A tagatose 4-epimerase and its use in the production of tagatose

Abstract

This invention discloses a tagatose 4-epimerase and its application in tagatose production, belonging to the fields of genetic engineering and modern enzyme technology engineering. This invention provides a novel tagatose 4-epimerase, the amino acid sequence of which is shown in SEQ ID NO.1. This enzyme is strictly Ni² dependent. + The metalloenzyme was synthesized. Using the recombinant E. coli whole-cell catalytic system of this invention, with fructose as a substrate, the conversion rate reached 24.89% under optimal conditions for 12 h. The product was identified as tagatose by HPLC. This method eliminates the need for enzyme separation and purification, features mild reaction conditions, simple operation, and is environmentally friendly, showing great promise for the industrial production of the functional sweetener tagatose.

Description

Technical Field

[0001] This invention relates to a tagatose 4-epimerase and its application in the production of tagatose, belonging to the fields of genetic engineering and modern enzyme technology engineering. Background Technology

[0002] D-tagatose is a rare natural hexose and a diastereomer of fructose. Its sweetness is approximately 92% that of sucrose, and its taste is very similar, but its calorie content is only 1.5 kcal / g (compared to 4 kcal / g for sucrose), far lower than traditional sugars. Numerous studies have shown that tagatose possesses various physiological activities, including lowering glycemic response, not causing drastic insulin fluctuations, a low glycemic index (GI≈3), anti-caries properties, improving gut microbiota, and antioxidant effects. Therefore, tagatose, as an ideal functional sweetener, has broad application prospects in diabetic foods, weight management products, oral care, and pharmaceuticals.

[0003] Currently, the enzymatic production of tagatose largely relies on L-arabinose isomerase (L-AI) to catalyze the conversion of D-galactose to tagatose. This route typically requires the prior preparation of D-galactose from lactose, involving numerous process steps. Furthermore, the reported activity, stability, and conversion efficiency of some L-AIs still fall short of the requirements for large-scale production. Therefore, developing biocatalytic systems with more direct catalytic pathways and superior performance using inexpensive and readily available raw materials remains a significant need in this field.

[0004] In recent years, a novel tagatose 4-epimerase has been discovered that can directly catalyze the conversion of inexpensive and abundant fructose into tagatose. Compared with the L-AI route, this reaction uses fructose as a substrate, eliminating the step of obtaining galactose from lactose hydrolysis, significantly reducing raw material costs and making the reaction pathway more efficient. However, very few tagatose 4-epimerases have been identified in nature, and they generally suffer from low enzyme activity, poor thermal stability, and low catalytic efficiency, falling far short of the requirements for industrial applications. Therefore, discovering and developing novel tagatose 4-epimerases with high activity and high stability has become a pressing technical challenge in this field. Summary of the Invention

[0005] To address the lack of highly catalytically active tagatose 4-epimerases in existing technologies, this invention provides a tagatose 4-epimerase obtained through a five-point mutation, the amino acid sequence of which is shown in SEQ ID NO.1. Furthermore, this protein was discovered for the first time to possess catalytic activity for the efficient conversion of fructose into tagatose, thereby enabling the construction of a whole-cell catalytic production technology based on this enzyme.

[0006] The first technical solution provided by the present invention is a tagatose 4-epimerase, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The second technical solution provided by the present invention is a gene that encodes the tagatose 4-epimerase described in the first technical solution.

[0008] In some embodiments, the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0009] The third technical solution provided by the present invention is a recombinant plasmid, wherein the recombinant plasmid carries the gene described in the second technical solution.

[0010] In some embodiments, the expression vector of the recombinant plasmid is the pET-22b (+) plasmid.

[0011] The fourth technical solution provided by the present invention is to express the tagatose 4-epimerase described in the first technical solution, or to contain the gene described in the second technical solution, or to transform a host cell with the recombinant plasmid described in the third technical solution.

[0012] In some embodiments, the host cell is Escherichia coli or Bacillus subtilis.

[0013] In some embodiments, the host cell is E. coli BL21 (DE3).

[0014] The fifth technical solution provided by the present invention is a catalyst for producing tagatose, wherein the catalyst contains the tagatose 4-epimerase described in the first technical solution or the host cell described in the fourth technical solution.

[0015] In some embodiments, the catalyst includes, but is not limited to, enzyme preparations, engineered microbial agents, or immobilized cell products.

[0016] The sixth technical solution provided by the present invention is a method for producing tagatose. The method involves first adding the tagatose 4-epimerase described in the first technical solution, the host cell described in the fourth technical solution, or the catalyst described in the fifth technical solution to a reaction system containing fructose to carry out the reaction, thereby obtaining a reaction solution, and then separating the tagatose from the reaction solution.

[0017] In some embodiments, the reaction conditions are: pH 8.0-9.0, temperature 60-70 °C, and the substrate in the reaction system is fructose with a concentration range of 1-15% (w / v).

[0018] In some embodiments, the reaction conditions in the reaction system are: pH 8.5, 65 °C; and the fructose concentration in the reaction system is 100 g / L.

[0019] The seventh technical solution provided by the present invention is the application of the tagatose 4-epimerase described in the first technical solution, the gene described in the second technical solution, the recombinant plasmid described in the third technical solution, the host cell described in the fourth technical solution, the catalyst described in the fifth technical solution, or the method described in the sixth technical solution in the production of tagatose or products containing tagatose.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention provides a novel tagatose 4-epimerase, the amino acid sequence of which is shown in SEQ ID NO.1.

[0021] (2) This invention utilizes the enzyme to directly produce tagatose using fructose as a substrate. The reaction system is simple, does not produce harmful byproducts, and is environmentally friendly. Under optimal conditions, the conversion rate of tagatose can reach 24.89%, showing extremely high potential for industrial application.

[0022] (3) The recombinant engineered bacteria constructed in this invention have high expression levels and are easy to cultivate, which can significantly reduce the production cost of enzymes and lay a solid foundation for the large-scale enzymatic production of tagatose. Attached Figure Description

[0023] Figure 1 SDS-PAGE analysis of recombinant Escherichia coli expression products.

[0024] Figure 2 The effect of pH on the catalytic activity of tagatose 4-epimerase.

[0025] Figure 3 The effect of temperature on the catalytic activity of tagatose 4-epimerase.

[0026] Figure 4 The effect of different substrate concentrations on conversion rate.

[0027] Figure 5 The effect of different enzyme dosages on conversion rate.

[0028] Figure 6 The HPLC chromatogram of the reaction solution shows the formation of the product tagatose. Detailed Implementation

[0029] Reference Appendix Figures 1-6 The preferred embodiments of the present invention will be described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0030] Test method: Standard enzyme activity assay method: Prepare a 100 g / L fructose substrate solution using 50 mmol / L Tris-HCl buffer (pH 8.5), and add NiSO4 to make Ni²⁻ + The final concentration was 5 mmol / L. Take 2 mL of the fermentation broth obtained in Example 2, centrifuge at 8000 rpm for 5 min to collect the wet cells, add the above substrate solution to a total reaction volume of 1 mL, resuspend thoroughly, and react at 65 ℃ and 200 rpm for 1 h. Immediately after the reaction, boil for 5 min to terminate the reaction, centrifuge at 12000 rpm for 5 min, filter the supernatant through a 0.22 μm filter membrane, and determine the amount of tagatose produced using the following HPLC method, and calculate the enzyme activity accordingly. Unless otherwise specified below, the standard enzyme activity test method is used.

[0031] Enzyme activity unit (U) definition: Under the above standard test conditions, the amount of enzyme required to catalyze the production of 1 μmol tagatose per minute is 1 U. Relative enzyme activity (%) is the ratio of enzyme activity under specific conditions to enzyme activity under reference conditions; enzyme activity under reference conditions is defined as 100%.

[0032] The amount of tagatose produced was determined by HPLC. The HPLC analysis conditions were as follows: a Waters Sugar-Pak™ I sugar analysis column (6.5 mm inner diameter, 300 mm length, product number WAT085188) was used, the column temperature was 85 ℃, the mobile phase was 50 mg / L EDTA calcium salt aqueous solution, the flow rate was 0.4 mL / min, the detector was a refractive index detector (RID), and the injection volume was 10 μL.

[0033] Raw materials used in the examples: LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH natural.

[0034] LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 15 g / L.

[0035] Example 1: Construction of a recombinant strain of tagatose 4-epimerase The specific steps are as follows: 1. Commercially synthesized recombinant plasmids (1) Enzyme screening Adopted from ThermotogaThe original enzyme sequence was obtained from the tagaguron acid epimerase family protein of sp. RQ7 (protein sequence accession number WP_041843930.1), and the amino acid sequence of the enzyme protein that may have tagaguron 4-epimerase activity was obtained by computer-aided screening, as shown in SEQ ID NO.1.

[0036] (2) Synthesis of the target gene The commissioned company performed codon optimization and whole-gene synthesis based on the amino acid sequence shown in SEQ ID NO.1, and introduced a sequence encoding a 6×His tag at the 3' end of the gene to obtain the coding gene (SEQ ID NO.2), which was then directly constructed into the NdeI / XhoI site of the expression vector pET-22b(+). The resulting plasmid was named pET-22b(+)-T4E.

[0037] 2. Construction of engineered bacteria The recombinant plasmid pET-22b(+)-T4E obtained in step 1 was transformed into E. coli BL21 (DE3) competent cells by thermal shock chemical transformation to obtain the corresponding recombinant engineered bacteria E. coli BL21 (DE3) / pET-22b(+)-T4E.

[0038] Example 2: Induction and purification of recombinase 1. Induced expression (1) The obtained recombinant engineered bacteria E. coli BL21 (DE3) / pET-22b(+)-T4E was streaked from a glycerol tube onto an LB agar plate (containing 50 μg / mL Amp, incubated upside down at 37°C for 12 h).

[0039] (2) Pick a single colony from LB solid medium and culture it in 5 mL LB liquid medium containing 50 μg / mL Amp at 37℃ and 200 r / min for 12 h to prepare seed liquid.

[0040] (3) The prepared seed culture was transferred to 50 mL LB liquid medium containing Amp at an inoculation rate of 2% (v / v) and cultured at 37°C and 200 r / min until OD. 600 The pH value was around 0.6. IPTG was added as an inducer to bring the final IPTG concentration in the culture medium to 0.1 mmol / L. Induction was carried out at 25℃ and 200 r / min for 24 h, yielding the fermentation broth. 1 mL of the fermentation broth was reserved for subsequent SDS-PAGE analysis.

[0041] (4) Centrifuge the fermentation broth at 4℃ and 6000 rpm for 10 min. Resuspend the cells in cell lysis buffer, then sonicate for 15 min (2 s sonication, 3 s rest). Centrifuge the lysed liquid at 4℃ and 6000 rpm for 10 min, and collect the supernatant, which is the crude enzyme solution. Perform SDS-PAGE analysis on both the whole cells and the lysed supernatant (120 V, 1 h). Figure 1 As shown, the molecular weight of tagatose 4-epimerase is around 55 kDa.

[0042] 2. Protein purification and SDS-PAGE analysis The crude enzyme solution obtained in step 1 (4) was passed through a 0.45 μm aqueous membrane to remove impurities, and then connected to a constant flow pump and Ni² in the protein purification system. + For the tubing connecting the affinity chromatography column, UV detector, and other components, set the constant flow pump to 1 mL / min and check for leaks using deionized water. After the protein purification system is ready, equilibrate Ni²⁺ with two column volumes of binding buffer (50 mmol / L Tris, 500 mmol / L NaCl, pH 7.0). + Affinity chromatography column was used, and the crude protein solution was pumped into the column at a flow rate of 0.5 mL / min. After all the crude protein solution had entered the column, unadsorbed protein and other impurities were washed away with binding buffer. Once the detector reading stabilized, wash buffer (50 mmol / L imidazole, 50 mmol / L Tris, 500 mmol / L NaCl, pH 7.0) was pumped in to wash away weakly binding proteins. Once the detector reading stabilized again, elution buffer (500 mmol / L imidazole, 50 mmol / L Tris, 500 mmol / L NaCl, pH 7.0) was pumped in to elute the adsorbed recombinant protein. The eluent was collected based on the UV detector signal value; this was the target recombinant protein. The target recombinant protein solution obtained above was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa. After clamping with dialysis clamps, the bag was placed in dialysis solution A (10 mmol / L EDTA·2Na, 50 mmol / L Tris, pH 7.0) and dialyzed in a chromatography cabinet at 4 ℃ for 18 h, with the dialysis solution replaced every 6 h to remove imidazole and other metal ions from the target recombinant protein solution. The dialysis bag was then transferred to dialysis solution B (50 mmol / L Tris, pH 7.0) and dialyzed for another 18 h, with the dialysis solution replaced every 6 h. After dialysis, the target recombinant protein solution was collected into a tube to obtain the pure enzyme solution.

[0043] Whole cells, lysed supernatant, and purified enzyme solution were analyzed by SDS-PAGE. The results are as follows: Figure 1As shown, the target protein molecular weight is approximately 55 kDa, consistent with the theoretical value, and a single band was obtained after purification, indicating successful acquisition of purified tagatose 4-epimerase. Further enzyme activity assays were performed on the purified enzyme solution: In a 1 mL reaction system, a fructose substrate solution with a final concentration of 100 g / L was prepared using 50 mmol / L Tris-HCl buffer (pH 8.5), and Ni²⁺ was added. + The final concentration was 5 mmol / L, and 100 μL of pure enzyme solution was added. The reaction was carried out at 65 ℃ and 200 rpm for 30 min. The reaction was immediately terminated by boiling for 5 min, followed by centrifugation at 12000 rpm for 5 min. The supernatant was collected, and the amount of tagatose produced was determined by HPLC. The protein concentration of the pure enzyme solution was determined using the BCA method, and the specific enzyme activity was calculated based on the protein content of the pure enzyme. The results showed that the specific enzyme activity was 3.56 U / mg.

[0044] Example 3: Effect of metal ions on tagatose 4-epimerase activity To investigate the enzyme's dependence on metal ions, different metal ions were added to the following standard systems to determine whole-cell enzyme activity. The specific steps are as follows: 1. Preparation of metal ion solutions: Weigh out the K-containing samples respectively + Na + Mg 2+ Fe 3+ Fe 2+ Ni 2+ Co 2+ Ca 2+ Zn 2+ Cu 2+ NH4 + Mn 2+ The inorganic salts were dissolved and diluted to volume with 50 mmol / L Tris-HCl buffer (pH 8.5) to prepare stock solutions with a metal ion concentration of 1 mol / L, which were then stored at 4 ℃ for later use.

[0045] 2. Whole-cell reaction and enzyme activity assay: Whole-cell enzyme activity was determined according to the standard enzyme activity assay method described above, with the difference being that 5 μL of the aforementioned metal ion stock solution was added to bring the final metal ion concentration to 5 mmol / L; an equal volume of buffer was added to the blank control. Ni²⁺ was used as the standard method. + The enzyme activity is 100% when it is present. Calculate the relative enzyme activity when other metal ions are present.

[0046] The results showed that among the twelve metal ions tested, only when Ni² was added to the reaction system... +A distinct tagatose product peak was detected at the time, and the fermentation enzyme activity measured using whole-cell assay was 0.08 U / mL, with this enzyme activity representing 100%; while at K + Na + Mg 2 + Fe 3+ Fe 2+ Co 2+ Ca 2+ Zn 2+ Cu 2+ NH4 + Mn 2+ In the presence of Ni, the relative enzyme activity is less than 1%. This indicates that the tagatose 4-epimerase provided by this invention is a Ni-dependent enzyme. 2+ Ni is a metalloenzyme that cannot be replaced by other common metal ions. 2+ It plays an activating role.

[0047] Example 4: Determination of the optimal reaction pH and temperature for tagatose 4-epimerase Using the bacterial cells obtained by centrifugation of the fermentation broth described in Example 2 as a catalyst, the effects of pH 4.0-9.0 and temperature ranges of 20 ℃-80 ℃ on the catalytic activity of tagatose 4-epimerase were investigated. The specific steps are as follows: 1. Preparation methods for substrate solutions with different pH values: Prepare buffer solutions with pH values ​​of 4.0, 5.0, and 5.5 using HAc-NaAc; prepare buffer solutions with pH values ​​of 5.5, 6.0, 7.0, and 7.5 using KH₂PO₄-NaOH; and prepare buffer solutions with pH values ​​of 7.5, 8.0, and 9.0 using Tris-HCl. Accurately weigh fructose, dissolve it in each of the above buffer solutions, and dilute to volume to obtain fructose solutions with a concentration of 100 g / L. Store the prepared solutions at 4 °C for later use.

[0048] 2. Whole-cell catalytic reaction and enzyme activity assay: The recombinant engineered strain E. coli BL21(DE3) / pET-22b(+)-T4E obtained in Example 1 was fermented according to the method in Example 2. Whole-cell enzyme activity was determined at each pH using fructose substrate solutions of different pH values ​​according to standard enzyme activity testing methods, and relative enzyme activity was calculated with the highest enzyme activity as 100%.

[0049] The results show that ( Figure 2 ): The relative enzyme activities under various pH conditions were as follows: pH 4.0, 2.3%; pH 5.0, 5.8%; pH 5.5 (HAc-NaAc), 7.17%; pH 5.5 (KH2PO4-NaOH), 5.76%; pH 6.0, 23.7%; pH 7.0, 55.2%; pH 7.5 (KH2PO4-NaOH), 69.74%; pH 7.5 (Tris-HCl), 52.8%; pH 8.0, 84.23%; pH 8.5, 100%; pH 9.0, 85.09%.

[0050] The optimal reaction pH for this tagatose 4-epimerase is 8.5 (50 mmol / L Tris-HCl buffer). Its relative enzyme activity exceeds 84% ​​in the pH range of 8.0-9.0, showing a significant alkalinity preference; it has almost no activity at pH below 5.5.

[0051] 3. Based on the optimal pH of 8.5 determined above, a 100 g / L fructose substrate solution was prepared using 50 mmol / L Tris-HCl buffer. Whole-cell enzyme activity was measured at different temperatures according to the standard enzyme activity assay method described above. The test temperatures were 20 ℃, 30 ℃, 40 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, and 80 ℃. The highest measured enzyme activity of 0.38 U / mL was taken as 100%, and the relative enzyme activity under each temperature condition was calculated.

[0052] The results show that ( Figure 3 ): The relative enzyme activities under various temperature conditions were as follows: 20 ℃, 1.03%; 30 ℃, 0.68%; 40 ℃, 10.72%; 50 ℃, 38.95%; 55 ℃, 58.38%; 60 ℃, 74.66%; 65 ℃, 100%; 70 ℃, 97.40%; 80 ℃, 20.95%.

[0053] The results showed that the optimal pH for tagatose 4-epimerase to catalyze the conversion of fructose to tagatose was 8.5, and the optimal temperature was 65 ℃.

[0054] Example 5: Effect of substrate concentration on whole-cell catalytic conversion of tagatose The specific steps are as follows: 1. Effect of substrate concentration on conversion rate: Substrate solutions with fructose concentrations of 25 g / L, 50 g / L, 100 g / L, 200 g / L, and 400 g / L were prepared using 50 mmol / L Tris-HCl buffer at pH 8.5. Enzyme-containing wet bacterial cells were added at a ratio of 4 U / g substrate, and the reaction was carried out at 65 °C and 200 rpm. The conversion rate was measured at regular intervals until the conversion rate tended to stabilize.

[0055] The results are as follows Figure 4 As shown, when the substrate concentration is 100 g / L, the reaction reaches equilibrium relatively quickly, and the conversion rate is highest at equilibrium, reaching 15.18%.

[0056] 2. Effect of enzyme dosage on conversion rate The concentration of fructose as a substrate was fixed at 100 g / L. Enzyme addition ratios of 16 U / g, 32 U / g, 50 U / g, and 100 U / g were set. The reaction was carried out at 65 ℃ and 200 rpm. The conversion rate was measured at regular intervals until the conversion rate tended to stabilize.

[0057] The results are as follows Figure 5 As shown, when the enzyme dosage is 32 U / g, the maximum conversion rate of 24.89% is reached after about 12 hours of reaction.

[0058] Example 6: HPLC identification of the product Whole-cell catalytic reaction was carried out under optimal reaction conditions, and the product was analyzed by HPLC to confirm the formation of tagatose and determine the conversion rate. The specific steps are as follows: Prepare a 100 g / L fructose substrate solution (containing 5 mmol / L Ni²) using 50 mmol / L Tris-HCl buffer (pH 8.5). + Take 8 mL of the fermentation broth obtained in Example 2 (corresponding to an enzyme addition ratio of 32 U / g fructose), centrifuge at 8000 rpm for 5 min to collect the wet cells, add the above substrate solution to a total volume of 1 mL, resuspend thoroughly, and react at 65 ℃ and 200 rpm for 12 h. Immediately after the reaction, boil for 5 min to inactivate the enzyme, centrifuge at 12000 rpm for 5 min, filter the supernatant through a 0.22 μm aqueous filter membrane, and perform HPLC analysis.

[0059] The tagatose standard was tested under the same conditions, and its retention time was recorded. The results are as follows: Figure 6 As shown, a distinct chromatographic peak appeared at a retention time of 16.40 min in the reaction solution, consistent with the retention time of the tagatose standard. The peak shape was symmetrical, and there were no other significant impurity peaks, indicating that the whole-cell catalytic product was indeed tagatose. Calculations using the external standard method based on peak area showed that the fructose conversion rate under these conditions reached 24.89%.

[0060] Comparative Example 1 A tagaguron acid epimerase family protein (protein sequence accession number WP_041843930.1) derived from Thermotoga sp. RQ7 was used as a comparative enzyme. Following the methods in Examples 1 and 2, codon optimization, recombinant expression, and Ni were performed on the coding gene corresponding to this original enzyme. 2+ Affinity chromatography was used to purify the enzyme solution.

[0061] Referring to the pure enzyme activity assay method in Example 2, a fructose substrate solution with a final concentration of 100 g / L was prepared in a 1 mL reaction system using 50 mmol / L Tris-HCl buffer (pH 8.5), and Ni was added. 2+ The final concentration was 5 mmol / L, and 100 μL of the above-mentioned purified enzyme solution was added. The reaction was carried out at 65 ℃ and 200 rpm for 30 min. The reaction was terminated by boiling for 5 min immediately after the reaction was completed. The supernatant was collected by centrifugation, and the amount of tagatose produced was determined by HPLC. The protein concentration was determined by BCA method, and the specific enzyme activity was calculated based on the purified enzyme protein content.

[0062] The results showed that, under the above conditions, the original enzyme derived from WP_041843930.1 could be detected to produce tagatose, and its pure enzyme specific activity was 0.030 U / mg, which was lower than the pure enzyme specific activity of the tagatose 4-epimerase of the present invention (3.56 U / mg).

[0063] 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 tagatose 4-epimerase, characterized in that, The amino acid sequence of the tagatose 4-epimerase is shown in SEQ ID NO.

1.

2. A gene characterized in that, The gene encodes the tagatose 4-epimerase as described in claim 1.

3. A recombinant plasmid, characterized in that, The recombinant plasmid carries the gene described in claim 2.

4. The recombinant plasmid according to claim 3, characterized in that, The expression vector for the recombinant plasmid is pET-22b(+) plasmid.

5. A host cell expressing the tagatose 4-epimerase of claim 1, or containing the gene of claim 2, or transformed with the recombinant plasmid of any one of claims 3 to 4.

6. The host cell according to claim 5, characterized in that, The host cell is either Escherichia coli or Bacillus subtilis.

7. A catalyst for producing tagatose, characterized in that, The catalyst contains the tagatose 4-epimerase as described in claim 1 or the host cell as described in any one of claims 5 to 6.

8. A method for producing tagatose, characterized in that, The method involves first adding the tagatose 4-epimerase described in claim 1, the host cell described in any one of claims 5-6, or the catalyst described in claim 7 to a reaction system containing fructose to obtain a reaction solution, and then separating the tagatose from the reaction solution.

9. The method according to claim 8, characterized in that, The reaction conditions are: pH 8.0-9.0, temperature 60-70℃, and the substrate in the reaction system is fructose.

10. The use of the tagatose 4-epimerase of claim 1, the gene of claim 2, the recombinant plasmid of any one of claims 3-4, the host cell of any one of claims 5-6, the catalyst of claim 7, or the method of any one of claims 8-9 in the production of tagatose or products containing tagatose.

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