Use of a tagatose-4-epimerase in the preparation of D-tagatose

Abstract

The application belongs to the technical field of biological manufacturing, and relates to application of tagatose-4-epimerase in preparation of D-tagatose, wherein the amino acid sequence of the tagatose-4-epimerase comprises a sequence as shown in SEQ ID No: 1 or a sequence with at least 60% sequence identity with the sequence shown in SEQ ID No: 1. The application successfully mines and screens a novel tagatose-4-epimerase with excellent catalytic performance from natural microbial resources through systematic structure alignment and sequence alignment analysis, and effectively solves the key bottleneck of insufficient activity of a core catalyst in the current enzymatic synthesis of D-tagatose.

Description

Technical Field

[0001] This invention belongs to the field of biomanufacturing technology and relates to the application of a tagatose-4-epimerase in the preparation of D-tagatose. Background Technology

[0002] Rare sugars, as novel functional sweeteners that are low in calories and have low absorption, and possess multiple physiological functions such as anti-caries, blood sugar regulation, prebiotics, and antioxidants, have significant application prospects in the food, pharmaceutical, and health product fields, and their development has become an international research hotspot. D-tagatose, as a representative rare sugar, has a sweetness close to sucrose but significantly lower calories, and possesses multiple physiological functions such as regulating blood sugar and improving gut microbiota. It has been approved for use by the US FDA and the National Health Commission of my country, making it an ideal sucrose substitute, and market demand is growing rapidly.

[0003] Among the methods for synthesizing D-tagatose, biosynthesis has become a research focus due to its advantages such as mild conditions, fewer byproducts, and environmental friendliness. The process route utilizing tagatose-4-epimerase to catalyze the synthesis of D-tagatose from inexpensive D-fructose has significant advantages in terms of economical raw materials, stable supply, and suitability for industrial production. However, the number of reported tagatose-4-epimerases is currently limited, and they generally suffer from low catalytic activity and low conversion efficiency, failing to meet the urgent need for efficient biocatalysts in industrial production and severely hindering the industrialization process of this route. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide an application of tagatose-4-epimerase in the preparation of D-tagatose.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides the application of tagatose-4-epimerase in the preparation of D-tagatose, wherein the amino acid sequence of the tagatose-4-epimerase includes the sequence shown in SEQ ID No:1 or a sequence having at least 60% sequence identity with the sequence shown in SEQ ID NO:1.

[0007] For example, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0008] SEQ ID No:1:

[0009] MPAIGIRIPPVFLPGILRAFKLRRTVGTLMLSYGRETAPEYVINAPPGKYEITRGHTGTSIKEYLSLAANYAFTEGVVVELEADHVSVSVSSIAAVKRISGVKSEYEISDEDIDKAL KYIEDEVEEAVSTGVIRFYTLDTCELIRYEADKMDKKDLDAAFEGIDGWKRILERYLDKRFTVIGESGKSYNFRFREEEIKRIAVKYWRSIEVAERVYKIFQEKTPWEFGIEIAFDET PHVTEPKEMLFYLNELWERGIPVDYIAPNVGFEKKKDYSGSLEELYRRVEIMSAIARRYGALLSFHSGSGSTPWTGKGPGVYETLLEATGYKLKYKVSGVYFELLMEIFARQPKGSK ARKLFEDIYDSVIEYVRKEIEKKGPLYSKVMEEQLREYENLVERTKDPYLPWADIFRYYSFLALNLRDEGGERPFRRRRIIELYEEDVALRNVIDREVAELTLRLIDGLDFKDNIDLL.

[0010] This invention, through systematic structural and sequence alignment analysis, successfully identified and screened a novel tagatose-4-epimerase (T4E) with excellent catalytic performance from natural microbial resources. This enzyme exhibits high catalytic efficiency for the conversion of D-fructose to D-tagatose even in its natural state, with activity significantly superior to most known natural T4E enzymes. It provides a natural biocatalyst for the efficient biosynthesis of D-tagatose. This discovery effectively solves the key bottleneck of insufficient core catalyst activity in current enzymatic synthesis of D-tagatose, providing an important enzyme resource and technological foundation for its industrial production.

[0011] Preferably, the application specifically includes: mixing and reacting tagatose-4-epimerase with a substrate solution containing D-fructose to obtain D-tagatose.

[0012] Preferably, the concentration of D-fructose in the substrate solution is 10 g / L or higher, such as 10 g / L, 12 g / L, 15 g / L, 18 g / L, 20 g / L, 22 g / L, 25 g / L, 28 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0013] Preferably, the substrate solution further comprises, in molar concentrations, 0.1-6 mM nickel sulfate and 50-100 mM Tris-HCl.

[0014] The molar concentration of nickel sulfate can be selected from 0.1 mM, 0.2 mM, 0.5 mM, 0.8 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, etc., and the molar concentration of Tris-HCl can be selected from 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, etc. Other specific values ​​within the above ranges can be selected, which will not be elaborated here.

[0015] The molar concentration of Tris-HCl refers to the molar concentration of Tris, while HCl is used for pH adjustment.

[0016] The pH of the Tris-HCl is 8.

[0017] Preferably, the tagatose-4-epimerase is added in the form of engineered bacterial cells, and the OD of the bacterial cells in the reaction system... 600 The range is 30-50, for example, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0018] The specific procedure involves mixing and resuspending the engineered bacterial strain's bacterial sludge with the substrate solution until the solution's OD value reaches a certain level. 600 It is 30-50.

[0019] Preferably, the reaction temperature is 60-90℃ and the time is 1-8 h.

[0020] Temperatures can be selected from 60℃, 65℃, 70℃, 75℃, 80℃, 85℃, 90℃, etc., and time can be selected from 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0021] Preferably, the engineered strain includes a strain expressing a fusion protein comprising tagatose-4-epimerase and a protein tag.

[0022] Preferably, the protein tag is a SUMO tag.

[0023] Preferably, the chassis strain of the engineered strain is an Escherichia coli strain.

[0024] Preferably, after the mixing reaction, the process further includes inactivation and centrifugation to collect the supernatant;

[0025] Preferably, the inactivation temperature is 100℃ and the time is 10-15 min, such as 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention, through systematic structural and sequence alignment analysis, successfully identified and screened a novel tagatose-4-epimerase (T4E) with excellent catalytic performance from natural microbial resources. This enzyme exhibits high catalytic efficiency for the conversion of D-fructose to D-tagatose even in its natural state, with activity significantly superior to most known natural T4E enzymes. It provides a natural biocatalyst for the efficient biosynthesis of D-tagatose. This discovery effectively solves the key bottleneck of insufficient core catalyst activity in current enzymatic synthesis of D-tagatose, providing an important enzyme resource and technological foundation for its industrial production. Attached Figure Description

[0028] Figure 1 It is a full-atom structure model of five representative tagatose-4-episodes.

[0029] Figure 2 This is the result of phylogenetic tree construction. Thar-T4E4 in the figure is Bath-T4E of this invention.

[0030] Figure 3 This is the result of agarose gel electrophoresis verification of the PCR products.

[0031] Figure 4 This is an image of the SDS-PAGE results.

[0032] Figure 5 These are the results of the catalytic activity test. Detailed Implementation

[0033] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0034] Example 1

[0035] Structure-guided homology mining of tagatose-4 epimerases

[0036] (1) Using the AlphaFold3 protein structure prediction platform, three-dimensional structural models of five reported representative tagatose-4-epimerases (T4E), including TpT4E, TsT4E, TharT4E, InuzT4E, and DRJ43-T4E, were performed to obtain high-confidence all-atom structure models. Using the above five T4E structures as probes, the Foldseek structure similarity comparison algorithm was used to search databases such as AFDB (AlphaFold ProteinStructure Database) and PDB (Protein Data Bank) to identify six key sites, including metal ion binding, catalysis, and substrate binding groups, and to screen candidate genes.

[0037] (2) The obtained sequences were sequence aligned to screen for candidate genes with less than 60% homology to the probe enzyme, and sequences that were too long or too short were excluded, ultimately narrowing the target to 45. A phylogenetic tree was constructed using MEGA, and four target sequences were finally identified: Thar-T4E3 (Uniprot ID: A0A7C4M980), Thar-T4E4 (Bath-T4EUniprot ID: A0A7C0XER7), Thar-T4E5 (Uniprot ID: A0A7J3F111), and TpT4E27 (Uniprot ID: A0A1V6CDD1).

[0038] Through topological analysis and functional motif conservation assessment, a novel T4E homologous sequence belonging to the phylum Candidatus Bathyarchaeota was identified from the developmental tree and named Bath-T4E, with its amino acid sequence shown in SEQ ID No:1. This sequence possesses a complete catalytic domain and metal ion binding motif in its native state, and exhibits significant sequence differences (sequence similarity <60%) compared to existing characterized T4E enzymes.

[0039] SEQ ID No:1:

[0040] MPAIGIRIPPVFLPGILRAFKLRRTVGTLMLSYGRETAPEYVINAPPGKYEITRGHTGTSIKEYLSLAANYAFTEGVVVELEADHVSVSVSSIAAVKRISGVKSEYEISDEDIDKAL KYIEDEVEEAVSTGVIRFYTLDTCELIRYEADKMDKKDLDAAFEGIDGWKRILERYLDKRFTVIGESGKSYNFRFREEEIKRIAVKYWRSIEVAERVYKIFQEKTPWEFGIEIAFDET PHVTEPKEMLFYLNELWERGIPVDYIAPNVGFEKKKDYSGSLEELYRRVEIMSAIARRYGALLSFHSGSGSTPWTGKGPGVYETLLEATGYKLKYKVSGVYFELLMEIFARQPKGSK ARKLFEDIYDSVIEYVRKEIEKKGPLYSKVMEEQLREYENLVERTKDPYLPWADIFRYYSFLALNLRDEGGERPFRRRRIIELYEEDVALRNVIDREVAELTLRLIDGLDFKDNIDLL.

[0041] Example 2

[0042] Construction of genetically engineered bacteria containing tagatose-4-epimerase fusion expression vector

[0043] A tagatose-4-epimerase expression vector fused with the highly soluble chaperone protein SUMO was constructed and transformed into host cells to achieve efficient soluble expression of Bath-T4E.

[0044] Construction method:

[0045] 1. Primer Design: Specific primers were designed using SnapGene software based on the Bath-T4E sequence and the SUMO vector sequence. The upstream primer for amplifying the Bath-T4E gene is shown in SEQ ID No:2, and the downstream primer is shown in SEQ ID No:3; the upstream primer for amplifying the SUMO gene is shown in SEQ ID No:4, and the downstream primer is shown in SEQ ID No:5.

[0046] SEQ ID No: 2: ATGCCGGCAATCGGTATCCGT.

[0047] SEQ ID No: 3: GTGGTGCTCGAGCAGCAGATCG.

[0048] SEQ ID No: 4: TACCGATTGCCGGCATGGATCCACCAATCTGTTCTCTGTGA.

[0049] SEQ ID No:5:

[0050] GCTGCTCGAGCACCACCACCACCACCACTGACTCGAGGATCCGGCTGCT.

[0051] 2. Preparation of PCR reaction system: Using Bath-T4E gene template (pET-Bath-T4E plasmid) and SUMO gene template (pET-SUMO plasmid) as templates, prepare 50 μL PCR reaction systems respectively: 2×ApexHF FS PCR Master Mix 25 μL, upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL, template DNA (10 μg / mL) 1 μL, DMSO 2.5 μL, and sterile water 19.5 μL.

[0052] 3. PCR reaction procedure:

[0053] Stage 1: Pre-denaturation treatment at 94℃ for 8 min, followed by denaturation at the same temperature for 15 s;

[0054] Phase Two: Includes annealing at 59℃ for 5 seconds and extending at 72℃ for 1 minute and 30 seconds;

[0055] Stage 3: 94℃ denaturation for 15 s, 57℃ annealing for 5 s and 72℃ extension for 1 min 30 s;

[0056] Phase 4: Enter the main amplification phase of 28 cycles, each cycle consisting of annealing at 55°C for 5 s and extension at 72°C for 1 min 30 s;

[0057] Stage 5: Final extension was carried out at 72°C for 8 minutes, and the product was stored at 4°C after the reaction was completed.

[0058] 4. PCR product validation:

[0059] 50 μL of the product was subjected to agarose gel electrophoresis to verify whether the PCR product band size was correct. After verification, the target fragment was cut out and the fragment was recovered using Omega's DNA purification kit (D2500-02). The steps are as follows: Place the gel containing the target DNA band into a 1.5 mL centrifuge tube, add an equal volume of solution PC, and incubate at 50°C for 10 min to completely dissolve the gel; add 500 μL of equilibration buffer BL to the adsorption column CB2, centrifuge at 12000 rpm for 1 min, discard the liquid, and return the adsorption column to the tube; transfer the solution into the adsorption column CB2, centrifuge at 12000 rpm for 1 min, discard the liquid, and place CB2 into a collection tube; add 600 µL of Buffer PW to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid; repeat the above step once; return the adsorption column to the empty collection tube, centrifuge at 12000 rpm for 2 min, and allow the adsorption column to air dry at room temperature; place the adsorption column in a clean 1.5 mL centrifuge tube, add 30 µL of DDH2O to the middle of the adsorption membrane, incubate at room temperature for 2 min, centrifuge at 12000 rpm for 2 min, collect the purified PCR product, and determine the concentration using Nanodrop.

[0060] 5. Construction of the fusion expression vector: The purified linearized vector pET-28a-SUMO was ligated with the Bath-T4E fragment: Ligation system (10 μL): 2× ClonExpress Mix 5 μL, pET-28a-SUMO vector (120 ng) 1 μL, Bath-T4E gene (65 ng) 2 μL, sterile water 2 μL, ligation at 50℃ for 15 min. The ligation solution was transformed into Trelief® 5α competent cells, plated on LB solid medium containing kanamycin, and cultured at 37℃ for 16 h. Single clones were picked for sequencing to verify the correctness of the fusion sequence.

[0061] The agarose gel electrophoresis results of the fusion expression vector are as follows: Figure 3 As shown, pET-28a-SUMO-Bath-T4E-1 and pET-28a-SUMO-Bath-T4E-2 are two parallel samples, and their fusion sequences are shown in SEQ ID No:6.

[0062] SEQ ID No:6:

[0063]

[0064] 6. Transformation of competent cells: Add 1.5 μL of the correctly sequenced pET-28a-SUMO-Bath-T4E plasmid to 50 μL of LE.coli BL21(DE3) competent cells and incubate on ice for 30 min; heat shock at 42℃ for 90 s and incubate on ice for 2 min; add 800 μL of LB liquid medium and revive at 37℃ and 200 rpm for 30 min; take 80 μL of revival solution and spread it on LB solid medium containing 50 μg / mL kanamycin and incubate upside down at 37℃ for 16 h.

[0065] Example 3

[0066] Induction of pET-28a-SUMO-Bath-T4E expression, bacterial cell collection and SDS-PAGE analysis

[0067] Pick one pET-28a-SUMO-Bath-T4E monoclonal antibody and inoculate it into 10 mL of LB liquid medium containing 50 μg / mL kanamycin. Incubate at 37°C and 220 rpm for 8 h. Take 1 mL of the bacterial culture and transfer it to 100 mL of LB liquid medium containing 50 μg / mL kanamycin. Incubate at 37°C and 220 rpm for 2.5 h until the OD value reaches 0.5-0.6. Add IPTG to a final concentration of 0.2 mM and induce incubation at 16°C and 220 rpm for 16 h. Collect the bacterial cells by centrifugation at 4500 rpm for 10 min, wash twice with deionized water, and collect the bacterial cells by centrifugation again.

[0068] Take 4 mL of bacterial culture, centrifuge, and resuspend in 1 mL of buffer (50 mM pH 8.0 Tris-HCl, 5% glycerol, 300 mM NaCl). Sonicate to disrupt the bacterial suspension (100 W, 2 s operation, 4 s interval, total 4 min). Centrifuge at 4℃ and 12000 rpm for 5 min. Collect the supernatant and precipitate for SDS-PAGE electrophoresis (12% separating gel, 5% stacking gel). Results are as follows: Figure 4 As shown, both the supernatant and the precipitate exhibited distinct protein bands at approximately 65 kDa, but the amount of protein in the precipitate was slightly greater than that in the supernatant. Compared to the precipitate without the SUMO tag, the protein solubility was significantly increased. (pET-28a-Bath-T4E was prepared using a pET plasmid without the SUMO tag; the preparation method is described in Example 2.)

[0069] Example 4

[0070] Catalytic activity determination of pET-28a-SUMO-Bath-T4E

[0071] Prepare a mixed substrate solution containing 10 g / L D-fructose, 1.5 mM nickel sulfate, and 50 mM Tris-HCl (pH=8.0). Resuspend an appropriate amount of pET-28a-SUMO-Bath-T4E cells in 1 mL of the above mixed substrate solution to OD200. 600 =50, and the resuspended solution was placed in an 80 ℃ constant temperature water bath for 6 h. After the reaction, it was inactivated in a 100 ℃ water bath for 10 min, and the supernatant was collected by centrifugation at 12000 rpm for 5 min for high performance liquid chromatography analysis (detection conditions and parameters: MARS MCa 10u 300×7.8mm column; RID-20A differential refractive index detector; mobile phase: double deionized water; flow rate: 0.5 mL / min; injection volume: 10 μL; column temperature: 80 ℃). The results are as follows. Figure 5 As shown, by Figure 5 It can be seen that the peak elution time of tagatose is 22 min. Among them, SUMO-Bath-T4E-1 and SUMO-Bath-T4E-2 are two parallel samples, and the final conversion rate of D-tagatose is 22.0%.

[0072] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. The application of a tagatose-4-epimerase in the preparation of D-tagatose, characterized in that, The amino acid sequence of the tagatose-4-epimerase includes the sequence shown in SEQ ID No:1 or a sequence having at least 60% sequence identity with the sequence shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The specific application includes: mixing tagatose-4-epimerase with a substrate solution containing D-fructose to obtain D-tagatose.

3. The application according to claim 2, characterized in that, The concentration of D-fructose in the substrate solution is above 10 g / L.

4. The application according to claim 2 or 3, characterized in that, The substrate solution also includes, in molar concentrations, 0.1-6 mM nickel sulfate and 50-100 mM Tris-HCl.

5. The application according to any one of claims 2-4, characterized in that, The tagatose-4-epimerase was added in the form of engineered bacterial cells, and the OD of the bacterial cells in the reaction system was... 600 It is 30-50.

6. The application according to any one of claims 2-5, characterized in that, The reaction is carried out at a temperature of 60-90℃ for 1-8 hours.

7. The application according to claim 5 or 6, characterized in that, The engineered strains include strains that express fusion proteins containing tagatose-4-epimerase and protein tags.

8. The application according to claim 7, characterized in that, The protein tag is a SUMO tag.

9. The application according to any one of claims 5-8, characterized in that, The chassis strain of the engineered strain is an Escherichia coli strain.

10. The application according to any one of claims 2-9, characterized in that, The mixture reaction also includes inactivation and centrifugation to collect the supernatant. Preferably, the inactivation temperature is 100°C and the time is 10-15 minutes.

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