A D-psicose 3-epimerase, its recombinant vector, strain and application

Abstract

The present invention discloses a D - psicose 3 - epimerase, its recombinant vector, strain and application, belonging to the technical field of functional enzymes. The present invention firstly provides a D - psicose 3 - epimerase derived from halotolerant Bacillus sp. ( Halotolerant Bacillus ), SYNY - 019. The protein sequence of the D - psicose - 3 - epimerase is as shown in SEQ ID No.1, and the nucleotide sequence obtained after codon optimization of this protein sequence is as shown in SEQ ID No.2. This D - psicose 3 - epimerase can be used for efficiently catalyzing the synthesis of D - psicose from D - fructose.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional enzymes, and particularly relates to a D-allulose 3-epimerase and its recombinant vector, strain and application. Background Art

[0002] D-allulose is a rare sugar, and it has been nearly 30 years since its first report. D-allulose is an "epimer" of fructose and is a safe sweetener. Although its sweetness is only 70% of that of sucrose, the energy it generates is only 0.3% of that of sucrose, making it a veritable low-energy sugar. Worldwide, D-allulose is widely used in foods and beverages, and its market scale is constantly increasing.

[0003] Initially, the chemical synthesis methods of D-allulose included selective aldol condensation synthesis, catalytic hydrogenation, addition reaction, etc. Although D-allulose can be prepared by chemical synthesis methods, there are problems such as poor economy and serious environmental pollution. Therefore, the chemical synthesis of D-allulose has not been industrialized. Compared with chemical synthesis methods, the biological synthesis of D-allulose not only has strong reaction specificity and a single product, but also has a simple separation and purification method and less environmental pollution. The biological transformation method is not only conducive to reducing industrial costs, but also conforms to the principle of green and environmental protection production, and is the main method for the industrial production of D-allulose at home and abroad.

[0004] The biological method mainly prepares D-allulose by enzymatic catalysis of fructose under the action of D-allulose 3-epimerase. In large-scale industrial production, fermentation or production environments such as high temperature, high sugar, and high ionic strength are extremely common, but the stability of most D-allulose 3-epimerases is weak, which limits their industrial application. In order to meet the complex requirements of industrial production, it is necessary to find D-allulose 3-epimerases with high enzyme activity and good stability. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a D-allulose 3-epimerase and its recombinant vector, strain and application. The aim is to solve the problem of enzyme instability when the existing D-allulose 3-epimerase catalyzes the synthesis of D-allulose from D-fructose, so that the D-allulose 3-epimerase has good environmental adaptability and multi-faceted stability.

[0006] In order to achieve the above technical objectives, the present invention provides the following technical solutions:

[0007] As the first aspect of the present invention, it lies in providing a D-allulose 3-epimerase, and the amino acid sequence of the D-allulose 3-epimerase is as shown in SEQ ID No.1.

[0008] As the second aspect of the present invention, it lies in further specifically optimizing the codons of its amino acid sequence, and providing an optimized nucleotide sequence encoding D-allulose 3-epimerase as shown in SEQ ID No. 2.

[0009] As the third aspect of the present invention, it lies in providing a recombinant vector expressing the D-allulose 3-epimerase, which contains a nucleotide sequence encoding D-allulose 3-epimerase.

[0010] Optionally, the recombinant vector comprises the pUC980 expression plasmid.

[0011] As the fourth aspect of the present invention, it lies in providing a strain, which is a strain expressing D-allulose 3-epimerase, and the strain contains a nucleotide sequence encoding D-allulose 3-epimerase, and the sequence is as shown in SEQ ID No. 2; the amino acid sequence of the D-allulose 3-epimerase is as shown in SEQ ID No. 1.

[0012] Optionally, the host bacterium of the strain expressing the D-allulose 3-epimerase is Bacillus subtilis.

[0013] As the fifth aspect of the present invention, it lies in providing a preparation method of the D-allulose 3-epimerase, which is to ferment and prepare the D-allulose 3-epimerase in a liquid fermentation medium by using the recombinant vector or the strain.

[0014] Preferably, the formula of the liquid fermentation medium is: 20 g of glycerol, 50 g of peptone, 5 g of acid-hydrolyzed casein, 1 g of KH2PO4, 0.5 g of KCl, 0.5 g of MgSO4·7H2O, 10 mg of FeSO4·7H2O, adjust the pH to 7.2, and make up the volume to 1 L with ddH2O. Sterilize at 121 °C for 20 min under high temperature and high pressure. Note that MgSO4·7H2O and FeSO4·7H2O cannot be sterilized under high temperature and high pressure. Prepare the mother liquor separately and filter-sterilize it.

[0015] Preferably, the inoculation amount of the recombinant vector or the host bacterium is 1-5% of the total volume of the liquid fermentation medium.

[0016] As the sixth aspect of the present invention, it lies in providing the application of the D-allulose 3-epimerase in the production of D-allulose.

[0017] In an embodiment of the present invention, the production of D-allulose by using D-allulose-3-epimerase comprises the following steps: using D-fructose as a substrate, adding the D-allulose 3-epimerase, and generating D-allulose through a catalytic reaction.

[0018] Preferably, in the reaction system, the pH is 6.0 - 8.0, and a more suitable pH is 7.0.

[0019] Preferably, the reaction temperature is 50 - 65 °C, and a more suitable temperature is 60 °C.

[0020] Preferably, metal ions are added to the reaction system, and the metal ions are selected from at least one of magnesium ions, cobalt ions, and manganese ions, and a more preferable metal ion is manganese ion.

[0021] Preferably, the final concentration of the metal ions is 0.1 - 1 mM, and a more suitable final concentration of the metal ions is 0.4 mM.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] (1) The present invention discovers a new D - psicose 3 - epimerase derived from halotolerant Bacillus sp. Bacillus halotolerans (SYNY - 019), optimizes its wild - type protein sequence by codons, and heterologously expresses it in the engineered Bacillus subtilis.

[0024] (2) The present invention also provides a preparation method of D - psicose 3 - epimerase, laying a foundation for the subsequent industrial production of D - psicose 3 - epimerase.

[0025] (3) In the present invention, using D - fructose as a substrate and D - psicose 3 - epimerase as a catalyst, directly enzymatically catalyzes the conversion of D - fructose into D - psicose, saving industrial production costs.

[0026] (4) The D - psicose 3 - epimerase provided by the present invention solves the problem of instability of the existing D - psicose 3 - epimerase when catalyzing the synthesis of D - psicose from D - fructose, enables the D - psicose 3 - epimerase to maintain good activity within a relatively wide pH range and a relatively wide temperature range, has good stability, and can adapt to the fluctuations of complex production environments and conditions in large - scale production; has high fructose tolerance performance and high conversion rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0028] Figure 1SDS-PAGE analysis after the expression of D-allulose 3-epimerase in Bacillus subtilis in Example 1; where M is the protein marker, 1 is the fermentation supernatant sample, 2 is the whole cell lysate sample, and 3 is the crude enzyme solution sample in the supernatant after cell disruption and centrifugation.

[0029] Figure 2 Liquid chromatogram of D-fructose reference substance (20 mg / mL);

[0030] Figure 3 Liquid chromatogram of D-allulose reference substance (20 mg / mL);

[0031] Figure 4 Liquid chromatogram of the reaction process of D-allulose 3-epimerase catalyzing the synthesis of D-allulose. Detailed implementation manners

[0032] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following descriptions are usually only a part of the embodiments of the present invention, rather than all of the embodiments. Therefore, all other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention shall fall within the scope of protection of the present invention.

[0033] Strains and expression vectors involved in the following examples: Bacillus subtilis strain B.subtilis 168, pUC980 expression plasmid, and all the enzymes used in the following examples are expressed by the above strains and vectors.

[0034] Media involved in the following examples:

[0035] LB liquid medium, the composition of which is: yeast powder 5.0 g / L, peptone 10.0 g / L, and NaCl 10.0 g / L.

[0036] M1 fermentation medium, the composition of which is: 20 g glycerol, 50 g peptone, 5 g acid-hydrolyzed casein (without vitamins), 1 g KH2PO4, 0.5 g KCl, 0.5 g MgSO4·7H2O, 10 mg FeSO4·7H2O, adjust the pH to 7.2, and make up the volume to 1 L with ddH2O. Sterilize at 121 °C for 20 min by high-temperature high-pressure sterilization. Note that MgSO4·7H2O and FeSO4·7H2O cannot be sterilized by high-temperature high-pressure sterilization. Prepare the mother liquor separately and filter sterilize.

[0037] The preparation method of phosphate buffer (PB) buffer with different pH values is as follows:

[0038] 1. Solution A (0.2 M aqueous solution of sodium dihydrogen phosphate): Dissolve 27.6 g of NaH2PO4·H2O in distilled water and dilute to 1000 mL.

[0039] 2. Solution B (0.2 M aqueous solution of disodium hydrogen phosphate): Dissolve 53.6 g of Na2HPO4·7H2O (or 71.6 g of Na2HPO4·12H2O or 35.6 g of Na2HPO4·2H2O) in distilled water and add water to 1000 mL.

[0040] Add Y mL of Solution B to X mL of Solution A (refer to Table 1) to obtain 0.2 M PB. If distilled water is further added to 800 mL, it becomes 50 mM PB buffer.

[0041] Table 1. Preparation system of phosphate buffer (PB) with different pH values

[0042]

[0043] The detection methods involved in the following examples are as follows: High-performance liquid chromatography (HPLC) detection: Ca 2+ Chromatographic column; The mobile phase is pure water, the flow rate of the mobile phase is 0.6 mL / min; The column temperature is 80 °C; The detector is a differential refractive index detector.

[0044] Example 1, a salt-tolerant Bacillus sp. ( Bacillus halotolerans ) SYNY-019

[0045] Salt-tolerant Bacillus sp. ( Bacillus halotolerans

[0046] This strain was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 26, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 26285.

[0047] Example 2, Construction of Recombinant Strain

[0048] Bacillus halotolerans ( Bacillus halotolerans ) SYNY-019. According to the sequencing results and genomic analysis, the gene encoding D-allulose 3-epimerase was obtained as follows: GenScript Biotech Corporation optimized the codons of Bacillus subtilis based on the amino acid sequence shown in SEQ ID NO: 1. After codon optimization, the optimized nucleotide sequence of D-allulose 3-epimerase was obtained, as shown in SEQ ID NO: 2, and the full gene was synthesized into the pUC980 vector plasmid to obtain the recombinant expression vector plasmid pUC980-DAE.

[0049] In this experiment, the Spizizen transformation method was used for all transformations involving Bacillus subtilis:

[0050] Preparation of Spizizen transformation medium: Each reagent was prepared separately, and the GM1 and GM2 solutions were mixed when in use. The system is shown in Table 2.

[0051] 1) 0.5% Tryptophan: Weigh 0.25 g of tryptophan, dissolve it in ddH2O, make up the volume to 50 mL, and filter sterilize.

[0052] 2) 2% Acid Hydrolyzed Casein (Vitamin-Free): Weigh 2 g of acid hydrolyzed casein (vitamin-free), dissolve it in ddH2O, make up the volume to 100 mL, and sterilize at 115 °C for 30 min.

[0053] 3) 40% Glucose: Weigh 40 g of glucose, dissolve it in ddH2O, make up the volume to 100 mL, and sterilize at 115 °C for 30 min.

[0054] 4) 20% MgSO4·7H2O: Weigh 10 g of MgSO4·7H2O, dissolve it in ddH2O, make up the volume to 50 mL, and filter sterilize.

[0055] 5) 10× Spizizen Minimal Salt Medium: Weigh 0.2 g of (NH4)2SO4, 1.83 g of K2HPO4, 0.6 g of KH2PO4, and 0.12 g of sodium citrate, dissolve them in ddH2O, make up the volume to 100 mL, adjust the pH to 7.2, and sterilize at 121 °C for 20 min.

[0056] 6) 5% Yeast extract: Weigh 2.5 g of yeast extract and dissolve it in ddH2O, make up the volume to 50 mL, sterilize at 121 °C for 20 min.

[0057] Table 2, Preparation system of Spizizen transformation medium

[0058]

[0059] Preparation and transformation of competent cells of Bacillus subtilis:

[0060] 1) Streak and activate the cryopreserved B. subtilis 168 on LB solid medium, culture overnight at 37 °C;

[0061] 2) Pick a single colony and inoculate it into 5 mL of GM1, place it in a shaker at 37 °C and 200 rpm for 14 h;

[0062] 3) Transfer 500 μL of the bacterial solution to 5 mL of GM1, place it in a shaker at 37 °C and 200 rpm for 4.5 h;

[0063] 4) Transfer 750 μL of the bacterial solution to 5 mL of GM2, place it in a shaker at 37 °C and 200 rpm for 1.5 h;

[0064] 5) Take out the test tube and dispense it into 1.5 mL centrifuge tubes, with 1 mL of the bacterial solution in each centrifuge tube, and the competent cells are made. Immediately use for transformation, do not store. Add 1 μg of plasmid and mix with the competent cells, resuscitate at 37 °C for 1.5 h. Spread the transformation product on a LB solid medium plate with kanamycin resistance, and culture overnight at 37 °C (about 16 h). Pick the transformants on the LB solid medium to obtain the recombinant Bacillus subtilis containing the recombinant plasmid B.subtilis 168 / pUC980-DAE.

[0065] Example 3, Preparation of enzyme

[0066] Pick the recombinant Bacillus subtilis B.subtilis 168 / pUC980-DAE monoclonal colony, inoculate (directly insert the pipette tip into the test tube) into a test tube containing 5 mL of LB liquid medium with kanamycin resistance, and culture overnight at 37 °C and 200 rpm (about 16 h).

[0067] Take 10 mL of the overnight cultured seed bacterial solution, transfer it to a shake flask containing 500 mL of M1 fermentation medium with resistance, and ferment at 37 °C for 48 h.

[0068] The cultured bacterial cells were centrifuged using a centrifuge to collect the cells. The cell pellet was weighed and resuspended according to the ratio of 0.1 g of cells to 1 mL of reaction buffer. The cell suspension was disrupted using an ultrasonic cell disruptor, with ultrasonic treatment for 3 s followed by a 2 s pause, for a total of 20 min (the time can be adjusted according to the volume of the ultrasonic treatment). After centrifuging the ultrasonic system, the supernatant obtained was the crude enzyme solution of the recombinant strain. The fermentation broth supernatant, whole cells, and supernatant after cell disruption were subjected to SDS-PAGE analysis, as Figure 1 shown, the target band was around 36 KDa.

[0069] Example 4, Method for determining the activity of D-allulose 3-epimerase

[0070] DAE can catalyze the conversion of D-fructose to D-allulose. Therefore, D-fructose was selected as the substrate for the activity detection of DAE. The total volume of the enzyme reaction system was 1 mL. The reaction system contained 50 g / L of D-fructose, 20 μL of the crude enzyme solution prepared in Example 3 above, and 50 mM PB 7.0 buffer was added to make up the reaction system to 1 mL. After reacting at 55 °C for 10 min, the reaction was stopped by heating in a metal bath at 100 °C for 10 min. After centrifuging at 12000 rpm for 5 min, the supernatant was taken and filtered through a 0.22 μm filter membrane for high performance liquid chromatography detection. The enzyme activity was defined by detecting the production amount of D-allulose.

[0071] Preparation of reference solutions: D-fructose reference solution (20 mg / mL, solvent is ultrapure water), D-allulose reference solution (20 mg / mL, solvent is ultrapure water). The liquid chromatography diagrams are respectively as Figure 2 and Figure 3 shown.

[0072] Example 5, Determination of the optimum pH of D-allulose 3-epimerase

[0073] The reaction system contained 50 g / L of D-fructose, 20 μL of the crude enzyme solution prepared in Example 3 above, and 50 mM PB 6.0, 6.5, 7.0, 7.5, 8.0 buffers were added to make up the reaction system to 1 mL. After reacting at 50 °C for 10 min, the reaction was stopped by heating in a metal bath at 100 °C for 10 min. After centrifuging at 12000 rpm for 5 min, the supernatant was taken and filtered through a 0.22 μm filter membrane for high performance liquid chromatography detection. The results are shown in Table 3. When the pH was 7, the reaction proceeded more favorably.

[0074] Table 3, Effect of pH on D-allulose 3-epimerase

[0075]

[0076] Example 6, Optimal Temperature of D-psicose 3-epimerase

[0077] The reaction system contained 50 g / L of D-fructose, 20 μL of the crude enzyme solution prepared in Example 3 above, and 50 mM PB 7.0 buffer was added to make the reaction system up to 1 mL. After reacting at 50, 55, 60, 65, 70 °C for 10 min, the reaction was stopped by heating in a metal bath at 100 °C for 10 min. Centrifuge at 12000 rpm for 5 min, take the supernatant, filter it through a 0.22 μm filter membrane and perform high performance liquid chromatography detection. The results are shown in Table 4. When the reaction temperature was 60 °C, the reaction proceeded more appropriately.

[0078] Table 4, Effect of Temperature on D-psicose 3-epimerase

[0079]

[0080] Example 7, Suitable Ions and Ion Concentrations for D-psicose 3-epimerase

[0081] The reaction system contained 50 g / L of D-fructose, 20 μL of the crude enzyme solution prepared in Example 3 above, 0.1 mM of magnesium ions, cobalt ions, manganese ions, and calcium ions, and 50 mM PB 7.0 buffer was added to make the reaction system up to 1 mL. After reacting at 60 °C for 10 min, the reaction was stopped by heating in a metal bath at 100 °C for 10 min. Centrifuge at 12000 rpm for 5 min, take the supernatant, filter it through a 0.22 μm filter membrane and perform high performance liquid chromatography detection. The results are shown in Table 5. Manganese ions were the most suitable reaction ions.

[0082] Table 5, Effect of Different Ions on D-psicose 3-epimerase

[0083]

[0084] The reaction system contained 50 g / L of D-fructose, 20 μL of the crude enzyme solution prepared in Example 3 above, 0.1, 0.2, 0.4, 0.6, 0.8, 1 mM of manganese ions, and 50 mM PB 7.0 buffer was added to make the reaction system up to 1 mL. After reacting at 60 °C for 10 min, the reaction was stopped by heating in a metal bath at 100 °C for 10 min. Centrifuge at 12000 rpm for 5 min, take the supernatant, filter it through a 0.22 μm filter membrane and perform high performance liquid chromatography detection. The results are shown in Table 6. The optimal addition amount of manganese ions was 0.4 mM.

[0085] Table 6, Effect of Different Ion Concentrations on D-psicose 3-epimerase

[0086]

[0087] Example 8, determination of reaction equilibrium

[0088] Since the reaction of epimerization has a reaction equilibrium, on the basis of the above optimized system, in this example, the enzyme amount and reaction time were increased to determine the reaction equilibrium point. The reaction system was 50 g / L D-fructose, 100 μL of the crude enzyme solution prepared in Example 3 above was added, 0.4 mM manganese ion was added, and the reaction system was made up to 1 mL with 50 mM PB 7.0 buffer. After reacting at 60 °C for 6, 8, and 10 h, the reaction was stopped by heating in a metal bath at 100 °C for 10 min. Centrifuge at 12,000 rpm for 5 min, take the supernatant, filter through a 0.22 μm filter membrane, and perform high performance liquid chromatography detection. The results are shown in Table 7. The conversion rate did not increase with the increase of time, indicating that the reaction had reached equilibrium. The conversion rate was about 33%. The liquid chromatogram of the reaction results is as Figure 4 shown.

[0089] Table 7, determination of reaction equilibrium

[0090]

[0091] Example 9, determination of the maximum conversion ability of the enzyme

[0092] Since this D-allulose 3-epimerase is derived from halotolerant Bacillus, its ability to tolerate high sugar is relatively higher than that of general D-allulose 3-epimerase. In this example, the highest fructose concentration that this D-allulose 3-epimerase can tolerate was determined. The reaction system was 100, 300, 400, 500, 600, 700 g / L D-fructose, 100 μL of the crude enzyme solution prepared in Example 3 above was added, 0.4 mM manganese ion was added, and the reaction system was made up to 1 mL with 50 mM PB 7.0 buffer. After reacting at 60 °C for 6, 8, and 10 h, the reaction was stopped by heating in a metal bath at 100 °C for 10 min. Centrifuge at 12,000 rpm for 5 min, take the supernatant, filter through a 0.22 μm filter membrane, and perform high performance liquid chromatography detection. The results are shown in Table 8. When the fructose concentration was 600 g / L, it did not affect the conversion rate, which remained at about 33%. When the concentration was further increased, the conversion rate decreased. Therefore, the highest tolerance concentration of D-allulose 3-epimerase was 600 g / L.

[0093] Table 8, conversion rates of D-allulose 3-epimerase at different fructose concentrations

[0094]

[0095] Example 10, stability of D-allulose 3-epimerase

[0096] Due to the complex production environment in large-scale production, when feeding large reactors, there may be short-term increases or decreases in conditions such as pH and temperature, which poses requirements for the stability of enzymes.

[0097] 1. pH stability detection

[0098] In this example, the stability that this D-allulose 3-epimerase can tolerate was determined. The crude enzyme solution prepared in Example 3 above was added and placed in buffer solutions with pH values of 5.8, 6.2, 6.6, 7.0, 7.4, 7.8, and 8.0 respectively, and treated at 4 °C for 16 h. Then, 20 μL of the treated crude enzyme solution was added to a reaction system where the D-fructose was 50 g / L, the manganese ion was 0.4 mM, and the reaction system was made up to 1 mL with 50 mM PB 7.0 buffer. After reacting at 60 °C for 10 min, the reaction was stopped by heating in a 100 °C metal bath for 10 min. After centrifuging at 12000 rpm for 5 min, the supernatant was taken and filtered through a 0.22 μm filter membrane for high-performance liquid chromatography detection. As shown in Table 9, from the detection results of the reaction, when the enzyme was placed in an environment of 5.8 - 8.0 for 16 h and then reacted under the optimal reaction conditions, it did not cause enzyme inactivation, indicating good pH stability.

[0099] Table 9. Stability of D-allulose 3-epimerase treated at different pH values

[0100]

[0101] 2. Temperature stability detection

[0102] In this example, the stability that this D-allulose 3-epimerase can tolerate was determined. The crude enzyme solution prepared in Example 3 above was added and placed in water baths at temperatures of 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, and 80 °C respectively, and treated for 30 min. Then, 20 μL of the treated crude enzyme solution was added to a reaction system where the D-fructose was 50 g / L, the manganese ion was 0.4 mM, and the reaction system was made up to 1 mL with 50 mM PB 7.0 buffer. After reacting at 60 °C for 10 min, the reaction was stopped by heating in a 100 °C metal bath for 10 min. After centrifuging at 12000 rpm for 5 min, the supernatant was taken and filtered through a 0.22 μm filter membrane for high-performance liquid chromatography detection. As shown in Table 10, from the detection results of the reaction, when the enzyme was placed under high-temperature conditions for 30 min and then reacted under the optimal reaction conditions, except for a slight effect on enzyme activity at 80 °C, there was no effect on enzyme activity below 70 °C, indicating good temperature stability.

[0103] Table 10. Stability of D-allulose 3-epimerase treated at different temperatures

[0104]

[0105] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A D-psicose 3-epimerase, characterized in that, The amino acid sequence of the D-psicose 3-epimerase is as shown in SEQ ID No.

1.

2. A nucleic acid, characterized in that, Encoding the D-psicose 3-epimerase described in claim 1, the nucleotide sequence thereof is as shown in SEQ ID No.

2.

3. A recombinant vector, characterized in that, Containing the nucleotide sequence encoding the D-psicose 3-epimerase described in claim 1.

4. The recombinant vector according to claim 3, wherein The recombinant vector contains the pUC980 expression plasmid.

5. A strain, characterized in that, It is a strain expressing D-psicose 3-epimerase, and the strain contains the nucleotide sequence encoding D-psicose 3-epimerase, and the sequence is as shown in SEQ ID No.2; the amino acid sequence of the D-psicose 3-epimerase is as shown in SEQ ID No.

1.

6. The strain according to claim 5, characterized in that, The host bacterium of the strain expressing D-psicose 3-epimerase is Bacillus subtilis.

7. A method for preparing D-psicose 3-epimerase, characterized in that, Using the strain described in any one of claims 5 to 6 to ferment and prepare D-psicose 3-epimerase in a liquid fermentation medium.

8. The preparation method of D-psicose 3-epimerase according to claim 7, characterized in that, The formula of the liquid fermentation medium is: 20 g of glycerol, 50 g of peptone, 5 g of acid-hydrolyzed casein, 1 g of KH2PO4, 0.5 g of KCl, 0.5 g of MgSO4·7H2O, 10 mg of FeSO4·7H2O, adjusting the pH to 7.2, and making up the volume to 1 L with ddH2O.

9. The application of the D-psicose 3-epimerase described in claim 1 in the production of D-psicose.

10. The application according to claim 9, wherein Using D-psicose-3-epimerase to produce D-psicose, including the following steps: using D-fructose as a substrate, after adding the D-psicose 3-epimerase, generating D-psicose through a catalytic reaction; In the reaction system, the pH is 6.0 to 8.0; The reaction temperature is 50 to 65 °C; A metal ion is added to the reaction system, and the metal ion is selected from at least one of magnesium ion, cobalt ion, and manganese ion; The final concentration of the metal ion is 0.1 to 1 mM.

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