Tagolose-6-phosphate phosphatase and application thereof

Abstract

The invention provides tagatose-6-phosphate phosphatase and an application of the tagatose-6-phosphate phosphatase. According to the invention, the fact that A0A101X5R59CREN of Thermoprotein sp.CIS19 in thermophilic bacteria has the activity of tagatose-6-phosphate phosphatase is found for the first time. The tagatose-6-phosphate phosphatase has high thermal stability and activity and high tagatose yield, and the tagatose yield reaches 4.3 g / L after 5 h with 10 g / L maltodextrin as a substrate under the conditions of 50 DEG C, pH 6.5 and 20 mM Mg < 2 + >, so that the tagatose-6-phosphate phosphatase has higher commercial application value.

Description

Technical Field

[0001] The present invention relates to the technical field of phosphatase, in particular to tagatose-6-phosphate phosphatase and application thereof. Background Art

[0002] Tagatose (D-Tagatose) is a rare, naturally occurring monosaccharide, the ketose form of galactose and a diastereomer of fructose. Its sweetness is similar to that of sucrose, but with only one-third the caloric content, earning it the nickname "low-calorie sweetener." Tagatose boasts excellent nutritional properties, including low caloric value, a zero glycemic index, blood sugar-lowering properties, a non-caries-causing effect, prebiotic properties, and antioxidant activity.

[0003] Tagatose production methods primarily include chemical synthesis and biosynthesis. Chemical synthesis uses D-galactose as a raw material. Under the catalysis of alkaline or alkaline earth metals, an isomerization reaction occurs, generating a metal hydroxide and a D-tagatose complex intermediate precipitate. This intermediate is then neutralized with acid to yield D-tagatose. While chemical synthesis is a cost-effective method for producing D-tagatose, it requires high temperature and high pressure during production and is prone to the formation of miscellaneous sugars such as sorbose and mannose, which are difficult to separate and purify. Furthermore, its application is limited by high energy consumption, complex products, difficulty in purification, numerous side reactions, and chemical pollution. Therefore, the production of tagatose using polysaccharides such as maltose and sucrose as raw materials using a phosphorylation-dephosphorylation multienzyme catalytic system is of great significance.

[0004] Currently, researchers have selected low-cost raw materials such as lactose, maltodextrin, and milk whey as substrates to produce tagatose using a multi-enzyme synthesis pathway. In 2022, Dai Yiwei et al. [1] constructed a multi-enzyme synthesis pathway consisting of α-glucan phosphorylase (αGP), phosphoglucomutase (PGM), glucose 6-phosphate isomerase (PGI), D-tagatose 1,6-bisphosphate aldolase (GatZ) and phosphoglycolate phosphatase (PGP). Using 10g / L maltodextrin as substrate, the reaction was carried out for 3h to obtain 3.383g / L D-tagatose, with a conversion rate of 33.83g / L [2]. Literature reports that the activity of the phosphatase (AfT6PP) from A. fulgidus is higher than that of other enzymes in the in vitro synthesis pathway of D-tagatose[3]. Patent CN106399427A uses AfT6PP as the last step of the multi-enzyme synthesis pathway. A one-pot reaction is carried out using 100g / L starch treated with starch hydrolyzing enzyme as the substrate. After 40 hours, 73g / L of tagatose can be produced, with a conversion rate of 73%. However, there is still a need to develop new tagatose-6-phosphate phosphatases to solve the problem of low utilization value in actual industrial production.

[0005] [1]Dai,Y.,Li,C.,Zheng,L.,et al.Enhanced biosynthesis of d-tagatosefrom maltodextrin through modular pathway engineering of recombinantEscherichia coli[J].Biochem.Eng.J.,2022,178:108303.

[0006] [2] Dai Y W. Biosynthesis of D-tagatose from maltodextrin by multi-enzymecatalytic system[D].Wuxi:Jiangnan University,2021.

[0007] [3]Hu C, Wei Summary of the Invention

[0008] The present invention provides a new tagatose-6-phosphate phosphatase, and the enzyme has high thermal stability, which lays a foundation for its application in tagatose production.

[0009] In a first aspect, the present invention provides a use of tagatose-6-phosphate phosphatase in tagatose synthesis, wherein the tagatose-6-phosphate phosphatase is derived from thermophilic bacteria.

[0010] In one embodiment of the present invention, the tagatose-6-phosphate phosphatase is derived from Thermoproteus sp. CIS_19 or a variant thereof.

[0011] In one embodiment of the present invention, the amino acid sequence of the tagatose-6-phosphate phosphatase is as shown in SEQ ID NO: 1, or an amino acid sequence that is at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.8%, or at least 99.9% identical to the sequence shown in SEQ ID NO: 1.

[0012] In the present invention, the tagatose-6-phosphate phosphatase has high enzyme activity. In one embodiment of the present invention, the kinetic constant kcat of the tagatose-6-phosphate phosphatase is 102.5±8.6min -1 , Km is 2.4±0.98mmol / L, and the maximum reaction rate vmax is 0.044±0.004mol / min.

[0013] In a second aspect, the present invention provides a method for synthesizing tagatose, comprising the step of converting tagatose-6-phosphate into tagatose under the action of the above-mentioned tagatose-6-phosphate phosphatase, a microorganism expressing the tagatose-6-phosphate phosphatase, and / or a culture of the microorganism.

[0014] In one embodiment of the present invention, the method comprises using hexose or a substance that can be converted into hexose as a raw material, adding a multi-enzyme system including tagatose-6-phosphate phosphatase, a microorganism expressing the tagatose-6-phosphate phosphatase and / or a culture of the microorganism to react and synthesize tagatose.

[0015] In one embodiment of the present invention, the hexose includes at least one of D-fructose, fructose-6-phosphate, tagatose-6-phosphate, D-galactose, D-galactose-6-phosphate, glucose, glucose-6-phosphate, glucose-1-phosphate, etc. The substance that can be converted into hexose includes at least one of disaccharides or polysaccharides such as sucrose, maltose, lactose, D-glyceraldehyde-3-phosphate, starch, dextrin, and maltodextrin.

[0016] In one embodiment of the present invention, the multi-enzyme system further comprises one or more of the following enzymes: α-glucan phosphorylase (αGP), phosphoglucomutase (PGM), glucose-6-phosphate isomerase (PGI), fructose-6-phosphate epimerase (TPE), 6-phosphate tagatose isomerase, tagatose kinase, galactitol 2-dehydrogenase, α-amylase, pullulanase, glucoamylase, isoamylase, sucrase, starch phosphorylase, maltodextrin phosphorylase, sucrose phosphorylase, glucokinase, hexokinase, and phosphoglucomutase.

[0017] In a specific embodiment of the present invention, the method comprises using fructose-6-phosphate or its salt as a raw material, adding tagatose-6-phosphate isomerase and tagatose-6-phosphate phosphatase to react and synthesize tagatose.

[0018] In a specific embodiment of the present invention, the method comprises using maltodextrin as raw material and synthesizing D-tagatose by a one-pot method using αGP, PGM, PGI, TPE, and TsPase.

[0019] In one embodiment of the present invention, the reaction is carried out in the presence of metal ions, including magnesium ions, manganese ions, calcium ions, zinc ions, nickel ions, and cobalt ions, preferably magnesium ions and / or manganese ions.

[0020] In one embodiment of the present invention, the pH of the reaction is 2-8, preferably 4-6.5, and more preferably 4.

[0021] In one embodiment of the present invention, the reaction temperature is 40-90°C, preferably 50-80.3°C, more preferably 70°C.

[0022] In one embodiment of the present invention, the hexokinase includes but is not limited to hexokinase I, hexokinase II, hexokinase III, and hexokinase IV (also known as glucokinase). For example, hexokinase from Saccharomyces cerevisiae such as AAA34698.1, hexokinase I from humans such as AAA52646.1, hexokinase II such as CAA86511.1, hexokinase III such as AAC50732.1, and hexokinase IV such as AAA51824.1.

[0023] In one embodiment of the present invention, the method for preparing the microorganism expressing the tagatose-6-phosphate phosphatase comprises: transferring a gene encoding the tagatose-6-phosphate phosphatase or an expression vector expressing the tagatose-6-phosphate phosphatase into a host cell.

[0024] In one embodiment of the present invention, the host cell comprises Escherichia coli. Preferably, the Escherichia coli comprises E. coli BL21 (DE3).

[0025] In one embodiment of the present invention, the expression vector is pET28a.

[0026] In one embodiment of the present invention, the method for preparing the culture of the microorganism comprises: culturing the above-mentioned microorganism and inducing expression to obtain the same.

[0027] In a third aspect, the present invention provides use of a composition in tagatose synthesis, wherein the composition comprises the tagatose-6-phosphate phosphatase, a microorganism expressing the tagatose-6-phosphate phosphatase, and / or a culture of the microorganism.

[0028] In one embodiment of the present invention, the composition further comprises one or more of the following enzymes: α-glucan phosphorylase, phosphoglucomutase, glucose-6-phosphate isomerase, fructose-6-phosphate isomerase, 6-phosphate tagatose isomerase, tagatose kinase, galactitol 2-dehydrogenase, α-amylase, pullulanase, glucoamylase, isoamylase, sucrase, starch phosphorylase, maltodextrin phosphorylase, sucrose phosphorylase, glucokinase, hexokinase, and phosphoglucomutase.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1) This study first discovered that A0A101X5R5_9CREN from Thermoproteus sp. CIS_19, a thermophilic bacterium, possesses tagatose-6-phosphate phosphatase activity. Furthermore, the tagatose-6-phosphate phosphatase exhibits high thermostability and activity, making it commercially valuable.

[0031] 2) The enzyme has high catalytic activity towards tagatose-6-phosphate and can be used to synthesize tagatose in vitro through a multi-enzyme cascade reaction, providing a theoretical basis and foundation for the highly economical in vitro synthesis of tagatose.

[0032] 3) The method of the present invention has a high tagatose yield at 50°C, pH 6.5, 20mM Mg 2+ Under the condition of 10 g / L maltodextrin as substrate, the yield of tagatose reached 4.3 g / L after 5 h. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 To construct a phylogenetic tree using AfT6PP as a template.

[0034] Figure 2 This is the SDS-PAGE electrophoresis diagram of T6PP-7 expression and purification. In the left figure, 1: pET28a(+); 2: AfT6PP; 3: T6PP-7; 4: Protein Marker; in the right figure, 1: AfT6PP; 2: T6PP-7; 3: Protein Marker.

[0035] Figure 3 Determination of T6PP-7 catalytic activity with different substrates.

[0036] Figure 4 This is the curve of T6PP-7 catalytic activity changing with reaction temperature.

[0037] Figure 5 This is the curve showing the change of T6PP-7 catalytic activity with pH.

[0038] Figure 6 This is a graph showing the changes in the catalytic activity of T6PP-7 with different metal ions.

[0039] Figure 7 Schematic diagram of the multi-enzyme pathway for synthesizing tagatose using fructose 6-phosphate as substrate.

[0040] Figure 8A Peak diagram and Figure 8B This is the product peak diagram.

[0041] Figure 9 Schematic diagram of the multi-enzyme synthesis pathway of tagatose using maltodextrin as substrate. DETAILED DESCRIPTION

[0042] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0043] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0044] T m The Tm value refers to the midpoint temperature of protein thermal denaturation (melting temperature), that is, the temperature at which the protein unfolds 50%. The Tm value is often used to evaluate the stability and thermal decomposition of proteins.

[0045] Km is the concentration of the substrate at which the initial rate of an enzyme reaction is half Vmax. Under certain conditions, it can be used to indicate the affinity of an enzyme for its substrate. A larger Km value indicates a lower affinity for the enzyme; conversely, a smaller Km value indicates a higher affinity for the enzyme.

[0046] Vmax represents the maximum reaction rate at a certain amount of enzyme, that is, the reaction rate when the enzyme is completely saturated with substrate, and is proportional to the enzyme concentration.

[0047] kcat is known as the catalytic constant, turnover number, or turnover number of an enzyme. It refers to the total number of substrate molecules converted into product by one enzyme molecule per unit time. The unit of kcat is s⁻¹. If an enzyme obeys the Michaelis-Menten equation, then kcat = k² = Vmax / Et.

[0048] In the present invention, T6PP-7 and TsPase are used interchangeably and both represent tagatose-6-phosphate phosphatase.

[0049] Example 1 Gene mining and cloning of tagatose-6-phosphate phosphatase

[0050] Using the amino acid sequence of AfT6PP from A. fulgidus as a template, we searched the Uniprot non-redundant protein sequence database using the Blastp function, and selected and downloaded the top 120 sequences with the highest sequence identity. We used MEGA to perform multiple sequence alignment and deleted alignment gaps. We constructed a phylogenetic tree of the remaining sequences using the neighbor-joining method ( Figure 1A novel gene, A0A101X5R5_9CREN, from Thermoproteus sp. CIS_19, which is closely related to the template sequence AfT6PP (PGP_ARCFU), was selected as a candidate gene (its amino acid sequence is shown in SEQ ID NO: 1). A0A101X5R5_9CREN was named T6PP-7 and has a theoretical molecular weight of 23.3 kDa.

[0051] The tagatose-6-phosphate phosphatase gene A0A101X5R5_9CREN was synthesized by the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, and ligated into the pET-28a(+) vector (New England Biolabs). The gene was then transformed into Escherichia coli BL21 Gold (DE3). Positive clones were screened and cultured on LB plates containing kanamycin resistance. The plasmid was extracted and sequenced to confirm the successful construction of the vector, which was named pET28a-TsPase.

[0052] Example 2 Expression of tagatose-6-phosphate phosphatase

[0053] Positive bacteria were picked with an inoculation needle and inoculated into 5 mL of LB medium. The cells were cultured at 37°C and 220 rpm for 12 hours. Subsequently, a 1% (v / v) inoculum was inoculated into 20 mL of LB medium and cultured at 37°C and 220 rpm. When the OD600 reached 0.6-0.8, IPTG was added to a final concentration of 0.1 mM to induce expression. Expression conditions were low-temperature induction at 20°C and 220 rpm for 24 hours. The cells were then harvested and disrupted, and the supernatant was obtained by centrifugation.

[0054] Purification was performed using a Ni-NAT pre-loaded gravity column. The protein purification column was first balanced with a phosphate buffer containing 50mmol / L imidazole (50mmol / LNaH2PO4, 300mmol / L NaCl, adjusted to pH=8 with sodium hydroxide), and then the collected crude enzyme solution was filtered through a 0.22μm filter membrane and loaded onto the column to allow the protein to fully bind to the column material. The filler was then washed with a phosphate buffer containing 50mmol / L imidazole (50mmol / L NaH2PO4, 300mmol / L NaCl, adjusted to pH=8 with sodium hydroxide) to remove impurities. Finally, the solution was eluted with a phosphate buffer containing 250mmol / L imidazole (250mmol / L NaH2PO4, 300mmol / L NaCl, adjusted to pH=8 with sodium hydroxide). The purification effect was detected and analyzed by SDS-PAGE, and the enzymatic properties of the purified enzyme solution were studied by testing the protein concentration using Bradford. The results are shown in the figure below. Figure 2The results show that T6PP-7, compared to the pET28a empty vector, exhibits a distinct specific band at less than 25 kDa, consistent with the theoretical molecular weight of T6PP-7 (23.3 kDa). Therefore, T6PP-7 is confirmed to be soluble in E.coil BL21 (DE3). Purification using a prepacked gravity column yielded a high-purity T6PP-7 enzyme for subsequent characterization.

[0055] Example 3 Parameter Detection of Tagatose-6-Phosphate Phosphatase Catalyzed Reaction

[0056] 1. Substrate specificity

[0057] Because the substrate spectrum of phosphatases is complex and confusing, clarifying the substrate spectrum of phosphatases is very important in their application. The following reaction system was used to perform reactions using glucose 1-phosphate (G1P), glucose 6-phosphate (G6P), tagatose 6-phosphate (T6P), and fructose 1-phosphate (F6P) as substrates.

[0058] The specific reaction system is as follows (200 μL):

[0059] Different substrates: final concentration 10mM; TPE: final concentration 2g / L; TsPase obtained in Example 2: final concentration 0.01g / L; magnesium chloride: final concentration 5mM. pH 6.5, 55℃ reaction for 60min, HPLC detection of product yield. The activity at the highest yield was set as 100%, and the catalytic activity for different substrates was compared to determine its substrate spectrum. The results are shown in Figure 2. Figure 3 shown.

[0060] The specific detection methods are as follows:

[0061] Mobile phase: 5 mM H2SO4; column: 87H; column temperature: 60°C; flow rate: 0.6 mL / min.

[0062] 2. Optimum reaction temperature

[0063] Referring to the reaction system in step 1, the reaction temperature was set at six temperatures within the range of 40-90°C. The substrate was tagatose-6-phosphate. The product yield was measured to determine its enzyme activity. A curve of enzyme activity versus temperature was drawn to determine the optimal reaction temperature of T6PP-7. The results are shown in Figure 2. Figure 4 The results show that the enzyme activity of T6PP-7 in catalyzing the conversion of tagatose-6-phosphate to tagatose increases with increasing temperature between 40°C and 70°C, reaching its maximum at 70°C. Subsequently, the enzyme activity decreases as the temperature rises. The results indicate that the optimal reaction temperature for T6PP-7 is 70°C.

[0064] 3. Optimum pH

[0065] Referring to the reaction system in step 1, sodium hydrogen phosphate-citrate buffer (pH 2-8) was prepared, enzyme activity was measured under different pH conditions, and the optimal pH curve was drawn to determine the optimal reaction pH of T6PP-7. Figure 5 The results show that the enzyme activity of T6PP-7 in catalyzing the conversion of tagatose-6-phosphate to tagatose increases with increasing pH between pH 2 and 4, reaching its maximum at pH 4. The enzyme activity then decreases with increasing pH. The optimal reaction pH for T6PP-7 is 4.

[0066] 4. Optimal metal ions

[0067] Referring to the reaction system in step 1, prepare 2+ Mg 2+ 、Zn 2+ 、Ni 2+ 、Co 2+ 、Mn 2+ The optimal metal ion for T6PP-7 was determined by adding different metal ions to the reaction system at a final concentration of 5 mM. The product yield was measured to determine the effect of metal ions on enzyme activity. The activity at the highest product yield was set as 100%, and the catalytic activity of T6PP-7 with different metal ions was analyzed. The results are shown in Figure 2. Figure 6 As shown in the results, tagatose-6-phosphatase is an enzyme that depends on metal ions, Mg 2+ The strongest activation factor for T6PP-7 enzyme activity was Mn. 2+ Therefore, the optimal metal ion for T6PP-7 to catalyze the conversion of tagatose-6-phosphate to tagatose is Mg. 2+ .

[0068] Example 4 Activity Detection of Tagatose-6-Phosphate Phosphatase

[0069] The pure enzyme obtained in Example 2 was reacted with F6P and tagatose 6-phosphate isomerase (TPE) according to the following system. The specific reaction process is as follows: Figure 7 As shown, the tagatose yield was detected by liquid chromatography, and the specific reaction system was as follows (200 μL):

[0070] Fructose 6-phosphate (F6P): final concentration 10 mM; TPE: final concentration 2 g / L; TsPase: final concentration 0.01 g / L; magnesium chloride: final concentration 5 mM. The reaction was carried out at pH 6.5, 60°C for 60 min, and tagatose production was determined by HPLC.

[0071] The specific detection methods are as follows:

[0072] Mobile phase: 5 mM H2SO4; column: 87H; column temperature: 60°C; flow rate: 0.6 mL / min.

[0073] The results are shown in Figure 8. The test results show that the new gene TsPase with tagatose-6-phosphatase activity was activated at 60°C, pH 6.5, 5mM Mg 2+ Under these conditions, 1.5 mM tagatose can be produced.

[0074] Example 5 Kinetic Detection of Tagatose-6-Phosphate Phosphatase

[0075] The pure enzyme obtained in Example 2 was reacted with F6P and tagatose-6-phosphate isomerase (TPE) according to the following system, and the tagatose yield was detected by liquid chromatography. The specific reaction system was as follows (200 μL):

[0076] Fructose 6-phosphate (F6P) at various final concentrations; TPE at 2 g / L; TsPase at 0.01 g / L; and magnesium chloride at 5 mM were used. The reaction was performed at pH 4 and 70°C for various reaction times. Tagatose production was determined by HPLC.

[0077] The specific detection methods are as follows:

[0078] Mobile phase: 5 mM H2SO4; column: 87H; column temperature: 60°C; flow rate: 0.6 mL / min.

[0079] The test results show that the kinetic constant K m is 2.40±0.98mmol / L, and the maximum reaction rate v max is 0.044±0.004mol / min, and the catalytic constant k cat 102.5±8.6min -1 .

[0080] Example 6 Thermal Stability Detection of Tagatose-6-Phosphate Phosphatase

[0081] Protein Thermal Shift TM The Dye Kit is used to measure the melting temperature (Tm) of proteins. The measurement system is as follows: 12.5 μl protein, 2.5 uL Protein Thermal Shift TM Dye(8×), 5μl ProteinThermal Shift TMBuffer. The temperature gradient was set to 25-99°C. The fluorescence signal was detected using a FAST7500 fluorescence quantitative PCR instrument to determine the Tm value. The Tm value was determined to be 80.3±1.3°C.

[0082] Example 7 One-pot biosynthesis of D-tagatose from maltodextrin

[0083] like Figure 9 As shown, D-tagatose was synthesized from maltodextrin using an enzymatic method involving αGP, PGM, PGI, TPE, and TsPase. D-tagatose was synthesized in one pot at 50°C in 10 mM phosphate buffer (pH 6.5) containing 10 g / L maltodextrin, 20 mM MgCl2, 1 U / mL αGP, 1 U / mL PGM, 1 U / mL PGI, 1 U / mL TPE, and 1 U / mL TsPase. The reaction was carried out at 50°C for 5 hours, and tagatose yield was determined by HPLC.

[0084] The specific detection methods are as follows:

[0085] Mobile phase: 5 mM H2SO4; column: 87H; column temperature: 60°C; flow rate: 0.6 mL / min.

[0086] The test results showed that the new gene TsPase with tagatose 6-phosphatase activity was detected at 50℃, pH 6.5, 20mM Mg 2 +, 4.3 g / L of tagatose can be produced.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

[0088] SEQ ID NO. 1

[0089]

Claims

1. Use of tagatose-6-phosphate phosphatase in tagatose synthesis, characterized in that: The tagatose-6-phosphate phosphatase is derived from thermophilic bacteria.

2. The use of a tagatose-6-phosphate phosphatase in tagatose synthesis according to claim 1, characterized in that: The tagatose-6-phosphate phosphatase is derived from Thermoproteus sp. CIS_19 or a variant thereof.

3. The use of a tagatose-6-phosphate phosphatase in tagatose synthesis according to claim 1, characterized in that: The amino acid sequence of the tagatose-6-phosphate phosphatase is as shown in SEQ ID NO: 1, or an amino acid sequence having at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.8%, or at least 99.9% identity to the sequence shown in SEQ ID NO:

1.

4. A method for synthesizing tagatose, characterized in that: The method comprises the step of converting tagatose-6-phosphate into tagatose under the action of the tagatose-6-phosphate phosphatase according to any one of claims 1 to 3, a microorganism expressing the tagatose-6-phosphate phosphatase and / or a culture of the microorganism.

5. The method for synthesizing tagatose according to claim 4, wherein: The method comprises using hexose or a substance that can be converted into hexose as a raw material, adding a multi-enzyme system including the tagatose-6-phosphate phosphatase, a microorganism expressing the tagatose-6-phosphate phosphatase and / or a culture of the microorganism to react and synthesize tagatose.

6. The method for synthesizing tagatose according to claim 5, wherein: The hexose includes one or more of D-fructose, fructose 6-phosphate, tagatose-6-phosphate, D-galactose, D-galactose-6-phosphate, glucose, glucose-6-phosphate, and glucose-1-phosphate; Preferably, the substance that can be converted into hexose includes disaccharides or polysaccharides, and preferably the disaccharides or polysaccharides include one or more of sucrose, maltose, lactose, D-glyceraldehyde triphosphate, starch, dextrin, and maltodextrin; Preferably, the multi-enzyme system further comprises one or more of the following enzymes: α-glucan phosphorylase, phosphoglucomutase, glucose-6-phosphate isomerase, fructose-6-phosphate isomerase, 6-phosphate tagatose isomerase, tagatose kinase, galactitol 2-dehydrogenase, α-amylase, pullulanase, glucoamylase, isoamylase, sucrase, starch phosphorylase, maltodextrin phosphorylase, sucrose phosphorylase, glucokinase, hexokinase, phosphoglucomutase; Preferably, the method comprises using fructose-6-phosphate or its salt as a raw material, adding 6-phosphate tagatose isomerase and tagatose-6-phosphate phosphatase to react and synthesize tagatose; Preferably, the method comprises using maltodextrin as raw material and synthesizing D-tagatose by a one-pot method using αGP, PGM, PGI, TPE and TsPase.

7. A method for synthesizing tagatose according to any one of claims 4 to 6, characterized in that: The reaction is carried out in the presence of metal ions; preferably, the metal ions include one or more of magnesium ions, manganese ions, calcium ions, zinc ions, nickel ions, and cobalt ions, more preferably magnesium ions and manganese ions; Preferably, the pH of the reaction is 2-8, more preferably 4-6.5; Preferably, the reaction temperature is 40-90°C, more preferably 50-80.3°C.

8. The method for synthesizing tagatose according to any one of claims 4 to 6, wherein: The preparation method of the microorganism comprises: transferring the gene encoding the tagatose-6-phosphate phosphatase or the expression vector expressing the tagatose-6-phosphate phosphatase into a host cell; Preferably, the host cell includes Escherichia coli; more preferably, the Escherichia coli includes E. coli BL21 (DE3). Preferably, the expression vector is pET28a.

9. The method for synthesizing tagatose according to any one of claims 4 to 6, wherein: The method for preparing the culture of the microorganism comprises: culturing the microorganism and inducing expression to obtain the culture.

10. Use of a composition in tagatose synthesis, characterized in that: The composition comprises the tagatose-6-phosphate phosphatase according to any one of claims 1 to 3, a microorganism expressing the tagatose-6-phosphate phosphatase, and / or a culture of the microorganism; Preferably, the composition further comprises one or more of the following enzymes: α-glucan phosphorylase, phosphoglucomutase, glucose-6-phosphate isomerase, fructose-6-phosphate isomerase, 6-phosphate tagatose isomerase, tagatose kinase, galactitol 2-dehydrogenase, α-amylase, pullulanase, glucoamylase, isoamylase, sucrase, starch phosphorylase, maltodextrin phosphorylase, sucrose phosphorylase, glucokinase, hexokinase, phosphoglucomutase.

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