Application of product in catalyzing dephosphorization of tagatose hexaphosphate to form tagatose

Abstract

The invention relates to a biological fermentation method of tagatose, in particular to application of a product in catalyzing dephosphorization of tagatose hexaphosphate to form tagatose. The product is tagatose hexaphosphate phosphatase, nucleotide for coding the tagatose hexaphosphate phosphatase or a microorganism for expressing the tagatose hexaphosphate phosphatase, and the amino acid sequence of the tagatose hexaphosphate phosphatase is as shown in SEQ ID NO.5. The tagatose provided by the invention has higher temperature stability, can highly selectively dephosphorize tagatose hexaphosphate under a higher temperature condition, and greatly reduces the generation of byproducts such as glucose, fructose and the like.

Description

[0001] The present invention is a divisional application with application number: 202410681025.6, application date: May 29, 2024, and the invention name is "High-efficiency catalytic synthesis of tagatase at high temperature and its application." Technical Field

[0002] The present invention relates to a biofermentation method for tagatose, and in particular to application of a product in catalyzing the dephosphorylation of tagatose hexaphosphate to form tagatose. Background Art

[0003] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0004] According to the inventors' research, starch is converted into tagatose in one step through the combined action of glycogen phosphorylase, phosphoglucomutase, glucose-6-phosphate isomerase, tagatose-6-phosphate synthase, and phosphatase. The specific process is as follows: starch and phosphate form glucose-1-phosphate (glucose monophosphate) under the catalytic action of glycogen phosphorylase, glucose-6-phosphate (glucose-6-phosphate) is converted by phosphoglucomutase to glucose-6-phosphate (glucose-6-phosphate), glucose-6-phosphate is converted to fructose-6-phosphate (fructose-6-phosphate) under the catalytic action of glucose-6-phosphate isomerase, fructose-6-phosphate is converted to tagatose-6-phosphate (tagatose-6-phosphate) under the catalytic action of tagatose-6-phosphate synthase, and tagatose-6-phosphate is dephosphorylated to tagatose under the action of phosphatase. However, the current method for converting starch into tagatose in one step using the above method produces a large number of byproducts and a low yield, thereby affecting its application in tagatose production. Summary of the Invention

[0005] In order to address the deficiencies of the prior art, the present invention aims to provide a product for use in catalyzing the dephosphorylation of tagatose hexaphosphate to form tagatose. The tagatase provided by the present invention has higher temperature stability.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] A product is used in catalyzing the dephosphorylation of tagatose hexaphosphate to form tagatose. The product is a tagatose hexaphosphate phosphatase, a nucleotide encoding the tagatose hexaphosphate phosphatase, or a microorganism expressing the tagatose hexaphosphate phosphatase. The amino acid sequence of the tagatose hexaphosphate synthase is shown in SEQ ID NO. 5.

[0008] The present invention screened and cloned a tagatose 6-phosphate phosphatase derived from Thermoanaerobacterales bacterium from the high-temperature hot spring metagenome. Compared with existing tagatose 6-phosphate phosphatases, the tagatose 6-phosphate phosphatase provided by the present invention can highly selectively dephosphorylate tagatose 6-phosphate at a higher temperature (70°C), greatly reducing the production of byproducts such as glucose and fructose.

[0009] Furthermore, the nucleic acid sequence of the nucleotide encoding tagatose hexaphosphate synthase is shown in SEQ ID NO.6.

[0010] Furthermore, the starting strain of the microorganism is Escherichia coli, specifically Escherichia coli BL21.

[0011] Furthermore, a method for constructing a microorganism capable of expressing tagatose 6-phosphate phosphatase comprises: using a second primer pair to amplify a nucleic acid sequence encoding a nucleotide sequence encoding tagatose 6-phosphate phosphatase, combining the amplified product with a vector to form a recombinant vector, and then transferring the recombinant vector into a starting bacterium; the base sequences of the second primer pair are shown in SEQ ID NO. 7 and SEQ ID NO. 8, respectively. Specifically, the vector is a pet41 vector.

[0012] Furthermore, the product is added to a reaction solution containing tagatose hexaphosphate, and a catalytic reaction is carried out at 65-70°C.

[0013] Furthermore, the reaction solution contains potassium dihydrogen phosphate and magnesium sulfate. Specifically, the molar ratio of potassium dihydrogen phosphate to magnesium sulfate is 9 to 11:1. Specifically, the concentration of potassium dihydrogen phosphate in the reaction system is 45 to 55 mM. In this invention, the unit "M" refers to mol / L, and the unit "mM" refers to mmol / L.

[0014] The beneficial effects of the present invention are:

[0015] The present invention screened a new tagatose six-phosphate phosphatase by using the high-temperature hot spring metagenome. The screened new tagatose six-phosphate phosphatase can more efficiently select tagatose six-phosphate as a substrate for dephosphorylation at 70°C. DETAILED DESCRIPTION

[0016] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0017] Example 1

[0018] Tagatose hexaphosphate synthase (T6PE-T) and its preparation:

[0019] A tagatose 6-phosphate synthase (T6PE-T) from Thermoflexus sp. was screened and cloned from the metagenome of a high-temperature hot spring. Currently, the synthase that is known to synthesize tagatose 6-phosphate is gatZ from Caldilinea aerophila (accession number: WP_014433578).

[0020] The amino acid sequence of tagatose hexaphosphate synthase T6PE-T is:

[0021] MKGYDVVAAGGGSCSAHVAARHGRASGKVATSNVNGGYTGMTAAGAAKMARGRRGGDHGWSHAMRKADVRAVAGTKHDCSMGGVARARARVAAAGRRDRYVVGSVAGGAAAGVTRDAAAAARRARRDTAWRV AVVGGDHYDRAAAAVRWGVGAHSTDYAARAVDHAKVGATARAVAADAAGSRAAAMARVHWKYYRGDARVRARRYSSDRRYYWAAVTARMRNGDRVGSYMYRRVRRGRNHRDGRVMAVGYAATVNGCR, as in SEQ Shown as ID NO.1.

[0022] Nucleic acid sequence:

[0023] ATGAAGGGTTATGATGTGGTTGCAGCAGGTGGTGGTAGCTGTAGCGCCCATGTGGCCGCACGTCATGGCCGTGCCAGTGGCAAAGTTGCAACCAGTAATGTGAATGGTGGTTATACCGGCATGACCGCAGCCGGCGCCGCCAAAATGGCACGCGGTCGTCGCGGCGGCGATCATGGTTGGAGTCATGCCATGCGTAAAGCCGATGTTCGTGCAGTGGCCGGTACCAAACATGATTGCAGCATGGGTGGTGTGGCCCGCGCACGTGCACGCGTTGCTGCTGCAGGCCGTCGTGATCGCTATGTGGTTGGCAGTGTGGCAGGCGGCGCAGCAGCCGGTGTGACACGTGATGCAGCCGCCGCCGCCCGTCGTGCAAGAAGAGATACCGCATGGCGCGTTGCAGTTGTTGGTGGTGACCATTATGATCGTGCAGCAGCCGCAGTGCGCTGGGGTGTGGGTGCACATAGCACCGATTATGCAGCCCGCGCAGTGGATCATGCCAAAGTGGGTGCAACCGCCCGTGCAGTGGCGGCTGATGCCGCAGGTAGTCGTGCCGCAGCCATGGCACGTGTTCATTGGAAATATTATCGCGGTGACGCCCGTGTTCGTGCACGTCGTTATAGTAGCGATCGTCGCTATTATTGGGCAGCAGTTACCGCCCGTATGCGCAATGGCGATCGCGTGGGTAGTTATATGTATCGTCGCGTGCGTCGCGGCCGTAATCATCGTGATGGTCGTGTTATGGCCGTGGGCTATGCAGCCACCGTGAATGGTTGTCGC, as shown in SEQ ID NO.2.

[0024] Primers P1: aactttaagaaggagatatacatATGAAGGGTTATGATGTGG (shown in SEQ ID NO. 3); P2: agtggtggtggtggtggtggtggtggtgctcgagGCGACAACCATTCACG (shown in SEQ ID NO. 4) were designed to amplify the T6PE-T sequence. The amplified product was separated, recovered, and concentrated by agarose gel electrophoresis. The pet41 vector was digested with NdeI and XhoI, and the product was separated, recovered, and concentrated by agarose gel electrophoresis. The digested pet41 vector and the amplified fragment were recombined using a DNA recombination kit and transformed into competent Escherichia coli DH5a. Positive clones were cultured, screened, and verified by sequencing. Positive clones were transformed into E. coli BL21 to construct an engineered strain capable of recombinantly expressing T6PE-T.

[0025] The recombinant strain was cultured in TB medium and induced for expression with IPTG. The cells expressing the protein were harvested by centrifugation and resuspended in PBS buffer containing 20 mM imidazole and 0.5 M sodium chloride. The cells were resuspended in 10 ml of buffer per 1 g of wet cells. The cells were ultrasonically disrupted and the supernatant was collected by centrifugation. The recombinant protein in the supernatant was adsorbed using a Ni column and eluted using PBS buffer containing 300 mM imidazole and 0.5 M sodium chloride. The eluted protein was desalted using a desalting column to obtain T6PE-T.

[0026] T6PE-T was purified using the His tag, and after desalting, its protein content was calibrated using the Coomassie Brilliant Blue G250 method.

[0027] Example 2

[0028] T6PE-T catalyzes the synthesis of tagatose hexaphosphate from fructose hexaphosphate:

[0029] 20 mg of T6PE-T was added to 100 ml of a reaction solution containing 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate, and 50 mM fructose hexaphosphate. The reaction was catalyzed at 70°C for 1 hour to obtain tagatose hexaphosphate with a yield of 8.5 mM and a yield of 17%.

[0030] Comparative Example 1

[0031] gatZ catalyzes the synthesis of tagatose hexaphosphate from fructose hexaphosphate:

[0032] gatZ catalyzes the synthesis of tagatose hexaphosphate from fructose hexaphosphate:

[0033] 20 mg of gatZ was added to a 100 ml reaction solution containing 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate, and 50 mM fructose hexaphosphate. The reaction was catalytically reacted at 70°C for 1 hour to obtain tagatose hexaphosphate with a yield of 4.9 mM and a yield of 9.8%.

[0034] Example 3

[0035] T6PE-T catalytic efficiency test:

[0036] T6PE-T was heat-treated at different temperatures (e.g., 55°C-70°C) for different times (e.g., 1500 minutes-5000 minutes). 20 mg of heat-treated T6PE-T and untreated T6PE-T were respectively added to 100 ml of a reaction solution containing 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate, and 50 mM fructose hexaphosphate. The reaction was catalyzed at 70°C for 1 hour. The catalytic efficiency was determined by measuring the amount of tagatose hexaphosphate produced by liquid chromatography. The amount produced is shown in Table 1.

[0037] Table 1 The amount of tagatose hexaphosphate produced by heat-treated T6PE-T and untreated T6PE-T

[0038]

[0039] Comparative Example 2

[0040] Detection of gatZ catalytic efficiency:

[0041] GatZ was heat-treated at different temperatures (e.g., 55°C-70°C) for different times (e.g., 1500 minutes-5000 minutes). 20 mg of heat-treated gatZ and untreated gatZ were respectively added to 100 ml of a reaction solution containing 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate, and 50 mM fructose hexaphosphate. The reaction was catalyzed at 70°C for 1 hour. The catalytic efficiency was determined by measuring the amount of tagatose hexaphosphate produced by liquid chromatography. The amount produced is shown in Table 2.

[0042] Table 2 The amount of tagatose hexaphosphate generated by heat-treated gatZ and untreated gatZ

[0043]

[0044]

[0045] By comparing the catalytic efficiency of Example 3 and Comparative Example 2, the half-lives of different tagatose hexaphosphate synthases under different heat treatments can be known, thereby knowing their thermal stability. The results are shown in Table 3.

[0046] Table 3 Thermal stability of T6PE-T and gatZ

[0047] T6PE-T gatZ Half-life at 70℃ 2300 minutes 1750 minutes Half-life at 65°C 3200 minutes 2100 minutes Half-life at 60℃ 4550 minutes 3380 minutes Half-life at 55°C 4990 minutes 3520 minutes

[0048] Table 3 shows that compared with gatZ, T6PE-T has stronger catalytic stability at high temperature conditions of 55-70°C and can produce more products through continuous catalysis at high temperature with the addition of less enzyme.

[0049] Example 4

[0050] Detection of T6PE-T and gatZ affinity for substrates:

[0051] Determination of 1. Fructose 6-phosphate Km Value: A catalytic reaction solution consisting of 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate, 20 mg of catalytic enzyme, and fructose 6-phosphate (0.5 mM-100 mM) was prepared and catalyzed at 70°C for 1 hour. The amount of tagatose 6-phosphate produced was determined by liquid chromatography. The Km values ​​of T6PE-T and gatZ for fructose 6-phosphate were determined using Linewaver-Burk plotting.

[0052] Determination 2. Fructose-1,6-bisphosphate (FRP) Km value: A catalytic reaction solution was prepared consisting of 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate, 20 mg of catalytic enzyme, and fructose-1,6-bisphosphate (0.5 mM-100 mM). The reaction was catalyzed at 70°C for 1 hour. The amount of tagatose-1,6-bisphosphate (TRP) produced was determined by liquid chromatography. The Km values ​​of T6PE-T and gatZ for FRP were determined using Linewaver-Burk plotting.

[0053] Determination 3. Km value of tagatose 1,6-diphosphate: A catalytic reaction solution was prepared with 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate, 20 mg of catalytic enzyme, and tagatose 1,6-diphosphate (0.5 mM-100 mM). The reaction was catalyzed at 70°C for 1 hour. The amount of fructose 1,6-diphosphate produced was determined by liquid chromatography. The Km values ​​of T6PE-T and gatZ for tagatose 1,6-diphosphate were determined using Linewaver-Burk plotting.

[0054] Determination 4. Km value of tagatose hexaphosphate: A catalytic reaction solution was prepared consisting of 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate, 20 mg of catalytic enzyme, and tagatose hexaphosphate (0.5 mM-100 mM). The reaction was catalyzed at 70°C for 1 hour. The amount of fructose hexaphosphate produced was determined by liquid chromatography. The Km values ​​of T6PE-T and gatZ for tagatose hexaphosphate were determined using Linewaver-Burk plotting.

[0055] The test results are shown in Table 4.

[0056] Table 4 Substrate affinity of T6PE-T and gatZ

[0057] T6PE-T gatZ Fructose 6-phosphate Km value (mmol / L) 1.84 36 Fructose 1,6-diphosphate Km value (mmol / L) 0.51 0.68 Tagatose 1,6-diphosphate Km value (mmol / L) 0.92 1.5 Tagatose hexaphosphate Km value (mmol / L) 189 124

[0058] Table 4 shows that T6PE-T has a lower Km value for fructose 6-phosphate during the catalytic process, so the enzyme can more specifically catalyze the formation of tagatose 6-phosphate and will not catalyze tagatose 6-phosphate to form other substances.

[0059] Example 5

[0060] T6PE-T catalyzes the synthesis of tagatose from starch:

[0061] A catalyst solution (pH 7.0) containing 50 mM potassium hydrogen phosphate, 5 mM magnesium sulfate, and 300 g / L starch was prepared. 10 U starch phosphorylase, 10 U glucose-6-phosphate isomerase, 10 U fructose-6-phosphate isomerase, 10 U T6PE-T, and 10 U T6PP were added to a 1 L reaction system and catalyzed at 70°C for 72 h. Liquid chromatography revealed 168 g of tagatose, with a yield of 56%.

[0062] Comparative Example 3

[0063] gatZ catalyzes the synthesis of tagatose from starch:

[0064] A catalyst solution (pH 7.0) containing 50 mM potassium phosphate dibasic, 5 mM magnesium sulfate, and 300 g / L starch was prepared. To a 1 L reaction system, 10 U starch phosphorylase, 10 U glucose-6-phosphate isomerase, 10 U fructose-6-phosphate isomerase, 10 U gatZ, and 10 U T6PP were added. The reaction was incubated at 70°C for 72 h. Liquid chromatography revealed 103 g of tagatose, yielding a 34.3% yield.

[0065] Example 6

[0066] Tagatose six-phosphate phosphatase (T6PP-T) and its preparation:

[0067] A novel tagatose 6-phosphate phosphatase (T6PP-T) was isolated from a high-temperature hot spring metagenome. The enzyme is from the bacterium Thermoanaerobacterales. The commonly used tagatose 6-phosphate phosphatase (T6PP) is from Sphaerobacter thermophilus, with sequence accession number WP_012872856.1.

[0068] The amino acid sequence of the tagatose six-phosphate phosphatase is:

[0069] MVFDLDGVLLDSEQAWDEARRALVRERGGRWRPEAQRAMMGMSSPEWSRYLRDELGVDMAPEDISRAVVERLVAGYRRHLPLLPGAVEAVRALAARWPLGLASSSNRPVIDLVLELAGLAGAFAATVSSEEVARGKPAPDVYLEAARRLGVAPEACVAVEDSTNGLRSAAAAGMRVVALPNRDYPPAADALALAAVRLGSLDELRPELIERLDRPGA, as shown in SEQ ID NO.5.

[0070] Nucleic acid sequence:

[0071] ATGGTTTTCGATCTGGATGGTGTTCTGCTGGATAGTGAACAGGCCTGGGATGAAGCACGCCGTGCACTGGTGCGCGAACGCGGTGGTCGTTGGCGTCCGGAAGCACAGCGCGCAATGATGGGTATGAGTAGCCCGGAATGGAGCCGCTATCTGCGTGATGAACTGGGCGTTGATATGGCACCGGAAGATATTAGTCGTGCAGTGGTGGAACGTCTGGTGGCCGGTTATCGTCGTCATCTGCCGCTGCTGCCGGGCGCAGTGGAAGCTGTGCGCGCTCTGGCCGCACGCTGGCCTTTAGGTCTGGCCAGCAGTAGTAATCGTCCGGTTATTGATCTGGTTCTGGAACTGGCCGGCCTGGCCGGCGCATTTGCAGCAACCGTTAGCAGTGAAGAAGTGGCACGTGGTAAACCGGCCCCGGATGTTTATCTGGAAGCAGCACGTCGTCTGGGTGTTGCCCCGGAAGCCTGCGTTGCAGTGGAAGATAGTACCAATGGTCTGCGTAGCGCAGCAGCCGCAGGCATGCGCGTGGTTGCCCTGCCTAATCGTGATTATCCGCCGGCCGCAGATGCACTGGCCCTGGCAGCAGTTCGCCTGGGTAGCCTGGATGAACTGCGTCCGGAACTGATTGAACGCCTGGATCGTCCGGGCGCC, as shown in SEQ ID NO.6.

[0072] Design primers P3: aactttaagaaggagatatacatATGGTTTTCGATCTGGATGG (as shown in SEQ ID NO.7); P4:

[0073] The amplified product agtggtggtggtggtggtggtggtggtgctcgagGGCGCCCGGACGATCCAGGCG (as shown in SEQ ID NO. 8) was amplified using the T6PP-T sequence as a template, and the product was separated, recovered, and concentrated by agarose gel electrophoresis. The pet41 vector was digested with NdeI+XhoI, and the product was separated, recovered, and concentrated by agarose gel electrophoresis. The digested pet41 vector and the amplified fragment were recombined using a DNA recombination kit and transformed into competent Escherichia coli DH5a. Positive clones were cultured, screened, and verified by sequencing. The positive clones were transformed into Escherichia coli BL21 to construct an engineered strain capable of recombinantly expressing T6PP-T.

[0074] The recombinant strain was cultured in TB medium and induced for expression with IPTG. The cells expressing the protein were harvested by centrifugation and resuspended in PBS buffer containing 20 mM imidazole and 0.5 M sodium chloride (10 ml of buffer per 1 g of wet cells). The cells were disrupted by sonication and the supernatant was collected by centrifugation. The recombinant protein in the supernatant was adsorbed using a Ni column and eluted using PBS buffer containing 300 mM imidazole and 0.5 M sodium chloride. The eluted protein was desalted using a desalting column to obtain T6PP-T.

[0075] T6PP-T was purified using the His tag, and after desalting, its protein content was calibrated using the Coomassie Brilliant Blue G250 method.

[0076] Example 7

[0077] T6PP-T catalyzes the dephosphorylation of tagatose hexaphosphate to synthesize tagatose:

[0078] 10 mg of T6PP-T was added to 100 mL of a reaction solution containing 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate, and 50 mM tagatose hexaphosphate. The reaction was catalytically reacted at 70°C for 1 hour to obtain tagatose with a yield of 48 mM and a yield of 96%.

[0079] Comparative Example 4

[0080] T6PP catalyzes the dephosphorylation of tagatose hexaphosphate to synthesize tagatose:

[0081] 10 mg of T6PP was added to 100 mL of a reaction solution containing 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate, and 50 mM tagatose hexaphosphate. The reaction was catalytically reacted at 70°C for 1 hour to obtain tagatose hexaphosphate with a yield of 33 mM and a yield of 66%.

[0082] Example 8

[0083] Detection of T6PP-T's substrate affinity:

[0084] A catalytic reaction solution was prepared containing 50 mM potassium dihydrogen phosphate (pH 7.2), 5 mM magnesium sulfate, 1 μmol / L catalytic enzyme, and substrates (tagatose hexaphosphate, fructose hexaphosphate, glucose hexaphosphate, and glucose monophosphate, 0.1 mM to 100 mM). The reaction was catalyzed at 70°C for 1 hour. The amount of dephosphorylated product produced was detected by liquid chromatography, and the Km value of T6PP-T for the substrate was determined using the Linewaver-Burk plot method. The results are shown in Table 5.

[0085] Table 5 Affinity of tagatose six-phosphate phosphatase (T6PP-T) for different sugar phosphate substrates

[0086]

[0087] Table 5 shows that the tagatose 6-phosphate phosphatase can more efficiently dephosphorylate tagatose 6-phosphate as a substrate at 70°C, while fructose 6-phosphate, glucose 6-phosphate, and glucose monophosphate in the same system cannot be dephosphorylated. Therefore, using the tagatose 6-phosphate phosphatase in the reaction can further improve the conversion rate of tagatose and greatly reduce the yield of byproducts (glucose and fructose).

[0088] Example 9

[0089] A catalytic solution (pH 7.0) containing 50 mM potassium hydrogen phosphate (K2PO4), 5 mM magnesium sulfate, and 300 g / L starch was prepared. 10 U starch phosphorylase, 10 U glucose-6-phosphate isomerase, 10 U fructose-6-phosphate isomerase, 10 U gatZ or 10 U T6PE-T, 10 U T6PP or T6PP-T were added to a 1 L reaction system. The reaction was catalyzed at 70°C for 72 hours. After completion of the catalysis, the tagatose and sorbose contents were determined by liquid chromatography, as shown in Table 6 below.

[0090] Table 6 Liquid chromatography detection of tagatose and sorbose content

[0091]

[0092]

[0093] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Use of a product in catalyzing the dephosphorylation of tagatose hexaphosphate to form tagatose, characterized in that: The product is tagatose six phosphate phosphatase, a nucleotide encoding the tagatose six phosphate phosphatase or a microorganism expressing the tagatose six phosphate phosphatase, and the amino acid sequence of the tagatose six phosphate synthase is shown in SEQ ID NO.

5.

2. The use according to claim 1, characterized in that: The nucleic acid sequence of the nucleotide encoding tagatose hexaphosphate synthase is shown in SEQ ID NO.

6.

3. The use according to claim 1, characterized in that: The starting strain of the microorganism is Escherichia coli.

4. The use according to claim 3, characterized in that: The starting strain of the microorganism is Escherichia coli BL21.

5. The use according to claim 3, characterized in that: The method for constructing a microorganism capable of expressing tagatose six-phosphate phosphatase is as follows: amplifying the nucleic acid sequence of the nucleotide encoding tagatose six-phosphate phosphatase with a second primer pair, preparing a recombinant vector with the amplified product and a vector, and transferring the recombinant vector into a starting bacterium; the base sequences of the second primer pair are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.

6. The use according to claim 5, characterized in that: The vector is a pet41 vector.

7. The use according to claim 1, characterized in that: The product is added to a reaction solution containing tagatose hexaphosphate and a catalytic reaction is carried out at 65 to 70°C.

8. The use according to claim 7, characterized in that: The reaction solution contains potassium dihydrogen phosphate and magnesium sulfate.

9. The use according to claim 8, characterized in that: The molar ratio of dipotassium hydrogen phosphate to magnesium sulfate is 9 to 11:

1.

10. The use according to claim 8, characterized in that: The concentration of dipotassium hydrogen phosphate in the reaction system is 45-55 mM.

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