Eanb thioesterase with ergothioneine synthesis activity and application thereof

Abstract

The application discloses a high-activity EanB sulfur transferase, a coding gene thereof and application of the high-activity EanB sulfur transferase in biosynthetic synthesis of ergothioneine, and belongs to the technical field of bioengineering; in view of the problems of low catalytic efficiency of an existing EanB enzyme and insufficient tolerance of a substrate and a sulfur donor, the application provides an EGT-ST7 sulfur transferase with an amino acid sequence as shown in SEQ ID NO:1 and a coding gene thereof, constructs a recombinant expression vector and an engineering bacterium, and establishes a catalytic synthesis method of ergothioneine under anaerobic conditions with histidine betaine as a substrate and inorganic polysulfide as a sulfur donor; the specific activity of the enzyme is 5.4 times that of a control enzyme, the catalytic efficiency kcat / Km is increased by 32.1 times, the tolerance to high-concentration substrates and polysulfides is significantly enhanced, and the enzyme can be adapted to various catalytic forms such as a purified enzyme and a resting cell, and is compatible with a chemical synthesis substrate pretreatment and ion exchange purification process, thereby providing a new functional gene and a technical scheme for efficient biological manufacturing of ergothioneine.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to an EanB thiotransferase with ergothioneine synthesis activity and its applications. Background Technology

[0002] Ergothioneine (EGT) is a natural sulfur-containing histidine derivative with biological functions such as antioxidation and cell protection, and has application value in food, cosmetics, nutritional health products, and pharmaceuticals. The human body cannot usually synthesize ergothioneine on its own and mainly relies on exogenous intake; therefore, establishing a stable, economical, and scalable ergothioneine preparation technology is of great significance.

[0003] Existing ergothioneine preparation technologies mainly include natural extraction, chemical synthesis, microbial fermentation, and enzymatic biocatalysis. Natural extraction methods are limited by the source of raw materials and the content of the target product, and usually suffer from low yield and high separation and purification costs. Chemical synthesis methods often involve multiple reaction steps and have high requirements for selectivity and post-processing. Although microbial fermentation has the advantages of biomanufacturing, it may still be constrained by factors such as product titer, substrate utilization, fermentation cycle, and downstream purification costs.

[0004] EanB-type thiotransferases can catalyze the production of ergothioneine under oxygen-independent conditions, using histidine betaine as the acceptor substrate and inorganic polysulfides as the sulfur donor. Compared with synthetic routes that rely on oxygen or organic sulfur sources, the EanB route has the advantages of simpler reaction components, milder reaction conditions, and lower sulfur donor costs.

[0005] However, EanB enzymes are known to have limitations in heterologous expression, substrate tolerance, sulfur donor tolerance, and high-load catalysis. In particular, enzyme activity tends to decrease at higher concentrations of histidine betaine or polysulfides, thus limiting the product concentration and production efficiency of ergothioneine. When using whole-cell or resting-cell catalysis, the soluble expression of the enzyme, cell integrity, and transmembrane mass transfer of substrate and sulfur donor also affect the actual conversion efficiency.

[0006] Therefore, there is a need in the field to obtain an EanB thiotransferase with higher intrinsic catalytic performance, stronger substrate affinity, better tolerance to inorganic polysulfides, and suitability for heterologous expression, and to further establish enzyme catalysis or whole-cell catalysis processes adapted to this enzyme. Summary of the Invention

[0007] To address the shortcomings of the existing technologies, this invention aims to provide an EanB thiotransferase with ergothionein synthesis activity and its applications, solving problems such as low catalytic efficiency, insufficient substrate and sulfur donor tolerance, decreased conversion rate under high substrate load, and insufficient heterologous expression and whole-cell catalytic adaptability of known EanB thiotransferases.

[0008] To solve the above problems, the present invention adopts the following technical solution: In a first aspect, the present invention provides an EanB thiotransferase having ergothioneine synthesis activity, the amino acid sequence of which is shown in SEQ ID NO:1.

[0009] Furthermore, the N-terminus of the EanB thiotransferase is fused with a solubilizing tag via a flexible linker peptide, and the solubilizing tag is either TrxA or SlyD.

[0010] In a second aspect, the present invention provides a nucleic acid molecule encoding the EanB thiotransferase, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0011] Thirdly, the present invention provides a recombinant expression vector comprising the aforementioned nucleic acid molecule.

[0012] Fourthly, the present invention provides a recombinant host cell comprising the aforementioned nucleic acid molecule or the aforementioned recombinant expression vector, wherein the host cell is Escherichia coli BL21(DE3).

[0013] Fifthly, the present invention provides a biocatalyst for the synthesis of ergothioneine, wherein the biocatalyst is the EanB thiotransferase, an enzyme preparation containing the thiotransferase, a recombinant host cell expressing the thiotransferase, or a resting cell prepared from the recombinant host cell.

[0014] Sixthly, the present invention provides a method for biocatalytic synthesis of ergothioneine, wherein histidine betaine, inorganic polysulfides and the aforementioned biocatalyst are added to a reaction system and a catalytic reaction is carried out under anaerobic conditions to convert histidine betaine into ergothioneine.

[0015] Furthermore, the inorganic polysulfide is sodium polysulfide (Na₂S). x Where x is 2-8; the inorganic polysulfides are added in a pulsed manner to maintain the concentration of free polysulfides in the reaction system at 15 mM; the initial concentration of histidine betaine in the reaction system is 13-45 g / L, the reaction pH is 7.0-8.5, the reaction temperature is 20-35℃, the reaction time is 1-36 hours, and the reaction is carried out in 50 mM Tris-HCl buffer.

[0016] Furthermore, when resting cells are used as biocatalysts, the cell density of resting cells is OD. 600 The concentration was 20-50, and 0.1-0.5% (w / v) of the cell membrane permeability agent Triton X-100 was added to the reaction system.

[0017] Furthermore, the histidine betaine is derived from a chemical synthesis solution. Before the reaction, the chemical synthesis solution is pretreated by neutralizing it to pH 7.0-8.0, removing organic solvents and water, reconstituted with a reaction buffer, and then filtered. After the reaction, cells and proteins are removed by centrifugation or ultrafiltration, and the target product is separated by a strong acid cation exchange resin. After washing with water to desalt, eluting with ammonia, concentration, crystallization, and drying, ergothioneine product is obtained.

[0018] The beneficial effects of this invention are as follows: The thiotransferase provided by this invention offers a new functional gene resource for ergothioneine biomanufacturing; compared with the control Cl-EanB-WT, the purified enzyme specific activity of the thiotransferase of this invention is increased by about 5.4 times, kcat by about 6.1 times, Km by about 5.3 times, and kcat / Km by about 32.1 times, indicating that its substrate affinity and intrinsic catalytic efficiency are significantly improved; it maintains high conversion capacity even at high histidine betaine concentrations, with conversion rates reaching 75.6% and 69.1% under substrate conditions of 30 g / L and 45 g / L, respectively, showing high substrate loading adaptability; it has strong tolerance to sodium polysulfide, retaining approximately 85% and 60% residual activity after treatment with 30 mM and 50 mM Na2S5, respectively, which is superior to the control enzyme and is beneficial for high-load reactions and sulfur donor fed-batch processes. Attached Figure Description

[0019] Figure 1 A schematic diagram of the reaction route for the EGT-ST7 thiotransferase to ergothioneine from histidine betaine.

[0020] Figure 2 Figures showing ergothionein yields of EGT-ST7 and Cl-EanB-WT at different substrate concentrations.

[0021] Figure 3 The effect of different sulfur donor addition methods on ergothionein production is shown in the figure.

[0022] Figure 4 The HPLC and mass spectrometry chromatograms show the identification of ergothionein. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments.

[0024] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention.

[0025] Example 1: Cloning of the EGT-ST7 encoding gene and construction of recombinant expression engineered bacteria 1. Experimental Materials Cloning host E. coli DH5α, expression host E. coli BL21(DE3), expression vector pET-28a(+), restriction endonucleases, T4 DNA ligase, plasmid extraction kit, gel extraction kit, kanamycin, IPTG, LB medium, etc.

[0026] 2. Experimental Methods Based on the EGT-ST7 amino acid sequence shown in SEQ ID NO:1, codon optimization was performed using E. coli codon preference to synthesize the coding nucleic acid sequence shown in SEQ ID NO:2, with NcoI and XhoI restriction sites introduced at both ends of the gene, respectively.

[0027] SEQ ID NO:1: MHKIRNISTRELRDHTEITTDSLLALLGSEKVKIELKALLGNPDVKIIDVRSTEAYFGYRENGEPVGGRIKGAISLPRSLLSNPNFLDIVKEKGIKPEDTIILYGYTPEESLE VASFFKSHGYTKVYIYPDYLPEWTGDPSFPREKVPNYERNIPAEWVKGIINGEEVDYYDGDKYLVVHAYYKNKSAYLSGHIPGSVPLDLTDLVSPETLRPKSPAELRAALEAH GIAADTTVILYGTWLNPDPAEALPLSKAGWMVAVRAAWVLRWAGVKDVRVLNGGLQAWVDAGFPLSTEDVPRTPVADFGAEIPANPELAVDLPEAKAMLKDPDALVVDVVPEA VYVGEVSGSPFLTRKGRIPGAVHFPNGKDAYTVDNLTNPDGTGFRDPQYIKEEAAKKGITPDKRIATYIVGVSGSVFAFQLLLLGWPNVSVAVGGLYEWFSDPNNPYLTGVPK.

[0028] SEQ ID NO:2:

[0029] The synthesized EGT-ST7 gene fragment and the pET-28a(+) vector were double-digested with NcoI and XhoI, respectively, to induce T7 promoter control. After gel purification, the fragments were ligated overnight at 16°C using T4 DNA ligase. The ligation product was transformed into E. coli DH5α competent cells and plated on LB agar plates containing 50 μg / mL kanamycin, and incubated overnight at 37°C. Single colonies were picked for colony PCR verification. After sequencing confirmation, the recombinant plasmid was extracted and named pET-28a-EGT-ST7.

[0030] The recombinant plasmid pET-28a-EGT-ST7 was chemically transformed into Escherichia coli BL21(DE3) competent cells, plated on LB agar plates containing 50 μg / mL kanamycin, and cultured at 37°C for 12 h to obtain the recombinant engineered strain BL21(DE3) / pET-28a-EGT-ST7.

[0031] Positive single clones were picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C with shaking at 220 rpm until OD (occurrence limit) was reached. 600 When the concentration reached 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and expression was induced at 16℃ and 180 rpm for 18 h. After induction, the cells were collected by centrifugation at 8000 rpm for 10 min, and washed twice with PBS buffer at pH 7.2.

[0032] The bacterial cells used for enzyme purification experiments were homogenized under high pressure, centrifuged at 12,000 rpm for 20 min at 4°C to collect the supernatant, purified the target protein using a Ni-NTA affinity chromatography column, and obtained purified EGT-ST7 enzyme solution after dialysis of the elution buffer. The protein concentration was determined by the BCA method.

[0033] Bacterial cells used for resting cell catalysis: directly resuspended in reaction buffer to prepare a resting cell suspension.

[0034] 3. Experimental Results A recombinant expression vector carrying the EGT-ST7 gene and engineered Escherichia coli were successfully constructed. Soluble expression of the target protein can be obtained after low-temperature induction. Electrophoretically pure EGT-ST7 protein can be obtained after Ni-NTA affinity purification, which can be used for subsequent enzymatic characterization and catalytic reactions.

[0035] Example 2: Comparison of transformation performance of EGT-ST7 and control EanB enzymes at gradient substrate concentrations 1. Experimental Materials Purified EGT-ST7 enzyme solution; control enzyme Cl-EanB-WT (derived from Chlorobium limicola, amino acid sequence as shown in SEQ ID NO:3, nucleotide as shown in SEQ ID NO:4), prepared using the same vector, host, and induction expression purification conditions as EGT-ST7; histidine betaine standard, disodium pentasulfide (Na2S5), 50 mM Tris-HCl buffer (pH 7.5), anaerobic workstation.

[0036] SEQ ID NO:3: MQNKNFRAPQSEAIGILYKLIETGSKHKNMYDHTEITTDSLLALLGSEKVKIIDVRSADAYNGWRMRGEVRGGHIKGAKSLPAKWLTDPEWLNIVRFKQIRPEDAIVLYGYTPE ECEQTATRFKENGYNNVSVFHRFHPDWTGNDAFPMDRLEQYNRLVPAEWVNGLISGEEIPEYDNDTFIVCHAHYRNRDAYLSGHIPGATDMDTLALESPETWNRRTPEELKKALE EHGITASTTVVLYGKFMHPDNADEFPGSAAGHIGAIRLAFIMMYAGVEDVRVLNGGYQSWTDAGFAISKDDVPKTTVPEFGAPIPSRPEFAVDIDEAKEMLQSEDSDLVCVRSY PEYIGEVSGYNYIKKKGRIPGAIFAECGSDAYHMENYRNHDHTTREYHEIEDIWAKSGIIPKKHLAFYCGTGWRGSEAWFNALLMGWPRVSVYDGGWFEWSNDPENPYETGVPK.

[0037] SEQ ID NO:4:

[0038] 2. Experimental Methods The publicly available EanB from Chlorobium limicola was selected as the control enzyme Cl-EanB-WT, whose amino acid sequence is shown in SEQ ID NO:3. Both enzymes were expressed and purified using the same vector, host, and induction conditions. The catalytic conversion abilities of the two enzymes at different substrate concentrations were investigated. The reaction route for the production of ergothionein is as follows: Figure 1 As shown.

[0039] The reaction system (1 mL) consisted of 50 mM Tris-HCl buffer (pH 7.5), a final concentration of purified enzyme of 0.5 mg / mL, a final concentration of Na2S5 of 20 mM, and final concentrations of histidine betaine of 5 g / L, 13 g / L, 20 g / L, 30 g / L, and 45 g / L, respectively.

[0040] The reaction system was placed in an anaerobic workstation and reacted at 30°C in the dark for 24 h. After the reaction was completed, an equal volume of methanol was added to terminate the reaction. The supernatant was collected by centrifugation at 12,000 rpm for 10 min, and the ergothioneine content was detected by HPLC to calculate the substrate conversion rate.

[0041] 3. Experimental Results The yield and conversion rates of ergothioneine for the two enzymes at gradient substrate concentrations are shown in Table 1 and 2. Figure 2 As shown, the conversion rate of wild-type Cl-EanB-WT decreased significantly with increasing substrate concentration, and it essentially lost its high-load conversion ability at a substrate concentration of 45 g / L; while the EGT-ST7 of this invention still achieved conversion rates of 75.6% and 69.1% under high substrate conditions of 30 g / L and 45 g / L, respectively, demonstrating excellent adaptability to high substrate loading.

[0042] Table 1. Comparison of conversion performance of EGT-ST7 and Cl-EanB-WT at gradient substrate concentrations

[0043] Example 3: Determination of specific activity and kinetic parameters of purified enzyme 1. Experimental Materials Purified EGT-ST7 and Cl-EanB-WT enzyme solution, histidine betaine, Na2S5, 50 mM Tris-HCl buffer (pH 7.5).

[0044] 2. Experimental Methods In a 50 mM Tris-HCl buffer system at 30℃ and pH 7.5, using 5 g / L histidine betaine as the substrate and 20 mM Na2S5 as the sulfur donor, the amount of ergothioneine produced after 5 min of reaction was measured, and the initial reaction rate was calculated. One unit of enzyme activity (U) was defined as the amount of enzyme required to produce 1 μmol of ergothioneine per minute, and the specific activity (U / mg) of the purified enzyme was calculated in conjunction with protein concentration.

[0045] A series of histidine betaine concentrations (0.05–5 mM) were set up to determine the initial reaction rates at different substrate concentrations. The Michaelis equation was used for nonlinear fitting, and the Michaelis constant K was calculated. m Catalytic constant kcat and catalytic efficiency kcat / K m .

[0046] 3. Experimental Results The specific activities and kinetic parameters of the two enzymes are shown in Table 2. Compared with the wild-type control enzyme, the EGT-ST7 of this invention exhibits a 5.4-fold increase in specific activity and a lower Kc. m A higher kcat value significantly enhances substrate affinity and catalytic conversion efficiency, resulting in a substantial improvement in overall catalytic efficiency (kcat / K). m It reached 32.1 times that of the control enzyme.

[0047] Table 2. Comparison of enzyme kinetic parameters between EGT-ST7 and Cl-EanB-WT

[0048] Example 4: Evaluation of EGT-ST7's tolerance to inorganic polysulfides 1. Experimental Materials Purified EGT-ST7 and Cl-EanB-WT enzyme solutions, Na2S5 solutions of different concentrations, and 50 mM Tris-HCl buffer (pH 7.5).

[0049] 2. Experimental Methods The two enzymes were diluted to a final concentration of 0.5 mg / mL with 50 mM Tris-HCl buffer (pH 7.5), and Na2S5 was added to a final concentration of 5, 10, 20, 30, 40 and 50 mM respectively. The solutions were then incubated at 30°C in the dark for 1 h.

[0050] After incubation, the treated enzyme solution was added to a standard reaction system (5 g / L HER, 20 mM Na2S5, pH 7.5, 30℃) to determine the residual enzyme activity; the residual activity percentage at different concentrations was calculated with the enzyme activity without Na2S5 incubation as 100%.

[0051] 3. Experimental Results The tolerance results of the two enzymes to different concentrations of Na2S5 are shown in Table 3 and Figure 3 As shown, with increasing Na2S5 concentration, the activity of wild-type enzymes decreases sharply, with residual activity of less than 20% at 30 mM; while the EGT-ST7 of this invention still retains about 85% residual activity after treatment with 30 mM Na2S5, and retains about 60% activity at 50 mM, demonstrating significantly enhanced tolerance to high concentrations of inorganic polysulfides and making it suitable for catalytic processes with high-load sulfur donor feedstock.

[0052] Table 3. Comparison of sulfur donor tolerance between EGT-ST7 and Cl-EanB-WT

[0053] Example 5: Attribution Analysis of the Contribution of Intrinsic Enzyme Properties to Process Optimization 1. Experimental Materials Recombinant engineered bacteria EGT-ST7 and Cl-EanB-WT, corresponding to engineered bacteria with N-terminal TrxA solubilizing tag, histidine betaine chemical synthesis solution, Na2S5, Tris-HCl buffer.

[0054] 2. Experimental Methods To differentiate the contributions of enzyme molecule modification and process optimization to catalytic performance enhancement, Cl-EanB-WT and EGT-ST7 were subjected to identical process optimization conditions, including: N-terminal fusion of TrxA solubilizing tag, pulsed addition of sulfur donor, pretreatment of chemically synthesized substrates, uniform control of resting cell density, and uniform control of reaction endpoint. The catalytic performance of the two was compared under the same conditions.

[0055] Soluble expression level: Under the same induction conditions, the concentration of soluble target protein in the lysed supernatant was determined.

[0056] Catalytic performance: Using 30 g / L and 45 g / L HER as substrates, the EGT yield was determined after 24 h of reaction in the same resting cell system.

[0057] Specific activity: After removing the solubilizing tag, the purified protein was measured for its specific activity.

[0058] Sulfur donor tolerance: Residual activity was determined after incubation with 30 mM Na2S5 for 1 h.

[0059] 3. Experimental Results Table 4 shows the performance comparison results of the two enzymes under the same complete process conditions. Even with identical process optimization methods, the performance improvement of wild-type Cl-EanB-WT is still very limited and cannot support high substrate loading reactions; while the EGT-ST7 of this invention shows orders of magnitude advantages in soluble expression level, product concentration, specific activity and sulfur donor tolerance, proving that the performance improvement mainly comes from the intrinsic functional improvement brought about by the modification of the enzyme molecule itself, rather than relying solely on external process optimization.

[0060] Table 4. Performance Comparison of Cl-EanB-WT and EGT-ST7 under Optimized Process Conditions

[0061] Example 6: Pretreatment and resting cell-catalyzed transformation of chemically synthesized histidine betaine 1. Experimental Materials L-histidine, iodomethane, sodium hydroxide, hydrochloric acid, EGT-ST7 resting cell suspension, Na2S5, Tris-HCl buffer, freeze dryer, 0.22 μm filter membrane.

[0062] 2. Experimental Methods Chemical synthesis of histidine betaine: Histidine betaine was synthesized by methylation reaction of L-histidine and iodomethane in an alkaline aqueous solution. After the reaction, HPLC analysis showed that the HER titer in the reaction solution was 74.3 g / L, and the pH of the reaction solution was 12.5.

[0063] Pretreatment of the synthesis solution: Take 100 mL of the above chemical synthesis solution and slowly add 6 M hydrochloric acid dropwise under stirring in an ice-water bath to adjust the pH to 7.5; after neutralization, the solution is freeze-dried to remove water and residual organic solvents to obtain a solid crude product; redissolve in 50 mM Tris-HCl buffer (pH 7.5) to make the HER concentration approximately 50 g / L, and filter through a 0.22 μm filter membrane for later use.

[0064] Resting cell catalysis: Pretreated HER was added to a 10 mL reaction system to a final concentration of 13 g / L. Na2S5 was added using a pulsed addition method to maintain the free polysulfide concentration of the system at approximately 15 mM. EGT-ST7 resting cells were then added to the OD level. 600 The concentration was 30, and the reaction was carried out under anaerobic conditions at 30℃ and 150 rpm for 24 h.

[0065] Set up a control group: The reaction system was prepared directly using unneutralized pH 12.5 chemical synthesis solution, with all other conditions remaining the same.

[0066] 3. Experimental Results The reaction was carried out using pretreated chemically synthesized substrates. After 24 h, the ergothioneine concentration reached 12.2 g / L, with an apparent yield of approximately 93.8%. In the control group, the enzyme was rapidly inactivated upon direct addition of the strongly alkaline synthesis solution, and no ergothioneine formation was detected. The results indicate that pretreatment involving neutralization, desolvation, and resolvation filtration effectively eliminates the adverse effects of the chemically synthesized solution on the enzyme, achieving a good integration between the chemical substrate and the biocatalytic system.

[0067] Example 7: Effect of solubilizing tags on the soluble expression and catalytic performance of EGT-ST7 1. Experimental Materials pET-28a-EGT-ST7 plasmid, TrxA gene fragment, flexible linker peptide coding sequence, E. coli BL21(DE3), SDS-PAGE electrophoresis reagent, Ni-NTA purification reagent.

[0068] 2. Experimental Methods Two expression plasmids were constructed: pET-28a-EGT-ST7 (tagless) and pET-28a-TrxA-EGT-ST7 (with a TrxA tag fused to the N-terminus, the tag being linked to the target protein via a (Gly4Ser)3 flexible linker peptide). Both plasmids were transformed into E. coli BL21(DE3) and induced for 18 h at 0.5 mM IPTG and 16 °C.

[0069] Equal amounts of bacterial cells were collected, and after sonication, the supernatant (soluble component) and precipitate (inclusion body component) were separated. SDS-PAGE electrophoresis was used for analysis, and grayscale scanning was used to quantify the content of the target protein in the supernatant. The soluble expression levels of the two constructs were compared.

[0070] The two proteins were purified by Ni-NTA and adjusted to the same concentration (1 mg / mL). They were then reacted under anaerobic conditions of 5 g / L HER, 20 mM Na2S5, pH 7.5, and 30℃ for 12 h. The amount of ergothionein produced was measured, and the catalytic effect was compared.

[0071] 3. Experimental Results SDS-PAGE grayscale quantification results showed that the soluble protein expression level of TrxA-EGT-ST7 was approximately 3.2 times that of untagged EGT-ST7. Catalytic activity assays showed that, based on 12 h ergothioneine production, the apparent catalytic effect of the TrxA fusion protein was approximately 1.5 times that of the untagged protein. These results indicate that the N-terminal fusion of a TrxA solubilizing tag significantly enhances the soluble expression level of EGT-ST7, thereby increasing the catalytic activity per unit volume of bacterial cells.

[0072] Example 8: HPLC quantification and mass spectrometry identification of the reaction product ergothioneine 1. Experimental Materials Ergothioneine standard, high performance liquid chromatograph, liquid chromatography-mass spectrometry, C18 reversed-phase column, methanol, formic acid (chromatographic grade).

[0073] 2. Experimental Methods HPLC quantification: Chromatographic conditions included a C18 column (4.6 mm × 250 mm, 5 μm), a mobile phase of 0.1% formic acid aqueous solution-methanol (95:5, v / v), a flow rate of 1.0 mL / min, a column temperature of 30℃, a detection wavelength of 254 nm, and an injection volume of 20 μL. A series of concentration standards were prepared to construct a standard curve, and the reaction samples were then quantified.

[0074] Mass spectrometry identification: Electrospray ionization (ESI) positive ion mode was used, with a scanning range of m / z 100~300, and the characteristic ion peaks of the standard and the reaction sample were compared.

[0075] 3. Experimental Results See Figure 4 (The upper curve represents ergothioneine, and the lower curve represents the sample.) The retention time of the target chromatographic peak in the reaction sample is completely consistent with that of the ergothioneine standard. Mass spectrometry analysis shows that the quasi-molecular ion peak of the target peak in the sample is m / z 230.1 [M+H]. + The results showed a perfect match with the standard quality spectrum characteristics, confirming that the catalytic reaction product was ergothioneine.

[0076] Example 9: Separation and purification process of ergothioneine catalytic reaction solution 1. Experimental Materials Catalytic reaction solution, ultrafiltration membrane with a molecular weight cutoff of 10 kDa, strongly acidic cation exchange resin, ammonia, anhydrous ethanol, and vacuum concentration device.

[0077] 2. Experimental Methods Clarification pretreatment: The reaction solution was centrifuged at 8000 rpm for 15 min to remove bacterial cells. The supernatant was then subjected to 10 kDa ultrafiltration membrane to remove soluble proteins and macromolecular impurities, resulting in a clarified crude liquid.

[0078] Cation exchange separation: The clarified crude liquid was loaded onto a strongly acidic cation exchange resin column, eluted with deionized water to remove inorganic salt impurities, and then eluted with 1 M ammonia water to collect the eluent containing ergothioneine.

[0079] Concentration, crystallization and drying: The eluent was concentrated under reduced pressure to a ergothioneine concentration of about 200 g / L, 3 times the volume of anhydrous ethanol was added, and the mixture was allowed to stand at 4°C for 12 h to crystallize. The crystals were collected by filtration and dried under vacuum at 60°C to obtain the final product.

[0080] 3. Experimental Results The above process can yield a white crystalline ergothioneine product from the catalytic reaction solution, with a purity of over 98.5% and a total separation yield of approximately 82%.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. An EanB thiotransferase with ergothioneine synthesis activity, characterized in that, The amino acid sequence of the thiotransferase is shown in SEQ ID NO:

1.

2. The EanB thiotransferase according to claim 1, characterized in that, The N-terminus of the EanB thiotransferase is fused with a solubilizing tag via a flexible linker peptide, and the solubilizing tag is either TrxA or SlyD.

3. A nucleic acid molecule encoding the EanB thiotransferase as described in claim 1 or 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

2.

4. A recombinant expression vector, characterized in that, It includes the nucleic acid molecule as described in claim 3.

5. A recombinant host cell, characterized in that, The present invention comprises the nucleic acid molecule of claim 3 or the recombinant expression vector of claim 4, wherein the host cell is Escherichia coli BL21(DE3).

6. A biocatalyst for the synthesis of ergothioneine, characterized in that, The biocatalyst is the EanB thiotransferase as described in claim 1 or 2, an enzyme preparation containing the thiotransferase, a recombinant host cell expressing the thiotransferase, or a resting cell prepared from the recombinant host cell.

7. A method for biocatalytic synthesis of ergothioneine, characterized in that, Histidine betaine, inorganic polysulfides, and the biocatalyst described in claim 6 are added to the reaction system, and a catalytic reaction is carried out under anaerobic conditions to convert histidine betaine into ergothioneine.

8. The method for biocatalytic synthesis of ergothioneine according to claim 7, characterized in that, The inorganic polysulfide is sodium polysulfide (Na₂S). x Where x is 2-8; the inorganic polysulfides are added in a pulsed manner to maintain the concentration of free polysulfides in the reaction system at 15 mM; the initial concentration of histidine betaine in the reaction system is 13-45 g / L, the reaction pH is 7.0-8.5, the reaction temperature is 20-35℃, the reaction time is 1-36 hours, and the reaction is carried out in 50 mM Tris-HCl buffer.

9. The method for biocatalytic synthesis of ergothioneine according to claim 7, characterized in that, When resting cells are used as biocatalysts, the cell density of resting cells is OD. 600 The concentration was 20-50, and 0.1-0.5% (w / v) of the cell membrane permeability agent Triton X-100 was added to the reaction system.

10. The method for biocatalytic synthesis of ergothioneine according to claim 7, characterized in that, The histidine betaine is derived from a chemical synthesis solution. Before the reaction, the chemical synthesis solution is pretreated by neutralizing it to pH 7.0-8.0, removing organic solvents and water, redissolving it with a reaction buffer, and then filtering it. After the reaction, cells and proteins are removed by centrifugation or ultrafiltration, and the target product is separated by a strong acid cation exchange resin. After washing and desalting with water, eluting with ammonia, concentration, crystallization, and drying, ergothioneine product is obtained.

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