D-psicose 3-epimerase as well as recombinant vector, strain and application thereof

Abstract

The invention discloses D-psicose 3-epimerase as well as a recombinant vector, a strain and application thereof, and belongs to the technical field of functional enzymes. The invention provides the D-psicose-3-epimerase derived from bacillus halotolerans SYNY-019 for the first time, the protein sequence of the D-psicose-3-epimerase is as shown in SEQ ID No.1, the nucleotide sequence obtained after the protein sequence is subjected to codon optimization is as shown in SEQ ID No.2, the nucleotide sequence is as shown in SEQ ID No.1, and the D-psicose-3-epimerase has the advantages that the nucleotide sequence is as shown in SEQ ID No.2; the D-psicose 3-epimerase can be used for efficiently catalyzing the synthesis of D-psicose from D-fructose.

Description

Technical Field

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

[0002] D-allulose is a rare sugar that has been reported for nearly 30 years. D-allulose is a diastereomer of fructose and a safe sweetener. Although its sweetness is only 70% of that of sucrose, the energy it produces is only 0.3% of that of sucrose, making it a truly low-energy sugar. D-allulose is used in large quantities in food and beverages around the world, and its market size is constantly increasing.

[0003] Initially, the chemical synthesis methods of D-psicose include selective alcohol-aldehyde condensation synthesis, catalytic hydrogenation, addition reaction, etc. Although chemical synthesis can prepare D-psicose, it has problems such as poor economic efficiency and serious environmental pollution. Therefore, the chemical synthesis of D-psicose has not been industrialized. Compared with chemical synthesis, biological synthesis of D-psicose not only has strong reaction specificity and single product, but also simple separation and purification methods and less environmental pollution. The biotransformation method is not only conducive to reducing industrialization costs, but also conforms to the production principle of green environmental protection. It is the main method for industrial production of D-psicose at home and abroad.

[0004] The biological method mainly prepares D-psicose from fructose by enzyme catalysis under the action of D-psicose 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-psicose 3-epimerase is weak, which limits its industrial application. In order to meet the complex needs of industrial production, it is necessary to find a D-psicose 3-epimerase 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-psicose 3-epimerase and a recombinant vector, strain and application thereof, aiming to solve the problem of enzyme instability of the existing D-psicose 3-epimerase when catalyzing D-fructose to synthesize D-psicose, so that the D-psicose 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: As a first aspect of the present invention, a D-psicose 3-epimerase is provided, and the amino acid sequence of the D-psicose 3-epimerase is shown in SEQ ID No. 1.

[0007] As a second aspect of the present invention, targeted codon optimization is further performed on the amino acid sequence, and an optimized nucleotide sequence encoding D-psicose 3-epimerase is provided as shown in SEQ ID No. 2.

[0008] As a third aspect of the present invention, a recombinant vector for expressing the D-psicose 3-epimerase is provided, which contains a nucleotide sequence encoding the D-psicose 3-epimerase.

[0009] Optionally, the recombinant vector comprises a pUC980 expression plasmid.

[0010] As a fourth aspect of the present invention, a strain is provided, which is a strain expressing D-psicose 3-epimerase, and the strain contains a nucleotide sequence encoding D-psicose 3-epimerase, the sequence of which is shown in SEQ ID No. 2; the amino acid sequence of the D-psicose 3-epimerase is shown in SEQ ID No. 1.

[0011] Optionally, the host bacteria of the strain expressing the D-psicose 3-epimerase is Bacillus subtilis.

[0012] As a fifth aspect of the present invention, a method for preparing the D-psicose 3-epimerase is provided, wherein the recombinant vector or strain is used to ferment in a liquid fermentation medium to prepare the D-psicose 3-epimerase.

[0013] Preferably, the formula of the liquid fermentation medium is: 20 g glycerol, 50 g peptone, 5 g acid hydrolyzed casein, 1 g KH 2 PO 4 , 0.5 g KCl, 0.5 g MgSO 4 7H 2 O, 10 mg FeSO 4 7H 2 O, adjust pH to 7.2, add ddH 2 O to 1 L. Autoclave at 121°C for 20 min. Note that MgSO 4 7H 2 O and FeSO 4 7H 2 O It cannot be sterilized by high temperature or high pressure. Prepare the mother solution separately and filter and sterilize.

[0014] Preferably, the inoculation amount of the recombinant vector or host bacteria is 1-5% of the total volume of the liquid fermentation medium.

[0015] As a sixth aspect of the present invention, provided is the use of the D-psicose 3-epimerase in the production of D-psicose.

[0016] In one embodiment of the present invention, producing D-psicose using D-psicose-3-epimerase comprises the following steps: taking D-fructose as a substrate, adding the D-psicose 3-epimerase, and generating D-psicose through a catalytic reaction.

[0017] Preferably, in the reaction system, the pH is 6.0 to 8.0, and the more preferred suitable pH is 7.0.

[0018] Preferably, the reaction temperature is 50-65°C, and a more preferred suitable temperature is 60°C.

[0019] 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 more preferably manganese ions.

[0020] Preferably, the final concentration of the metal ion is 0.1-1 mM, and more preferably the final concentration of the metal ion is 0.4 mM.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention has discovered a new Bacillus halotolerans ) D-psicose 3-epimerase of SYNY-019, the wild-type protein sequence was codon-optimized and heterologously expressed in Bacillus subtilis engineered bacteria; (2) The present invention also provides a method for preparing D-psicose 3-epimerase, which lays a foundation for the subsequent industrial production of D-psicose 3-epimerase; (3) In the present invention, D-fructose is used as a substrate and D-psicose 3-epimerase is used as a catalyst to directly enzymatically catalyze the conversion of D-fructose into D-psicose, thereby saving industrial production costs; (4) The D-psicose 3-epimerase provided by the present invention solves the problem that the existing D-psicose 3-epimerase is unstable when catalyzing the synthesis of D-psicose from D-fructose, so that the D-psicose 3-epimerase maintains good activity in a wider pH range and a wider temperature range, has good stability, and can adapt to the complex production environment and fluctuations in conditions in large-scale production; it has high fructose tolerance and high conversion rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0023] Figure 1 This is the SDS-PAGE analysis of D-psicose 3-epimerase expressed in Bacillus subtilis in Example 1; wherein M is a protein marker, 1 is a fermentation supernatant sample, 2 is a whole cell fluid sample, and 3 is a supernatant crude enzyme solution sample after crushing and centrifugation.

[0024] Figure 2 This is the liquid chromatography spectrum of D-fructose reference substance (20 mg / mL); Figure 3 This is the liquid chromatography spectrum of the reference substance of D-psicose (20 mg / mL); Figure 4 This is the liquid chromatography spectrum of the reaction process of D-psicose synthesis catalyzed by D-psicose 3-epimerase. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments of the specification. 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 description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention. The strains and expression vectors involved in the following examples are: Bacillus subtilis strain B. subtilis 168, pUC980 expression plasmid. The enzymes used in the following examples are all expressed by the above strains and vectors.

[0026] The culture medium involved in the following examples: LB liquid culture medium, whose composition is: yeast powder 5.0 g / L, peptone 10.0 g / L and NaCl 10.0 g / L.

[0027] M1 fermentation medium, which consists of: 20 g glycerol, 50 g peptone, 5 g acid hydrolyzed casein (without vitamins), 1 g KH 2 PO 4 , 0.5 g KCl, 0.5 g MgSO 4 7H 2 O, 10 mg FeSO 4 7H 2 O, adjust pH to 7.2, add ddH 2 O to 1L. Sterilize by high temperature and high pressure at 121℃ for 20 min. Note that MgSO 4 7H 2 O and FeSO 4 7H2 O It cannot be sterilized by high temperature or high pressure. Prepare the mother solution separately and filter and sterilize.

[0028] The preparation methods of phosphate buffer (PB) buffers with different pH values ​​are as follows: 1. Solution A (0.2 M sodium dihydrogen phosphate aqueous solution): NaH 2 PO 4 ·H 2 O 27.6 g, dissolved in distilled water, diluted to 1000 mL.

[0029] 2. Solution B (0.2 M sodium dihydrogen phosphate aqueous solution): Na 2 HPO 4 7H 2 O 53.6 g (or Na 2 HPO 4 ·12 H 2 O71.6 g or Na 2 HPO 4 ·2H 2 O 35.6 g) was dissolved in distilled water and the volume was increased to 1000 mL.

[0030] Add Y mL of solution B to X mL of solution A (see Table 1) to make 0.2 M PB. Add distilled water to 800 mL to make 50 mM PB buffer.

[0031] Table 1. Preparation system of phosphate buffer (PB) buffer with different pH values

[0032] 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 mobile phase flow rate is 0.6 mL / min; the chromatographic column temperature is 80 ℃; the detector is a differential refractive index detector.

[0033] Example 1, a salt-tolerant Bacillus ( Bacillus halotolerans )SYNY-019 Halotolerant Bacillus Bacillus halotolerans

[0034] The strain was deposited at the General Microbiology Center of China Microbiological Culture Collection Administration 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, with the deposit number CGMCC No.26285.

[0035] Example 2, Construction of recombinant strains Halotolerant Bacillus Bacillus halotolerans ) According to the sequencing results and genome analysis of SYNY-019, the gene encoding D-psicose 3-epimerase was obtained in the following manner: GenScript Biotech Co., Ltd. performed codon optimization of Bacillus subtilis based on the amino acid sequence shown in SEQ ID NO: 1. After codon optimization, the optimized nucleotide sequence of D-psicose 3-epimerase was obtained, as shown in SEQ ID NO: 2, and the whole gene was synthesized into the pUC980 vector plasmid to obtain the recombinant expression vector plasmid pUC980-DAE.

[0036] The transformation of Bacillus subtilis in this experiment adopts the Spizizen transformation method: Preparation of Spizizen transformation medium: Each reagent is prepared separately, and the culture medium is mixed to prepare GM1 and GM2 solutions when used. The system is shown in Table 2.

[0037] 1) 0.5% tryptophan: weigh 0.25 g tryptophan and dissolve it in ddHO 2 O, dilute to 50 mL, and filter sterilize; 2) 2% acid hydrolyzed casein (without vitamins): weigh 2 g of acid hydrolyzed casein (without vitamins) and dissolve in ddH 2 O, make up to 100 mL, sterilize at 115 °C for 30 min; 3) 40% glucose: weigh 40 g glucose and dissolve it in ddH 2 O, make up to 100 mL, sterilize at 115 °C for 30 min; 4) 20% MgSO 4 7H 2 O: weigh 10 g MgSO 4 7H 2 O dissolved in ddH 2 O, dilute to 50 mL, and filter sterilize; 5) 10× Spizizen Basic Salt Medium: weigh 0.2 g (NH 4 ) 2 SO 4 , 1.83 g K 2 HPO4 , 0.6 gKH 2 PO 4 , 0.12 g sodium citrate dissolved in ddH 2 O, make up to 100 mL, adjust pH to 7.2, sterilize at 121 °C for 20 min; 6) 5% yeast extract: weigh 2.5 g yeast extract and dissolve it in ddHO 2 O, make up to 50 mL, and sterilize at 121 °C for 20 min.

[0038] Table 2, Spizizen transformation medium preparation system

[0039] Preparation and transformation of Bacillus subtilis competent cells: 1) Streak the frozen B. subtilis 168 on LB solid medium and culture it at 37°C overnight; 2) Pick a single colony and inoculate it into 5 mL GM1, and culture it in a shaker at 37°C and 200 rpm for 14 h; 3) Transfer 500 μL of bacterial solution into 5 mL of GM1 and culture in a shaker at 37 °C and 200 rpm for 4.5 h; 4) Transfer 750 μL of bacterial solution into 5 mL of GM2 and culture in a shaker at 37 °C and 200 rpm for 1.5 h; 5) Take out the test tube and divide it into 1.5 mL centrifuge tubes, with 1 mL of bacterial solution in each centrifuge tube. The competent cells are ready. Use them for transformation immediately and do not store them. Add 1 μg of plasmid and mix with the competent cells, and resuscitate at 37 ℃ for 1.5 h. Spread the transformation product on a kanamycin-resistant LB solid medium plate and culture it at 37 ℃ overnight (about 16 h). Pick the transformant on the LB solid medium to obtain the recombinant Bacillus subtilis containing the recombinant plasmid. B. subtilis 168 / pUC980-DAE.

[0040] Example 3, Preparation of enzyme Pick recombinant Bacillus subtilis from the plate B. subtilis The 168 / pUC980-DAE single clone was inoculated (by directly injecting the pipette tip into the test tube) into a test tube containing 5 mL of LB liquid medium with kanamycin resistance and cultured overnight (about 16 h) at 37°C and 200 rpm.

[0041] Take 10 mL of overnight cultured seed culture and transfer it to a shake flask containing 500 mL of M1 fermentation medium with resistance and ferment it at 37 °C for 48 h.

[0042] The cultured bacteria were centrifuged and collected. The bacterial sludge was weighed and resuspended in the ratio of 0.1 g of bacteria to 1 mL of reaction buffer. The bacterial suspension was broken by ultrasonic disruption instrument, ultrasonic for 3 s, paused for 2 s, and the time was 20 min (the time can be adjusted according to the size of the ultrasonic volume). After the ultrasonic system was centrifuged, the supernatant obtained was the crude enzyme solution of the recombinant strain. The fermentation broth supernatant, bacterial whole cells, and the supernatant after bacterial disruption were subjected to SDS-PAGE analysis. Figure 1 As shown, the target band is about 36 KDa.

[0043] Example 4, Method for Determining the Activity of D-psicose 3-epimerase DAE can catalyze the conversion of D-fructose into D-psicose, so DAE activity detection uses D-fructose as the substrate. The volume of the entire enzyme reaction system is 1 mL, the reaction system is 50 g / L of D-fructose, 20 μL of the crude enzyme solution prepared in Example 3 above is added, and 50 mM PB 7.0 buffer is added to make the reaction system 1 mL. After reacting at 55 °C for 10 min, the reaction is stopped by heating in a 100 °C metal bath for 10 min. Centrifuge at 12000 rpm for 5 min, take the supernatant and filter through a 0.22 μm filter for high performance liquid chromatography detection, and define the enzyme activity by detecting the amount of D-psicose produced.

[0044] Preparation of reference solution: D-fructose reference solution (20 mg / mL, solvent is ultrapure water), D-psicose reference solution (20 mg / mL, solvent is ultrapure water), the liquid chromatograms are as follows: Figure 2 and Figure 3 shown.

[0045] Example 5, Determination of the Optimal pH for D-psicose 3-epimerase The reaction system was 50 g / L D-fructose, 20 μL of the crude enzyme solution prepared in Example 3 was added, and 50 mM PB 6.0, 6.5, 7.0, 7.5, 8.0 buffer was 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. Centrifuged at 12000 rpm for 5 min, the supernatant was filtered through a 0.22 μm filter membrane for high performance liquid chromatography detection. The results are shown in Table 3. When the pH is 7, it is more suitable for the reaction.

[0046] Table 3. Effect of pH on D-psicose 3-epimerase

[0047] Example 6, Optimal Temperature of D-psicose 3-epimerase The reaction system was 50 g / L D-fructose, 20 μL of the crude enzyme solution prepared in Example 3 was added, and 50 mM PB 7.0 buffer was added to make the reaction system 1 mL. After reacting at 50, 55, 60, 65, and 70 °C for 10 min, the reaction was stopped by heating in a 100 °C metal bath for 10 min. Centrifuged at 12000 rpm for 5 min, the supernatant was filtered through a 0.22 μm filter membrane for high performance liquid chromatography detection. The results are shown in Table 4. When the reaction temperature is 60 °C, the reaction is more suitable.

[0048] Table 4. Effect of temperature on D-psicose 3-epimerase

[0049] Example 7, Suitable ions and ion concentrations for D-psicose 3-epimerase The reaction system was 50 g / L D-fructose, 20 μL of the crude enzyme solution prepared in Example 3 was added, 0.1 mM magnesium ion, cobalt ion, manganese ion, and calcium ion were added, and 50 mM PB 7.0 buffer was added to make up the reaction system 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. Centrifuged at 12000 rpm for 5 min, the supernatant was filtered through a 0.22 μm filter membrane for high performance liquid chromatography detection. The results are shown in Table 5, and manganese ion is the most suitable reaction ion.

[0050] Table 5. Effects of different ions on D-psicose 3-epimerase

[0051] The reaction system was 50 g / L D-fructose, 20 μL of the crude enzyme solution prepared in Example 3 was added, 0.1, 0.2, 0.4, 0.6, 0.8, 1 mM manganese ions were added, and 50 mM PB 7.0 buffer was added to make up the reaction system to 1 mL. After reacting at 60°C for 10 min, the reaction was stopped by heating in a 100°C metal bath for 10 min. Centrifuged at 12000 rpm for 5 min, the supernatant was filtered through a 0.22 μm filter membrane for high performance liquid chromatography detection, and the results are shown in Table 6. The optimal amount of manganese ions added was 0.4 mM.

[0052] Table 6. Effects of different ion concentrations on D-psicose 3-epimerase

[0053] Example 8, Determination of Reaction Equilibrium Since there is a reaction equilibrium in the reaction of diastereoisomerization, this example increases the amount of enzyme and the reaction time on the basis of the above optimized system, and determines the reaction equilibrium point. The reaction system is 50 g / L of D-fructose, 100 μL of the crude enzyme solution prepared in the above Example 3 is added, 0.4 mM manganese ions are added, and 50 mM PB 7.0 buffer is added to make up the reaction system to 1 mL. After reacting at 60 °C for 6, 8, and 10 h, the reaction is stopped by heating in a 100 °C metal bath for 10 min. Centrifuge at 12000 rpm for 5 min, take the supernatant and filter it through a 0.22 μm filter membrane for high performance liquid chromatography. The results are shown in Table 7. The conversion rate does not increase with time, indicating that the reaction has reached equilibrium. The conversion rate is about 33%. The liquid phase diagram of the reaction results is shown in Table 7. Figure 4 shown.

[0054] Table 7, Determination of reaction equilibrium

[0055] Example 9, determination of the maximum conversion capacity of the enzyme Since this D-psicose 3-epimerase is derived from halotolerant Bacillus, its ability to tolerate high sugar is higher than that of general D-psicose 3-epimerase. In this example, the maximum fructose concentration that this D-psicose 3-epimerase can tolerate is determined. The reaction system is D-fructose at 100, 300, 400, 500, 600, and 700 g / L, 100 μL of the crude enzyme solution prepared in Example 3 above is added, 0.4 mM manganese ions are added, and 50 mM PB 7.0 buffer is added to make up the reaction system to 1 mL. After reacting at 60 °C for 6, 8, and 10 h, the reaction is stopped by heating in a 100 °C metal bath for 10 min. The mixture was centrifuged at 12000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane for high performance liquid chromatography. The results are shown in Table 8. When the fructose concentration was 600 g / L, the conversion rate was not affected and remained at around 33%. When the concentration increased further, the conversion rate decreased. Therefore, the maximum tolerable concentration of D-psicose 3-isomerase was 600 g / L.

[0056] Table 8. Conversion rate of D-psicose 3-epimerase at different fructose concentrations

[0057] Example 10, Stability of D-psicose 3-epimerase Due to the complex production environment in large-scale production and the feeding of materials into large reactors, the pH, temperature and other conditions may rise or fall for a short period of time, which puts forward requirements for the stability of the enzyme.

[0058] 1. pH stability test In this example, the stability that this D-psicose 3-epimerase can tolerate is determined. The crude enzyme solution prepared in the above Example 3 is placed in a buffer solution of pH 5.8, 6.2, 6.6, 7.0, 7.4, 7.8, and 8.0, respectively, and treated at 4 ° C for 16 h. 20 μL of the treated crude enzyme solution is added to a reaction system of 50 g / L D-fructose, 0.4 mM manganese ion, and 50 mM PB7.0 buffer to make up the reaction system to 1 mL of the reaction system. After reacting at 60 ° C for 10 min, the reaction is stopped by heating in a 100 ° C metal bath for 10 min. Centrifuge at 12000 rpm for 5 min, take the supernatant through a 0.22 μm filter membrane for high performance liquid chromatography detection, as shown in Table 9. From the test results of the reaction, it can be seen that placing the enzyme in an environment of 5.8 to 8.0 for 16 h and then placing it under the most suitable reaction conditions for reaction will not cause enzyme inactivation and has good pH stability.

[0059] Table 9. Stability of D-psicose 3-epimerase treated at different pH values

[0060] 2. Temperature stability test In this example, the stability of D-psicose 3-epimerase was determined. The crude enzyme solution prepared in Example 3 was placed in a water bath at 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C for 30 min. 20 μL of the crude enzyme solution was added to a reaction system containing 50 g / L D-fructose, 0.4 mM manganese ions, and 50 mM PB 7.0 buffer to make up the reaction system to 1 mL. After reacting at 60°C for 10 min, the reaction was stopped by heating in a 100°C metal bath for 10 min. The mixture was centrifuged at 12000 rpm for 5 min and the supernatant was filtered through a 0.22 μm filter membrane for high performance liquid chromatography detection. As shown in Table 10, from the reaction detection results, the enzyme was placed under high temperature conditions for 30 min and then placed under the most suitable reaction conditions for reaction. Except for a slight effect on the enzyme activity at 80 °C, there was no effect on the enzyme activity below 70 °C, indicating that the enzyme had good temperature stability.

[0061] Table 10. Stability of D-psicose 3-epimerase treated at different temperatures

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in 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 shown in SEQ ID No.

1.

2. A nucleic acid, characterized in that A gene encoding the D-psicose 3-epimerase according to claim 1, wherein the nucleotide sequence is shown in SEQ ID No.

2.

3. A recombinant vector, characterized in that: Contains a nucleotide sequence encoding the D-psicose 3-epimerase according to claim 1.

4. The recombinant vector according to claim 3, characterized in that The recombinant vector comprises a pUC980 expression plasmid.

5. A strain characterized by: The invention relates to a strain expressing D-psicose 3-epimerase, wherein the strain contains a nucleotide sequence encoding D-psicose 3-epimerase, the sequence of which is shown in SEQ ID No. 2; and the amino acid sequence of the D-psicose 3-epimerase is shown in SEQ ID No.

1.

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

7. A method for preparing D-psicose 3-epimerase, characterized in that: The recombinant vector according to any one of claims 3 to 4 or the strain according to any one of claims 5 to 6 is used to ferment and prepare D-psicose 3-epimerase in a liquid fermentation medium.

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

9. Use of the D-psicose 3-epimerase according to claim 1 in producing D-psicose.

10. The use according to claim 9, characterized in that: The method of producing D-psicose by using D-psicose-3-epimerase comprises the following steps: taking D-fructose as a substrate, adding the D-psicose-3-epimerase, and generating D-psicose by catalytic reaction; In the reaction system, the pH is 6.0 to 8.0; The reaction temperature is 50-65°C; Adding metal ions to the reaction system, wherein the metal ions are selected from at least one of magnesium ions, cobalt ions, and manganese ions; The final concentration of metal ions is 0.1-1 mM.

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