacterial cells were collected. The bacterial cells were then disrupted to obtain cell lysate. The cell lysate was centrifuged to obtain the supernatant and precipitate. The supernatant and precipitate were analyzed by SDS-PAGE, and the results are as follows: Figure 1 As shown.

[0044] The results showed that the protein soluble expression of the above three enzymes was good, and they could be directly purified.

[0045] (2) Enzyme purification

[0046] The crude enzyme solutions of sucrose phosphorylase (SP), fructokinase (FRK), and D-allulose-6-phosphate phosphatase (A6PP) obtained in step (1) were purified by nickel column affinity chromatography. The specific steps are as follows:

[0047] 1) Preparation of solution:

[0048] Solution A: 0.5 mol / L NaCl, 20 mmol / L imidazole, 25 mmol / L Tris-HCl, 1% glycerol (m / m), pH = 7.4.

[0049] Solution B: 0.5 mmol / L NaCl, 500 mmol / L imidazole, 25 mmol / L Tris-HCl, 1% glycerol (m / m), pH = 7.4.

[0050] Lysis buffer: 0.5 mmol / L NaCl, 25 mmol / L Tris-HCl, 50 mmol / L EDTA, pH 7.0.

[0051] NiSO4: 100 mmol / L NiSO4.

[0052] 2) Operation:

[0053] Column regeneration: A 1 mL Ni-NTA pre-packed gravity column was used for protein purification. First, 10 column volumes of solution A were added to the column to wash it. When solution A was almost completely drained, 10 column volumes of ultrapure water were quickly added for rinsing. Then, 10 column volumes of lysis buffer were added for rinsing, and finally, 10 column volumes of Watson's ultrapure water were added to ensure the column was thoroughly cleaned. Next, 10 column volumes of the prepared NiSO4 solution were added to the column, and the column was allowed to stand for 5 minutes. After observing a distinct blue-green color in the column packing material, the NiSO4 solution was drained, and 20 column volumes of solution A were added to the column to complete the preparation.

[0054] Sample loading: Load the supernatant obtained after removing impurities onto the column, and then add 10 column volumes of solution A into the column.

[0055] Elution: The target protein was eluted using different gradient elution methods. Different concentrations of imidazole (50 mmol / L, 200 mmol / L, 200 mmol / L, 400 mmol / L, and 500 mmol / L, respectively) were set by adjusting the ratio of solution A to solution B to elute the target protein adsorbed on the column. The eluents from each gradient were collected.

[0056] Storage: Clean the column with 10 column volumes of solution B, then rinse with 10 column volumes of ultrapure water, and finally store the column with 20% ethanol. Collect the eluents of each gradient in 10 mL centrifuge tubes and analyze the concentration of the target protein using SDS-PAGE to determine the appropriate eluent concentration for the target protein. Concentrate and replace the identified single coarse band using a 10 kDa ultrafiltration tube at 4°C and 4000 rpm. Finally, aliquot the obtained pure enzyme into 1.5 mL centrifuge tubes for subsequent enzymatic property studies.

[0057] Sucrose phosphorylase (SP), fructokinase (FRK), and D-alokulose-6-phosphate phosphatase (A6PP) were prepared separately. The enzyme concentrations were then detected using Nanodrop, and the results showed that the concentrations were 10 mg / mL, 8 mg / mL, and 10 mg / mL, respectively.

[0058] 2. Expression and purification of D-allulose-6-phosphate-3-epimerase

[0059] (1) Enzyme expression

[0060] Following the method in step 1, the cell lysis supernatant and precipitate were analyzed by SDS-PAGE, and the results are as follows: Figure 1 As shown in the figure, the results indicate that the enzyme has good protein soluble expression and can be directly purified.

[0061] A crude enzyme solution of D-alokulose-6-phosphate-3-epimerase was prepared.

[0062] (2) Enzyme purification

[0063] The D-allulose-6-phosphate 3-epimerase was obtained for GST-tagged protein purification. The specific steps are as follows:

[0064] 1) Preparation of solution:

[0065] Equilibrium solution: 150 mmol / L NaCl, 50 mmol / L Tris-HCl, pH = 8.0.

[0066] Eluent: 150 mmol / L NaCl, 50 mmol / L Tris-HCl, 10 mmol / L reduced glutathione, pH=8.0.

[0067] 2) Operation:

[0068] Equilibration: Glutathione agarose gel column was used for protein purification. First, the column was washed with 10 mL of ultrapure water, and then rinsed with 10 mL of equilibration buffer.

[0069] Sample loading: Load the supernatant obtained after removing impurities onto the column, and then add 20 column volumes of equilibration solution to the column.

[0070] Elution: Add 30-40 column volumes of eluent to the column to elute the target protein adsorbed on the column, and collect the eluent.

[0071] Storage: Clean the column with 20 column volumes of equilibration buffer, then rinse with 10 column volumes of ultrapure water, and finally store the column with 20% ethanol. Collect the eluents of each gradient in 10 mL centrifuge tubes and analyze the concentration of the target protein using SDS-PAGE to determine the appropriate eluent concentration for the target protein. Concentrate and replace the identified single coarse band using a 50 kDa ultrafiltration tube at 4°C and 4000 rpm. Finally, aliquot the obtained pure enzyme into 1.5 mL centrifuge tubes for subsequent enzymatic property studies.

[0072] A pure enzyme solution of -alokulose-6-phosphate-3-epimerase was prepared. The enzyme concentration was then detected using Nanodrop, and the results showed that the concentration was 5 mg / mL.

[0073] Example 3: The effect of different metal ions on cascade reactions

[0074] The specific steps are as follows:

[0075] To a reaction system containing a final concentration of 10 mM sucrose and 10 mM ATP in Tris-HCl (50 mM, pH 8.0), 0.4 μM sucrose phosphorylase (SP), 0.4 μM fructokinase (FRK), 0.4 μM D-allulose-6-phosphate phosphatase (A6PP), and 0.8 μM D-allulose-6-phosphate-3-epimerase (A6PE) prepared in Example 2 were added, followed by the addition of different metal ion solutions to a final concentration of 1 mM: Mg 2+ Mn 2+ Co 2+ Ca 2+ NH4 + Zn 2+ .

[0076] Control: Reaction system without the addition of metal ions.

[0077] The reaction was carried out at 40℃, and samples were taken at different times for high-performance liquid chromatography analysis. The results are shown in the figure. Figure 2 As shown in Table 1, the times vary because each reaction reaches equilibrium at a different time, as the yield of the product is the maximum.

[0078] Table 1 Reactions of different metal ions

[0079] metal ions Maximum product yield (%) Reaction time (h) Comparison 15.9 36 <![CDATA[Mg 2+ ]]> 26.3 36 <![CDATA[Mn 2+ ]]> 12.3 36 <![CDATA[Co 2+ ]]> 76.2 12 <![CDATA[Ca 2+ ]]> 14.2 36 <![CDATA[NH4 + ]]> 11.7 36 <![CDATA[Zn 2+ ]]> 18.6 36

[0080] Depend on Figure 1 As shown in Table 1, the addition of Mg 2+ Co 2+ and Zn 2+ The product yields of the reactions were all higher than those without the addition of metal ions, with the addition of Co being particularly significant. 2+ It can increase the product yield to five times the original level.

[0081] Example 4: Co 2+ Effect of concentration on cascade reaction

[0082] To a reaction system containing a final concentration of 10 mM sucrose and 10 mM ATP in Tris-HCl (50 mM, pH 8.0), 0.4 μM sucrose phosphorylase (SP), 0.4 μM fructokinase (FRK), 0.4 μM D-alulose-6-phosphate phosphatase (A6PP), and 0.8 μM D-alulose-6-phosphate-3-epimerase (A6PE) prepared in Example 2 were added, followed by the addition of CoCl2·6H2O to final concentrations of 0.5 mM, 1 mM, 2 mM, 5 mM, and 10 mM, respectively.

[0083] Comparison: No Co added 2+ The reaction.

[0084] The reaction was carried out at 40℃, and samples were taken at different times for high-performance liquid chromatography (HPLC) analysis. Since this represents the maximum yield of the product, the times varied because each reaction reaches equilibrium at different times. The results are shown in [Figure number missing]. Figure 3 As shown in Table 2.

[0085] Table 2 Different Co 2+ Concentration reaction

[0086] Concentration (mM) Maximum product yield (%) Reaction time (h) Comparison 15.9 36 0.5 68.5 36 1 76.2 12 2 79.3 6 5 83.3 6 10 84.4 6

[0087] Depend on Figure 2 As shown in Table 2, when Co 2+ When the concentration is higher than 2 mM (including 2 mM), the maximum product yield can be reached after 6 hours of reaction, and the product yield is around 80%. Based on the product separation and purification, 2 mM Co was selected. 2+ concentration.

[0088] Comparative Example 1: Effects of D-allulose-6-phosphate-3-epimerase from different sources on D-allulose yield

[0089] The specific implementation method is the same as in Examples 1-3, except that the D-allulose-6-phosphate-3-epimerase (hereinafter referred to as PspA6PE) from Pantoea sp. is adjusted to: D-allulose-6-phosphate-3-epimerase from Thermoanaerobacterium thermosaccharolyticum (NCBI accession number: WP_013298194.1, abbreviated as TtA6PE) and D-allulose-6-phosphate-3-epimerase from Escherichia coli (NCBI accession number: EDU66157.1) (abbreviated as EcA6PE).

[0090] (1) D-alokulose-6-phosphate-3-epimerase TtA6PE, EcA6PE and PspA6PE pure enzyme solutions were prepared according to the methods in Examples 1 and 2.

[0091] Meanwhile, pure enzyme solutions of sucrose phosphorylase (SP), fructokinase (FRK), and D-allulose-6-phosphate phosphatase (A6PP) were prepared.

[0092] (2) Following the method in Example 3, 0.4 μM sucrose phosphorylase (SP), 0.4 μM fructokinase (FRK), 0.4 μM D-allulose-6-phosphate phosphatase (A6PP), and 0.8 μM D-allulose-6-phosphate-3-epimerase (i.e., TtA6PE, EcA6PE, and PspA6PE enzymes were added respectively) to a Tris-HCl (50 mM, pH 8.0) buffer reaction system containing a final concentration of 10 mM sucrose and 10 mM ATP. No metal ions were added, and the reaction temperature was 40 °C to prepare different reaction products.

[0093] The results showed that after 48 hours of reaction, the maximum yield of the product was <5% in the reaction containing pure enzyme solution of TtA6PE or EcA6PE, while the maximum yield of the product was >10% in the reaction containing pure enzyme solution of PspA6PE.

[0094] Therefore, D-allulose-6-phosphate-3-epimerase derived from Pantoea sp. was selected.

[0095] Comparative Example 2: Effect of different carriers on the purification of D-allulose-6-phosphate-3-epimerase

[0096] The specific implementation method is the same as in Examples 1 and 2, except that pET28a is used as the expression vector to express D-allulose-6-phosphate-3-epimerase to prepare E.coli BL21(DE3) / pET28a-A6PE. Since the soluble expression of E.coli BL21(DE3) / pET28a-A6PE protein is poor, and the purification method in Example 2 cannot be used to prepare pure enzyme solution of D-allulose-6-phosphate-3-epimerase, the pET28a expression vector is replaced with the pET42b expression vector.

[0097] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for producing D-allulose via multi-enzyme catalysis, characterized in that, The method involves adding [a certain substance] to a reaction system containing 10 mM sucrose and 10 mM ATP. Bifidobacterium adolescentis Sucrose phosphorylase from source, Clostridium acetobutylicum Fructose kinase from source Clostridium thermocellum D-allulose-6-phosphate phosphatase from this source Pantoea The reaction system contains D-allulose-6-phosphate-3-epimerase derived from *Sp*, and simultaneously, 5.0–10.0 mmol / L of the metal ion Co is added to the reaction system. 2+ After solution preparation, a reaction is carried out to produce D-allulose. The sucrose phosphorylase, fructose kinase, D-allulose-6-phosphate phosphatase, and D-allulose-6-phosphate-3-epimerase are produced using recombinant Escherichia coli. E. coli The recombinant enzymes expressed by BL21(DE3), the amino acid sequence of the sucrose phosphorylase is shown in SEQ ID NO.1; the amino acid sequence of the fructose kinase is shown in SEQ ID NO.2; the amino acid sequence of the D-allulose-6-phosphate phosphatase is shown in SEQ ID NO.3; and the amino acid sequence of the D-allulose-6-phosphate-3-epimerase is shown in SEQ ID NO.

4.

2. The method as described in claim 1, characterized in that, The reaction conditions in the reaction system are: 40~50℃, pH 7.5~8.5, and reaction time is 6~48 h.

3. A method for increasing the yield of D-allulose, characterized in that, To contain 10 mM sucrose, 10 mM ATP, Bifidobacterium adolescentis Sucrose phosphorylase from source, Clostridium acetobutylicum Fructose kinase from source Clostridium thermocellum D-allulose-6-phosphate phosphatase from this source Pantoea In the reaction system of D-allulose-6-phosphate-3-epimerase derived from sp, 5~10.0 mmol / L of the metal ion Co is added. 2+ The solution was reacted to prepare D-allulose, wherein the sucrose phosphorylase, fructose kinase, D-allulose-6-phosphate phosphatase, and D-allulose-6-phosphate-3-epimerase were prepared using recombinant Escherichia coli. E. coli The recombinant enzymes expressed by BL21(DE3), the amino acid sequence of the sucrose phosphorylase is shown in SEQ ID NO.1; the amino acid sequence of the fructose kinase is shown in SEQ ID NO.2; the amino acid sequence of the D-allulose-6-phosphate phosphatase is shown in SEQ ID NO.3; and the amino acid sequence of the D-allulose-6-phosphate-3-epimerase is shown in SEQ ID NO.4.

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