A method for biosynthesis of d-tagatose based on multi-enzyme cascade isomerization-phosphorylation
Patent Information
- Application Number
- CN202611077753.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-20
- Publication Date
- 2026-08-21
AI Technical Summary
然而，常规β-GAL与L-AI之间存在温度适配性差的问题，导致半乳糖供应受限，成为级联反应的关键瓶颈
[0024]本发明在最优条件下经完整的磷酸化-去磷酸化反应后，以50 g/L乳糖为底物，D-塔格糖产率可达29.0%（14.5 g/L），较未引入磷酸化模块的对照体系提升约3倍。
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Figure CN122609660A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of synthetic biology, specifically relating to a method for the biosynthesis of D-tagatose based on multi-enzyme cascade isomerization-phosphorylation, which is particularly suitable for enzyme catalytic systems using lactose as a substrate. Background Technology
[0002] D-Tagatose, a rare natural ketose, has a sweetness approximately 92% that of sucrose and a caloric value only 38% of sucrose. It has been proven to possess multiple physiological functions, including inhibiting postprandial blood glucose elevation, improving lipid metabolism, and preventing tooth decay. In recent years, its demand has continued to grow in functional foods, diabetic foods, and pharmaceutical excipients. Currently, the industrial synthesis of D-tagatose mainly relies on the D-galactose isomerization pathway catalyzed by L-arabinose isomerase (L-AI). However, this process faces two constraints: firstly, the high cost of D-galactose raw materials increases production costs; secondly, the L-AI catalytic reaction is limited by thermodynamic equilibrium, with the theoretical conversion rate typically failing to exceed 30%, and product accumulation further inhibiting the reaction process in actual production. To reduce raw material costs, researchers have attempted to construct a cascade catalytic system of β-galactosidase (β-GAL) and L-AI using lactose as a substrate, achieving conversion through the "lactose → galactose → D-tagatose" pathway. However, there is a poor temperature compatibility issue between conventional β-GAL and L-AI, leading to limited galactose supply and becoming a key bottleneck in the cascade reaction. Although some studies have screened β-GAL from different sources to improve hydrolysis efficiency, the thermodynamic equilibrium limitations of the isomerization step have not been fundamentally resolved. Summary of the Invention
[0003] The technical problem to be solved by this invention is to provide a method for the biosynthesis of D-tagatose based on multi-enzyme cascade isomerization-phosphorylation, which is limited by thermodynamic equilibrium in the existing L-arabinose isomerase catalytic reaction.
[0004] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:
[0005] The present invention discloses a method for the biosynthesis of D-tagatose based on multi-enzyme cascade isomerization-phosphorylation, comprising the following steps:
[0006] S1. Using lactose as a substrate, three independent recombinant Escherichia coli crude enzyme solutions heterologously expressing β-galactosidase, L-arabinose isomerase and D-tagakase: β-GAL crude enzyme solution, L-AI crude enzyme solution and TagK crude enzyme solution were simultaneously added to the reaction system. In the presence of metal ions, sodium hexametaphosphate and ATP, the next step catalyzes the reaction to synthesize D-tagakase-6-phosphate.
[0007] S2. Using the fourth recombinant Escherichia coli crude enzyme solution of heterologous expression of phosphoglycolate phosphatase: PGP crude enzyme solution, the D-tagatose-6-phosphate obtained in step S1 was dephosphorylated in vitro to obtain D-tagatose.
[0008] The β-GAL encoding gene lacZ is derived from *Streptococcus thermophilus* (GenBank: AKH33819), the L-AI encoding gene araA is derived from *Lactococcus lactis* (GenBank: MH940215), the TagK encoding gene tagK is derived from *Escherichia coli* Nissle 1917 (GenBank: WP_000048611.1), and the PGP encoding gene pgp is derived from *Archaeoglobus profundus* DSM 5631 (GenBank: ARCPR_RS04055).
[0009] The crude enzyme solution was prepared by the following methods:
[0010] β-GAL crude enzyme solution: The lacZ gene was inserted between the BamHI and HindIII restriction sites of the pRSFDuet-1 vector to construct the recombinant plasmid pRSFDuet-1-lacZ; the recombinant plasmid pRSFDuet-1-lacZ was transformed into E. coli (E. coli BL21(DE3)) to obtain the engineered strain Ec01; the engineered strain Ec01 was used to prepare wet cells of the engineered strain Ec01, and the supernatant was collected after the wet cells of the engineered strain Ec01 were lysed to obtain the β-GAL crude enzyme solution;
[0011] L-AI crude enzyme solution: The araA gene was inserted between the BamHI and HindIII restriction sites of the pRSFDuet-1 vector to construct the recombinant plasmid pRSFDuet-1-araA; the recombinant plasmid pRSFDuet-1-araA was transformed into E. coli (E. coli BL21(DE3)) to obtain the engineered strain Ec02; the engineered strain Ec02 was used to prepare wet cells of the engineered strain Ec02, and the supernatant was collected after the wet cells of the engineered strain Ec02 were lysed to obtain L-AI crude enzyme solution;
[0012] TagK crude enzyme solution: The tagK gene was inserted between the BamHI and HindIII restriction sites of the pRSFDuet-1 vector to construct the recombinant plasmid pRSFDuet-1-tagK; the recombinant plasmid pRSFDuet-1-tagK was transformed into E. coli (E. coli BL21(DE3)) to obtain the engineered strain Ec03; the engineered strain Ec03 was used to prepare wet cells of the engineered strain Ec03, and the supernatant was collected after the wet cells of the engineered strain Ec03 were lysed to obtain the TagK crude enzyme solution;
[0013] PGP crude enzyme solution: The pgp gene was inserted between the BamHI and HindIII restriction sites of the pRSFDuet-1 vector to construct the recombinant plasmid pRSFDuet-1-pgp; the recombinant plasmid pRSFDuet-1-pgp was transformed into E. coli (E. coli BL21(DE3)) to obtain the engineered strain Ec04; the engineered strain Ec04 was used to prepare wet cells of the engineered strain Ec04, and the supernatant was collected after the wet cells of the engineered strain Ec04 were lysed to obtain the PGP crude enzyme solution.
[0014] The metal ion is at least Ni. 2+ Cu 2+ Co 2+ Mg 2+ Mn 2+ Fe 2+ Zn 2+ Ca 2+ Ba 2+ One of them has a metal ion addition amount of 1 mM; the ATP addition amount is 10-20 mM; and the sodium hexametaphosphate addition amount is 10 mM.
[0015] The catalytic reaction conditions described in step S1 are a substrate concentration of 50-100 g / L, 35-60℃, and pH of 6.0-9.0.
[0016] The conditions for the catalytic reaction in step S1 are: substrate concentration 50 g / L, 45℃, pH 8.0, and metal ion concentration of 1 mM Ni. 2+ The amount of ATP added was 20 mM, and the amount of sodium hexametaphosphate added was 10 mM.
[0017] The specific process of in vitro dephosphorylation in step S2 is as follows: Take 900 μL of the supernatant of the catalytic reaction, adjust the pH to 7.0 with solid NaOH, add crude PGP enzyme solution, and react at 50℃ for 12 h.
[0018] In step S1, the volume ratio of crude β-GAL enzyme solution, crude L-AI enzyme solution, and crude TagK enzyme solution in the reaction system is 3:3:3.
[0019] In step S2, the amount of PGP crude enzyme solution added is such that the volume ratio of supernatant to PGP crude enzyme solution is 9:1.
[0020] The in vitro dephosphorylation treatment described in step S2 also involves the addition of metal ions.
[0021] The method for preparing the wet bacterial cells is as follows: the engineered strain is inoculated into LB medium at a 1% inoculum and cultured at 37°C and 200 rpm until OD. 600 The concentration was 0.6–1.0, and IPTG was added to a final concentration of 0.1 mM. The cells were induced at 16°C for 24 h. The cells were collected by centrifugation at 8000 rpm and 4°C for 5 min. The cells were resuspended in 1×PBS buffer and washed twice. After centrifugation, wet cells were obtained.
[0022] The LB medium was prepared with the following components: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and pH adjusted to 7.0.
[0023] The beneficial effects of this invention are as follows:
[0024] Under optimal conditions, after a complete phosphorylation-dephosphorylation reaction, the yield of D-tagatose can reach 29.0% (14.5 g / L) using 50 g / L lactose as a substrate, which is about 3 times higher than the control system without the introduction of the phosphorylation module.
[0025] This invention successfully breaks the inherent thermodynamic equilibrium limitation of L-arabinose isomerase by constructing a phosphorylation-dephosphorylation cascade module, causing the reaction equilibrium to shift continuously to the right and significantly improving the conversion efficiency of lactose to D-tagatose.
[0026] Compared with the existing whole-cell phosphorylation D-tagatose synthesis system, the isomeric-phosphorylation multi-enzyme cascade catalytic system constructed in this invention significantly reduces the ineffective consumption of intermediate products and lowers the difficulty of pathway modification.
[0027] This invention uses lactose as a substrate with a concentration range of 50-100 g / L. It maintains high conversion efficiency even at high substrate concentrations, and the process is green and economical, with potential for industrial application.
[0028] This invention can effectively improve the conversion efficiency of lactose to D-tagatose. Attached Figure Description
[0029] Figure 1 This is a schematic diagram of the synthesis path of the synthesis method described in this invention;
[0030] Figure 2 This is a process flow diagram of the present invention;
[0031] In the figure, LacZ is the β-galactosidase gene; AraA is the L-arabinose isomerase gene; TagK is the tagatose kinase gene; and PGP is phosphate glycolate phosphatase. Detailed Implementation
[0032] The present invention will be further illustrated below with specific examples. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.
[0033] The raw materials, reagents, culture media, and instruments used in the embodiments of this invention are all conventional products in the art and can be purchased through commercial channels. LB liquid culture medium was prepared according to the following composition: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH adjusted to 7.0. 1×PBS buffer was prepared by diluting 20×PBS; the reaction buffer used was 50 mM Tris-HCl (pH 5.0–10.0) or 50 mM phosphate buffer. High-performance liquid chromatography (HPLC) detection conditions: Ultimate Sugar-Ca column (7.8 × 300 mm), mobile phase: ultrapure water, flow rate: 0.6 mL / min, column temperature: 80℃, differential refractive index detector, injection volume: 20 μL.
[0034] The synthesis method described in this invention has the following synthesis path: Figure 1 As shown. β-GAL is used for lactose hydrolysis, L-AI for galactose isomerization, TagK for phosphorylation, and PGP for dephosphorylation. The process flow of this invention is as follows. Figure 2 As shown, the process is divided into three main stages: construction of recombinant E. coli, preparation of crude enzyme, and two-step enzyme catalysis. First, four recombinant E. coli strains, Ec01, Ec02, Ec03, and Ec04, were constructed and uniformly cultured, induced, centrifuged, and washed to obtain wet cells. The wet cells were used to prepare crude enzyme solutions in two parallel branches: the left branch was for β-GAL crude enzyme solution, L-AI crude enzyme solution, and TagK crude enzyme solution, and the right branch was for PGP dephosphorylation crude enzyme solution. The left branch used lactose as a substrate and combined it with cofactors such as metal ions, ATP, and sodium hexametaphosphate to carry out phosphorylation reaction to synthesize D-tagatose-6-phosphate reaction solution. The PGP dephosphorylation crude enzyme solution was added to the D-tagatose-6-phosphate reaction solution to finally synthesize D-tagatose.
[0035] The following examples illustrate the process of constructing engineered bacteria, preparing crude enzyme solution, catalytically preparing D-tagatose-6-phosphate, and dephosphorylating to prepare D-tagatose. Example 1 describes the preparation of crude enzyme solution, Examples 2–8 describe the catalytic preparation of D-tagatose-6-phosphate, and Examples 9–12 describe the dephosphorylation preparation of D-tagatose.
[0036] Example 1: Preparation of crude enzyme solution
[0037] (1) Construction of recombinant plasmids
[0038] The purpose of this step is to construct four recombinant plasmids for subsequent transformation into engineered strains.
[0039]
[0040]
[0041]
[0042]
[0043] (2) Construction of engineered strains
[0044] pRSFDuet-1-lacZ, pRSFDuet-1-araA, pRSFDuet-1-tagK, and pRSFDuet-1-pgp were transformed into Escherichia coli (E. coli BL21(DE3)), plated on LB agar plates containing kanamycin (100 mg / L), and incubated at 37°C for 12 h. Single colonies were picked for colony PCR and plasmid digestion verification. The engineered strains Ec01, Ec02, Ec03, and Ec04 were successfully obtained.
[0045] (3) Preparation of wet mycelium:
[0046] The constructed engineered strains Ec01, Ec02, Ec03, and Ec04 were inoculated into 5 mL of LB liquid medium containing kanamycin (100 mg / L) and cultured at 37°C and 200 rpm for 12 h to obtain seed culture. Seed culture was then transferred at a 1% inoculum to 50 mL of LB liquid medium containing kanamycin (100 mg / L) and cultured at 37°C and 200 rpm until OD (dose retardation). 600 =0.6-0.8, add IPTG to a final concentration of 0.1 mM, and induce at 16℃ and 200 rpm for 24 h; centrifuge at 8000 rpm and 4℃ for 5 min, discard the supernatant, resuspend the bacterial cells in 1×PBS and centrifuge at 8000 rpm and 4℃ for 5 min, repeat twice to collect wet bacterial cells. Wet bacterial cells of four engineered strains, Ec01, Ec02, Ec03 and Ec04, were obtained.
[0047] (4) Preparation of crude enzyme solution:
[0048] The collected engineered bacterial strains Ec01, Ec02, Ec03, and Ec04 were thoroughly resuspended in 5 mL of 1×PBS buffer, and then sonicated (parameter settings: power 40%, sonication for 2 s, stop for 3 s, processing time approximately 15 min) until the bacterial solution was clear. The cell lysate was centrifuged at 8000 rpm and 4℃ for 20 min, and the supernatant was collected to obtain the crude enzyme solutions for the corresponding engineered strains Ec01, Ec02, Ec03, and Ec04. The crude enzyme solution for engineered strain Ec01 was β-GAL crude enzyme solution, the crude enzyme solution for engineered strain Ec02 was L-AI crude enzyme solution, the crude enzyme solution for engineered strain Ec03 was TagK crude enzyme solution, and the crude enzyme solution for engineered strain Ec04 was PGP crude enzyme solution.
[0049] Example 2: Catalytic preparation of D-tagatose-6-phosphate
[0050] Prepare a 1 mL catalytic system by mixing crude β-GAL enzyme solution, crude L-AI enzyme solution, crude TagK enzyme solution, and 50 mM Tris-HCl (pH 7.0) buffer. The volume ratio of the solutions added is β-GAL crude enzyme solution: L-AI crude enzyme solution: TagK crude enzyme solution: Tris-HCl buffer solution 3:3:3:1. The system also contains 50 g / L lactose, 20 mM ATP, and 10 mM sodium hexametaphosphate. A metal ion, 1 mM Ni, is added during the reaction. 2+ The reaction was carried out at 45℃ and 200 rpm in a water bath with shaking for 16 h. After the reaction, 100 μL of crude PGP enzyme solution was added directly to the reaction system, and the reaction was carried out at 45℃ for 12 h for dephosphorylation treatment to convert D-tagatose-6-phosphate to D-tagatose. The reaction was terminated by boiling for 10 min immediately after the reaction was completed. The supernatant was collected by centrifugation at 8000 rpm for 5 min, filtered through a 0.22 μm filter membrane, and the concentration of D-tagatose in the reaction solution was analyzed by high performance liquid chromatography (HPLC) to calculate the yield.
[0051] Example 3: Optimization of reaction temperature for the catalytic preparation of D-tagatose-6-phosphate
[0052] Following the procedure in Example 2, only the reaction temperature was adjusted to 35°C, 40°C, 45°C, 50°C, 55°C, and 60°C, while all other conditions remained unchanged.
[0053] Example 4: pH optimization experiment for the catalytic preparation of D-tagatose-6-phosphate
[0054] Following the procedure in Example 2, the temperature was adjusted to 45°C, and the pH was adjusted to 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, while keeping the other conditions unchanged.
[0055] Example 5: Optimization experiment of metal ions for the catalytic preparation of D-tagatose-6-phosphate
[0056] Following the procedure in Example 2, the temperature was adjusted to 45°C, the pH to 8.0, and the metal ion concentration was adjusted to 1 mM Ni. 2+ Cu 2+ Co 2+ Mg 2+ Mn 2+ All other conditions remain unchanged.
[0057] Example 6: Optimization experiment for the catalytic preparation of D-tagatose-6-phosphate ATP
[0058] Following the procedure in Example 2, the temperature was adjusted to 45°C, the pH to 8.0, and the metal ion was Ni. 2+The concentrations of ATP were adjusted to 1 mM, 5 mM, 10 mM, 15 mM, and 20 mM, while the other conditions remained unchanged.
[0059] Example 7: Optimization Experiment of Sodium Hexametaphosphate for Catalytic Preparation of D-Tragatose-6-Phosphate
[0060] Following the procedure in Example 2, the temperature was adjusted to 45°C, the pH to 8.0, and the metal ion was Ni. 2+ The concentration of ATP was adjusted to 20 mM, and the concentrations of sodium hexametaphosphate were adjusted to 1 mM, 5 mM, 10 mM, 15 mM, and 20 mM, while the other conditions remained unchanged.
[0061] Example 8: Optimization of substrate concentration for the catalytic preparation of D-tagatose-6-phosphate
[0062] The substrate concentration was adjusted to 100 g / L, the temperature to 45°C, and the pH to 8.0. All other operations were the same as those in Example 2.
[0063] Example 9: Preparation of D-tagatose by dephosphorylation (optimization of dephosphorylation conditions)
[0064] Unlike the simplified dephosphorylation method in Examples 2-8, which involved directly adding crude PGP enzyme solution to the reaction system, this example and subsequent Examples 10-12 systematically optimized the dephosphorylation step. 900 μL of the reaction supernatant from Example 2 was taken, the pH was adjusted to 7.0 with solid NaOH, and 100 μL of crude PGP enzyme solution was added. The reaction was carried out at 50°C for 12 h. The reaction was terminated by boiling for 10 min, and the supernatant was collected by centrifugation at 8000 rpm for 5 min. After filtration through a 0.22 μm filter membrane, the concentration of D-tagatose in the reaction solution was analyzed by high-performance liquid chromatography (HPLC) to calculate the yield.
[0065] Example 10: Optimization of metal ions for the dephosphorylation preparation of D-tagatose
[0066] Following the procedure in Example 9, the following metal ions were added to the dephosphorylation reaction system at a final concentration of 1 mM: Mg 2+ Mn 2+ Co 2+ Ni 2+ Ca 2+ Zn 2+A control group without any added metal ions was used. Other conditions were the same as in Example 9 (reaction system: 900 μL supernatant, pH 7.0, 100 μL PGP crude enzyme solution, reaction at 50℃ for 12 h). The reaction was terminated by boiling for 10 min, and the supernatant was collected by centrifugation at 8000 rpm for 5 min. After filtration through a 0.22 μm filter membrane, the concentration of D-tagatose in the reaction solution was analyzed by high-performance liquid chromatography (HPLC) to calculate the yield.
[0067] Example 11: Optimization of metal ion concentration for the dephosphorylation preparation of D-tagatose
[0068] This embodiment is based on Example 10, and focuses on Mg 2+ Optimize the optimal addition concentration. Set Mn 2+ The final concentrations were 0.5 mM, 1 mM, 2 mM, 5 mM, and 10 mM, respectively, compared with no Mg addition. 2+ For control purposes, the remaining reaction conditions were the same as in Example 9 (900 μL supernatant, pH 7.0, 100 μL crude PGP enzyme solution, reaction at 50℃ for 12 h). The reaction was terminated by boiling for 10 min, centrifuged at 8000 rpm for 5 min, and the supernatant was collected. After filtration through a 0.22 μm filter membrane, the concentration of D-tagatose in the reaction solution was analyzed by high-performance liquid chromatography (HPLC) to determine the yield.
[0069] Example 12: Temperature optimization for dephosphorylation preparation of D-tagatose
[0070] This example investigates the effect of reaction temperature on the dephosphorylation reaction for the preparation of D-tagatose. The reaction temperatures for the dephosphorylation reaction for the preparation of D-tagatose were set to 30℃, 40℃, 50℃, 60℃, and 70℃, with Mg added. 2+ The concentration was 2 mM, and the other conditions were the same as in Example 9 (900 μL supernatant, pH 7.0, 100 μL crude PGP enzyme solution, reaction time 12 h). The reaction was terminated by boiling for 10 min, centrifuged at 8000 rpm for 5 min, and the supernatant was collected. After filtration through a 0.22 μm filter membrane, the concentration of D-tagatose in the reaction solution was analyzed by high performance liquid chromatography (HPLC) to determine the yield.
[0071] After phosphorylation, all samples from Examples 2-8 of this invention underwent uniform dephosphorylation treatment (direct addition of crude PGP enzyme solution, reaction at 45°C for 12 h). Key parameters and the yield of D-tagatose after dephosphorylation are shown in Table 1.
[0072] Table 1. Comparison of key parameters and D-tagatose yield in Examples 2-8
[0073]
[0074]
[0075] The yield and conversion rate of D-tagatose in Examples 9-12 of this invention are shown in Table 2.
[0076] Table 2 Comparison of key parameters and D-tagatose yield in Examples 9-12
[0077]
[0078] Effect Analysis:
[0079] As shown in Table 1, in Example 2 (standard conditions) at 45℃ and pH 7.0, the yield of D-tagatose was 12.0 g / L, with a conversion rate of 24.0%. Through system optimization, the optimal reaction temperature was determined to be 45℃, the optimal pH to be 8.0, and the optimal metal ion to be Ni. 2+ Under these conditions, the D-tagatose yield increased to 14.5 g / L, with a conversion rate of 29.0%. Examples 6 and 7 further optimized the concentrations of ATP and sodium hexametaphosphate, finding that the optimal yield was maintained under the conditions of 20 mM ATP and 10 mM sodium hexametaphosphate. Example 8 used 100 g / L lactose as a substrate, and the D-tagatose yield reached 24.5 g / L, indicating that the method of the present invention can still effectively convert D-tagatose at higher substrate concentrations.
[0080] As shown in Table 2: In Example 9, the residual free D-tagatose after phosphorylation was only 2.5 g / L, indicating that TagK can effectively catalyze the phosphorylation of D-tagatose and drive the isomerization equilibrium to shift continuously to the right; after dephosphorylation with PGP, the total recovery of D-tagatose reached 14.0 g / L, verifying the effectiveness of the phosphorylation-dephosphorylation cascade strategy; Example 10 shows that adding metal ions in the dephosphorylation step can further increase the yield, when the added metal ion is 1 mM Mg 2+ The hourly yield was 14.5 g / L, a 3.6% increase compared to the yield without added metal ions (14.0 g / L); Example 11 further optimized Mg 2+ Concentration, found Mg 2+ At a concentration of 1–2 mM, the yield was stable at 14.5 g / L with a conversion rate of 29.0%; however, excessively high concentrations (≥5 mM) had an inhibitory effect; Example 12 confirmed that the optimal dephosphorylation temperature was 50°C, and high temperatures (≥60°C) led to a significant decrease in enzyme activity, with a yield of only 6.0 g / L at 70°C.
[0081] The above embodiments are merely preferred embodiments of the present invention. Those skilled in the art can make equivalent substitutions or improvements within the spirit and principles of the present invention (such as adjusting plasmid combinations, optimizing metal ion types, replacing buffer systems, etc.), all of which should be included within the protection scope of the present invention.
Claims
1. A method for the biosynthesis of D-tagatose based on multi-enzyme cascade isomerization-phosphorylation, characterized in that, Includes the following steps: S1. Using lactose as a substrate, three independent recombinant Escherichia coli crude enzyme solutions heterologously expressing β-galactosidase, L-arabinose isomerase and D-tagakase: β-GAL crude enzyme solution, L-AI crude enzyme solution and TagK crude enzyme solution were simultaneously added to the reaction system. In the presence of metal ions, sodium hexametaphosphate and ATP, the next step catalyzes the reaction to synthesize D-tagakase-6-phosphate. S2. Using the fourth recombinant Escherichia coli crude enzyme solution of heterologous expression of phosphoglycolate phosphatase: PGP crude enzyme solution, D-tagatose-6-phosphate obtained in step S1 was dephosphorylated in vitro to synthesize D-tagatose.
2. The method for biosynthesizing D-tagatose based on multi-enzyme cascade isomerization-phosphorylation according to claim 1, characterized in that, The β-galactosidase encoding gene lacZ is derived from *Streptococcus thermophilus*; the L-arabinose isomerase encoding gene araA is derived from *Lactococcus lactis*; the D-tagoglucosidase encoding gene tagK is derived from *Escherichia coli* Nissle 1917; and the phosphate glycolate phosphatase encoding gene pgp is derived from *Archaeoglobus profundus* DSM 5631.
3. The method for biosynthesizing D-tagatose based on multi-enzyme cascade isomerization-phosphorylation according to claim 1, characterized in that, The crude β-GAL enzyme solution, crude L-AI enzyme solution, crude TagK enzyme solution, and crude PGP enzyme solution were prepared by the following methods: β-GAL crude enzyme solution: The lacZ gene was inserted between the BamHI and HindIII restriction sites of the pRSFDuet-1 vector to construct the recombinant plasmid pRSFDuet-1-lacZ; the recombinant plasmid pRSFDuet-1-lacZ was transformed into Escherichia coli (E. coli BL21(DE3)) to obtain the engineered strain Ec01; Wet cells of engineered strain Ec01 were prepared using engineered strain Ec01. After the wet cells of engineered strain Ec01 were crushed, the supernatant was collected to obtain crude β-GAL enzyme solution. L-AI crude enzyme solution: The araA gene was inserted between the BamHI and HindIII restriction sites of the pRSFDuet-1 vector to construct the recombinant plasmid pRSFDuet-1-araA; the recombinant plasmid pRSFDuet-1-araA was transformed into E. coli (E. coli BL21(DE3)) to obtain the engineered strain Ec02; the engineered strain Ec02 was used to prepare wet cells of the engineered strain Ec02, and the supernatant was collected after the wet cells of the engineered strain Ec02 were lysed to obtain the L-AI crude enzyme solution; TagK crude enzyme solution: The tagK gene was inserted between the BamHI and HindIII restriction sites of the pRSFDuet-1 vector to construct the recombinant plasmid pRSFDuet-1-tagK. The recombinant plasmid pRSFDuet-1-tagK was transformed into Escherichia coli (E. coli BL21(DE3)) to obtain the engineered strain Ec03; the engineered strain Ec03 was used to prepare wet cells of the engineered strain Ec03, and the supernatant was collected after the engineered strain Ec03 was broken to obtain crude TagK enzyme solution. PGP crude enzyme solution: The pgp gene was inserted between the BamHI and HindIII restriction sites of the pRSFDuet-1 vector to construct the recombinant plasmid pRSFDuet-1-pgp; The recombinant plasmid pRSFDuet-1-pgp was transformed into Escherichia coli (E. coli BL21(DE3)) to obtain the engineered strain Ec04. Wet cells of engineered strain Ec04 were prepared using engineered strain Ec04. After the wet cells of engineered strain Ec04 were broken, the supernatant was collected to obtain crude PGP enzyme solution.
4. The method for biosynthesizing D-tagatose based on multi-enzyme cascade isomerization-phosphorylation according to claim 1, characterized in that, The metal ion is at least Ni. 2+ Cu 2+ Co 2+ Mg 2+ Mn 2+ One of them has a metal ion addition amount of 1 mM; the ATP addition amount is 10-20 mM; and the sodium hexametaphosphate addition amount is 10 mM.
5. The method for biosynthesizing D-tagatose based on multi-enzyme cascade isomerization-phosphorylation according to claim 1, characterized in that, The conditions for the catalytic reaction in step S1 are a substrate concentration of 50-100 g / L, 35-60℃, and pH of 6.0-9.
0.
6. The method for biosynthesizing D-tagatose based on multi-enzyme cascade isomerization-phosphorylation according to claim 1, characterized in that, The conditions for the catalytic reaction in step S1 are: substrate concentration 50 g / L, 45℃, pH=8.0, and metal ion concentration of 1 mM Ni. 2+ The amount of ATP added was 20 mM, and the amount of sodium hexametaphosphate added was 10 mM.
7. The method for biosynthesizing D-tagatose based on multi-enzyme cascade isomerization-phosphorylation according to claim 1, characterized in that, The specific process of in vitro dephosphorylation in step S2 is as follows: Take 900 μL of the supernatant of the catalytic reaction, adjust the pH to 7.0 with solid NaOH, add crude PGP enzyme solution, and react at 50℃ for 12 h.
8. The method for biosynthesizing D-tagatose based on multi-enzyme cascade isomerization-phosphorylation according to claim 1, characterized in that, In step S1, the volume ratio of crude β-GAL enzyme solution, crude L-AI enzyme solution, and crude TagK enzyme solution in the reaction system is 3:3:
3.
9. The method for biosynthesizing D-tagatose based on multi-enzyme cascade isomerization-phosphorylation according to claim 1, characterized in that, In step S2, the amount of PGP crude enzyme solution added is such that the volume ratio of supernatant to PGP crude enzyme solution is 9:
1.
10. The method for biosynthesizing D-tagatose based on multi-enzyme cascade isomerization-phosphorylation according to claim 1, characterized in that, The in vitro dephosphorylation treatment described in step S2 also involves the addition of metal ions.