Method for synthesizing ergothioneine based on chemical-enzyme method
The one-pot synthesis of ergothioneine using a chemical-enzymatic method solves the problems of low yield and high cost in existing technologies, achieving efficient and simplified ergothioneine production with a significant increase in yield.
Patent Information
- Application Number
- CN202510928196.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-07
- Publication Date
- 2025-11-14
Smart Images
Figure CN120944982A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for synthesizing ergothioneine based on a chemical-enzymatic process, belonging to the field of biotechnology. Background Technology
[0002] Ergothioneine (ERG) is a unique amino acid derivative of thiohistidine betaine, and its unique redox properties make it one of the best natural antioxidants. Ergothioneine is mainly synthesized by microorganisms such as fungi and actinomycetes, and it has unique physiological effects on plants and animals. Ergothioneine is equivalent to a rare vitamin in animals and has a good inhibitory effect on oxidative stress. Therefore, ergothioneine has great potential as an antioxidant and a nutritional food, and has significant application prospects in the food, cosmetics, and pharmaceutical industries.
[0003] Currently, the main methods for synthesizing ergothioneine include chemical synthesis, extraction, and microbial fermentation. These methods suffer from drawbacks such as long production cycles and complex extraction processes, hindering large-scale industrial production. Chemical synthesis of ergothioneine often fails to achieve the correct chirality, and bio-extraction capacity is insufficient. Bio-fermentation synthesis has become the mainstream development direction. However, although ergothioneine synthases derived from bacteria such as *Mycobacterium smegmatis* and fungi such as *Neurospora crassa* and *Trichoderma reesei* have been successfully elucidated, the yield of heterologous microbial synthesis of ergothioneine remains generally low. The direct precursor histidine betaine limits the synthesis efficiency of ergothioneine; even with the addition of excessive amino acids, the highest reported yield is only 9.3 g / L. Production costs remain high, and the production cycle is long, far from reaching the potential for industrial scale-up. Summary of the Invention
[0004] [Technical Issues]
[0005] Existing technologies for producing ergothioneine result in low yields, high production costs, and long production cycles.
[0006] [Technical Solution]
[0007] This invention provides a one-pot synthesis method for ergothioneine based on a chemical-enzymatic approach, aiming to bypass the rate limitations of traditional histidine betaine synthesis and provide a highly efficient ergothioneine synthesis system. L-histidine is synthesized as L-histidine betaine via iodomethane methylation under alkaline conditions, followed by the direct synthesis of ergothioneine with the addition of histidine betaine sulfonase and a sulfur donor. The intermediate processes eliminate the need for product separation and extraction, and the required enzyme solution does not require purification, simplifying the experimental steps and reducing production costs. Furthermore, the catalytic efficiency is further improved through truncation modification and point mutagenesis.
[0008] This invention provides a method for synthesizing ergothioneine, which utilizes chemical synthesis and histidine betaine sulfonase catalysis to synthesize ergothioneine using histidine, iodomethane, and potassium polysulfide as substrates.
[0009] In one embodiment of the present invention, the chemical synthesis uses L-histidine as a raw material, methanol and water as solvents, iodomethane as a methylating agent, and reacts at room temperature under alkaline conditions to prepare histidine betaine. The reaction formula is as follows:
[0010]
[0011] In one embodiment of the present invention, the molar ratio of iodomethane to L-histidine in the chemical synthesis reaction system is 3:(0.75-1.2), preferably 3:1.
[0012] In one embodiment of the present invention, the chemical synthesis time is 12-18 hours, preferably 16 hours.
[0013] In one embodiment of the present invention, the volume ratio of water to methanol in the chemical reaction system is 2:(1-2), preferably 2:1.
[0014] In one embodiment of the present invention, the alkaline environment is an environment containing an alkaline substance; the alkaline substance includes one or more of potassium carbonate, sodium hydroxide, potassium hydroxide, and ammonia water, preferably sodium hydroxide; the molar ratio of iodomethane to the alkaline substance in the reaction is 1:(2-3.5), preferably 3.
[0015] In one embodiment of the present invention, potassium polysulfide is added as a sulfur donor to the enzyme catalytic system, and is added appropriately during the reaction.
[0016] In one embodiment of the present invention, the buffer solution in the reaction system is 50-100 mM Tris-HCl, preferably 100 mM, and the pH is 7.0-8.0, preferably 7.4.
[0017] In one embodiment of the present invention, the enzyme catalytic reaction temperature is 20–40°C and the catalytic time is 1–10 h.
[0018] In one embodiment of the present invention, the histidine betaine sulfonase comprises the EanB enzyme or a mutant thereof with the amino acid sequence shown in SEQ ID NO:1.
[0019] In one embodiment of the present invention, the mutant is: based on the histidine betaine thiolase shown in SEQ ID NO:1, the first to third amino acids at the N-terminus are truncated, and any of the following mutations are performed:
[0020] (1) Mutate the 97th phenylalanine to alanine to obtain F97A;
[0021] (2) The threonine at position 387 was mutated to methionine to obtain T387M;
[0022] (3) Mutate the glutamic acid at position 277 to lysine to obtain E277K;
[0023] (4) Mutate the asparagine at position 354 to glutamine to obtain N354Q;
[0024] (5) Mutate the serine at position 335 to lysine to obtain S335K;
[0025] (6) Mutate the phenylalanine at position 441 to serine to obtain F441S;
[0026] (7) Mutate phenylalanine at position 97 to alanine, threonine at position 387 to methionine, and glutamic acid at position 277 to lysine to obtain F97A / T387M / E277K.
[0027] (8) Mutate phenylalanine at position 97 to alanine, threonine at position 387 to methionine, glutamic acid at position 277 to lysine, asparagine at position 354 to glutamine, phenylalanine at position 441 to serine, and serine at position 335 to lysine to obtain F97A / T387M / E277K / N354Q / F441S / S335K.
[0028] In one embodiment of the present invention, the histidine betaine sulfonase or its mutant is expressed using recombinant microbial cells; the host of the recombinant microbial cells includes Escherichia coli, Pichia pastoris or Saccharomyces cerevisiae, and the expression vector includes pET28a(+), pAO815 or pYC16.
[0029] In one embodiment of the present invention, the recombinant microbial cells are fermented in a culture medium, and OD 600 When the concentration reaches 0.6–1.0, add 0.2–0.5 mM IPTG and induce expression at 25–30 °C for 10–15 hours. Collect recombinant microbial cells for processing to obtain the catalyst. Add the catalyst to a reaction system containing histidine betaine and potassium polysulfide and catalyze at pH 7.2–7.4 and 20–40 °C for at least 1 hour.
[0030] In one embodiment of the present invention, the catalyst comprises the lysate of the recombinant microbial cells, or purified histidine betaine sulfonase or a mutant thereof.
[0031] In one embodiment of the present invention, the catalytic activity of the histidine betaine sulfonase or its mutant in the reaction system is 10000-30000 U / L.
[0032] In one embodiment of the present invention, the mass ratio of histidine betaine to potassium polysulfide in the reaction system is 1:(0.8-1.5).
[0033] This invention also protects the use of the method in the preparation of products containing ergothioneine.
[0034] This invention provides a histidine betaine sulfonase mutant, which, based on the histidine betaine sulfonase shown in SEQ ID NO:1, has its N-terminal amino acids 1-32 truncated and possesses any of the following mutations:
[0035] (1) Mutate the 97th phenylalanine to alanine to obtain F97A;
[0036] (2) The threonine at position 387 was mutated to methionine to obtain T387M;
[0037] (3) Mutate the glutamic acid at position 277 to lysine to obtain E277K;
[0038] (4) Mutate the asparagine at position 354 to glutamine to obtain N354Q;
[0039] (5) Mutate the serine at position 335 to lysine to obtain S335K;
[0040] (6) Mutate the phenylalanine at position 441 to serine to obtain F441S;
[0041] (7) Mutate phenylalanine at position 97 to alanine, threonine at position 387 to methionine, and glutamic acid at position 277 to lysine to obtain F97A / T387M / E277K.
[0042] (8) Mutate phenylalanine at position 97 to alanine, threonine at position 387 to methionine, glutamic acid at position 277 to lysine, asparagine at position 354 to glutamine, phenylalanine at position 441 to serine, and serine at position 335 to lysine to obtain F97A / T387M / E277K / N354Q / F441S / S335K.
[0043] In one embodiment of the present invention, the histidine betaine sulfonase is derived from Chlorobium limicola, GenBank number ACD90218.1.
[0044] This invention provides a gene encoding the mutant.
[0045] The present invention provides a recombinant vector that expresses the mutant or carries the gene.
[0046] The present invention provides recombinant microbial cells expressing the mutant or carrying the recombinant vector.
[0047] In one embodiment of the present invention, the expression host of the recombinant microbial cell is Escherichia coli, and pET28a(+) is used as the expression vector.
[0048] This invention provides a recombinant Escherichia coli, using pET-28a(+) as a vector to recombinantly express the mutant.
[0049] This invention provides a method for preparing histidine betaine sulfonase, wherein the recombinant microbial cells or the recombinant Escherichia coli are cultured in a culture medium for a period of time, and the expression of histidine betaine sulfonase is induced by IPTG as an inducer.
[0050] In one embodiment of the present invention, the concentration of the inducer is 0.4 to 0.6 mM.
[0051] In one embodiment of the present invention, after induction, the recombinant Escherichia coli is cultured at 16–25°C.
[0052] In one embodiment of the present invention, the cell lysate of the mutant or the recombinant Escherichia coli is added to a reaction system containing histidine betaine and catalyzed at 25–35°C.
[0053] The present invention also provides the application of the mutant or the recombinant Escherichia coli in the synthesis of ergothionein, wherein the cell lysate of the mutant or the recombinant Escherichia coli is added to a reaction system containing histidine betaine and potassium polysulfide, and catalyzed at 20-40°C for at least 1 hour.
[0054] The present invention also protects the use of the mutant, the recombinant microbial cell, or the recombinant Escherichia coli in the synthesis of ergothioneine or ergothioneine-containing products.
[0055] [Beneficial Effects]
[0056] 1. This invention provides a method for synthesizing ergothioneine. Using histidine as the starting substrate, ergothioneine is synthesized by a combination of chemical and enzymatic methods. The intermediate process does not require product separation and extraction, and the required enzyme solution does not need to be purified, which simplifies the experimental steps and reduces production costs. After 5 hours of reaction with histidine betaine thiosulfate enzyme EanB, the ergothioneine conversion rate reaches 60%, and the yield is 21 g / L.
[0057] 2. This invention constructed histidine betaine sulfonase EanB mutants F97A, T387M, E277K, N354Q, F441S, S335K, F97A / T387M / E277K, and F97A / T387M / E277K / N354Q / F441S / S335K. Compared with the truncated mutant Δ32EanB, the mutants exhibited the highest relative enzyme activity of 523.6±4.4%, significantly improving the catalytic level of histidine betaine sulfonase EanB. When these mutants were used to prepare ergothioneine, the conversion rate reached 95% after 5 hours of reaction, with a yield of 33.8 g / L. Attached Figure Description
[0058] Figure 1 This is a schematic diagram of ergothioneine synthesis.
[0059] Figure 2 This is a diagram showing the optimization of EanB enzyme fermentation and reaction conditions.
[0060] Figure 3 This is a schematic diagram of the construction of the EanB enzyme truncated mutant.
[0061] Figure 4 This shows the protein expression of wild-type EanB and Δ32EanB truncated mutant.
[0062] Figure 5 This is a diagram showing the results of single-point mutations and saturation mutations in the EanB enzyme.
[0063] Figure 6 This is a graph showing the results of chemical synthesis of histidine betaine using different methyl donors.
[0064] Figure 7 This is a graph showing the conversion rate of wild-type EanB enzyme and mutant EanB in the chemical reaction solution for the synthesis of ergothionein. Detailed Implementation
[0065] 1. Escherichia coli BL21(DE3), pET28a(+) were preserved in the laboratory.
[0066] 2. Plasmid construction reagents and sequencing verification were all purchased and completed at Shanghai BioBio Biotechnology Co., Ltd.
[0067] 3. All analytical grade reagents were purchased from Sinopharm Group.
[0068] 4. Culture medium:
[0069] LB medium: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract.
[0070] TB medium, 2.31 g / L KH2PO4, 12.54 g / L K2HPO4, 12 g / L tryptone, 24 g / L yeast extract, 4 mL / L glycerol.
[0071] EanB enzyme activity is defined as the amount of enzyme required to synthesize 1 μM ergothioneine per hour at 30°C.
[0072] The enzyme activity assay for EanB enzyme was performed as follows: The catalytic system consisted of 1 mL of a combination of 1 mM histidine, histidine betaine, 0.5 g / L potassium polysulfide, and 100 μL of enzyme, which was then brought to a final volume of 1 mL with Tris-HCl (pH 7.4) buffer. The reaction was carried out at 30 °C for 1 h. After the reaction was completed, the reaction was terminated by incubating in a water bath for 10 min. The reaction solution was then centrifuged and the ergothionein yield was determined using high-performance liquid chromatography (HPLC).
[0073] Ergothionein detection method: An Agilent 1260 HPLC system was used, with a C18 column (column temperature 30℃, detection wavelength 257nm), a mobile phase of 10% methanol, and an injection flow rate of 0.7 mL / min. -1 The injection volume was 5 μL, and the single sample detection time was 18 min.
[0074] Histidine betaine detection method: Take 1 mL of reaction solution, dilute it appropriately, and add 1 mL of EanB enzyme catalysis system to control the concentration of histidine betaine at 50-100 mg / L. Add 200 μL of EanB enzyme solution and control the concentration of potassium polysulfide at 5 g / L. React at 30℃ for 3 h. After the reaction is completed, stop the reaction by water bath for 10 min. Detect the produced ergothionein according to the ergothionein detection method. Calculate the histidine betaine in the reaction solution based on a 100% conversion rate.
[0075] Example 1: Heterologous expression of EanB enzyme and acquisition of crude enzyme solution
[0076] The ergothionein thiocyanate enzyme EanB (GenBank: ACD90218.1) gene was synthesized by Tianlin Biotechnology Co., Ltd. after codon optimization. Its nucleotide sequence is shown in SEQ ID NO:2. This sequence was linked to the vector pET-28a(+), with the EanB gene insertion site located between the restriction enzyme sites BamHI and HindIII, resulting in the recombinant plasmid pET-28a(+)-EanB. The synthesized plasmid was then transformed into Escherichia coli BL21(DE3) to obtain recombinant strains expressing the EanB enzyme.
[0077] Shake-flask fermentation: The recombinant *E. coli* strain was streaked onto a plate containing kanamycin (50 μg / L). Single colonies were picked and inoculated into LB medium. After incubation at 37°C for 8-12 h, the seed culture was transferred to 50 mL of TB medium at a volume fraction of 1 mL / 50 mL. The initial inoculum size was 2 × 10⁻⁶. 9 After incubating for 2 hours, 0.5 mM IPTG was added and the cells were induced at 25°C for 13 hours. After induction, the bacterial cells were collected in 50 mL centrifuge tubes, centrifuged at 8000 rpm for 10 minutes at 4°C, the supernatant was discarded, and the cells were resuspended in 50 mL of pH 7.5, 20 mM Tris-HCl buffer. The cells were then sonicated for approximately 25 minutes, and the lysate was centrifuged at 8000 rpm for 20 minutes at 4°C. The precipitate was discarded, and the supernatant was the crude enzyme solution. Enzyme activity testing showed that the EanB crude enzyme solution had an activity of only 12 U / L.
[0078] Example 2: Optimization of EanB enzyme fermentation and investigation of optimal reaction conditions
[0079] Microbial fermentation is a key technology for the large-scale production of enzymes, and its yield and activity directly affect the feasibility of its industrial application. Therefore, based on Example 1, experiments were conducted to investigate the optimal fermentation temperature and IPTG induction concentration of EanB enzyme. Following the principle of single variable, the catalytic activity of EanB was investigated at IPTG induction concentrations of 0.1 mM, 0.2 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.8 mM, and 1 mM, and at induction culture temperatures of 16℃, 20℃, 25℃, 30℃, and 37℃. Simultaneously, the reaction temperature and pH are directly related to the catalytic efficiency. Enzyme activity of EanB was measured, and following the principle of single variable, the temperature and pH in the enzyme activity detection method were adjusted to investigate the catalytic activity of EanB at reaction temperatures of 25℃, 28℃, 30℃, 35℃, and 37℃, and pH ranges of 6.8–8.4. The results are as follows: Figure 2 As shown, the optimal induction culture temperature for EanB enzyme is 25℃, and the final concentration of IPTG should be 0.4–0.6 mM; its optimal reaction pH range is 7.2–7.8, and the catalytic temperature is 20–30℃.
[0080] Example 3: Truncated modification of EanB enzyme
[0081] Given the low enzyme activity of EanB crude enzyme solution, in order to improve enzyme activity, such as... Figure 3As shown, the first 5, 10, 15, 16, 20, 25, 30, 32, 34, 36, 44, and 49 amino acids of the N-terminus were truncated. The PCR primers are listed in Table 1. Taking the mutant Δ32 with truncated N-terminus 32 as an example, the recombinant plasmid pET-28a(+)-EanB obtained in Example 3 was used as a template. Using 32-F and ΔEanB-R as primers, the target plasmid fragment pET-28a(+)-Δ32-EanB was amplified by reverse PCR. Homologous recombination was performed and the fragment was transformed into Escherichia coli BL21(DE3). Positive clones were screened and sent for testing. Sequencing confirmed that the mutant recombinant Escherichia coli BL21(DE3) / pET28a-Δ32-EanB was obtained.
[0082] Table 1 Primer sequences for constructing the EanB truncated mutant
[0083]
[0084]
[0085] The recombinant E. coli that was constructed was cultured in shake flasks and its enzyme activity was measured according to the method in Example 1. The results are shown in Table 2. The enzyme activity of the Δ32 mutant was significantly increased to 295.5±9.4 U / L, and the enzyme activity was also measured based on the protein electrophoresis results. Figure 4 It is evident that the EanB protein expression level in the △32 mutant was significantly increased compared to the initial strain.
[0086] Table 2 Enzyme activity of truncated mutants of EanB
[0087] name Enzyme activity (U / L) △5 13.4±1.1 △10 14.7±0.7 △15 16.6±0.5 △16 19.8±0.6 △20 112.6±4.8 △25 186.8±12.4 △30 231.6±17.6 △32 295.5±9.4 △34 209.9±7.7 △36 46.7±2.9 △44 42.9±1.2 △49 0.62±0.13
[0088] Example 4: Single-point mutations and saturation mutations of EanB enzymes
[0089] The Δ32-EanB enzyme was subjected to a single-point mutation. Using pET28a(+)-Δ32-EanB as the plasmid template and F97A-F and F97A-R as primers, the corresponding mutant plasmid fragment was amplified by PCR. The product was ligated and transformed into E. coli BL21(DE3). The transformed strain was correctly sequenced, yielding the mutant strain pET28a(+)-Δ32-EanB-F97A. Other mutant strains were obtained using the same method with the PCR primers listed in Table 3. The mutant strains were subjected to shake-flask fermentation according to the method in Example 1, and the enzyme activities were measured. The enzyme activities of F97A, T387M, and E277K were increased by 60%, 110%, and 50%, respectively, compared with the truncated mutant Δ32EanB. Figure 5 (Table 4).
[0090] The Δ32-EanB enzyme was subjected to saturation mutagenesis at three sites: 335, 354, and 441. The PCR primers are listed in Table 3. The enzyme activities of N354Q, F441S, and S335K were increased by 58%, 40%, and 32%, respectively, compared to Δ32EanB. Figure 5 (Table 4).
[0091] Following the methods described above, the effects of other single mutations on EanB enzyme activity, based on the truncation of amino acids 1-32, were also investigated. Figure 5 The results showed that mutants K357D, F123C, V70D, R136D, V183C, S132V, N156C, D140L, R67H, N190C, E103D, P255R, A409C, A259Y, A161C, R360C, Y375R, R266W, K303L, E171D, E394H, S400M, A105F, E333C, G263A, S236D, D54F, C339S, N283V, K405D, T234R, and N354Q could not achieve a significant increase in enzyme activity.
[0092] All sites were subjected to superposition mutations. Taking the construction of F97A / T387M / E277K / N354Q / F441S / S335K as an example, pET28a(+)-△32-EanB was used as the plasmid template, and F97A-F and F97A-R were used as primers for PCR. The PCR product was transformed into E. coli BL21(DE3) through homologous recombination. After obtaining the transformants, the plasmid pET28a(+)-F97A-△32-EanB was correctly obtained by sequencing. nB; then using plasmid pET28a(+)-F97A-△32-EanB as a template, PCR was performed using T387M-F and T387M-R primers. The PCR product was transformed into E. coli BL21(DE3) via homologous recombination. After obtaining transformants, the plasmid pET28a(+)-T387M / F97A-△32-EanB was correctly obtained through sequencing; then pET28a(+)-T387M / F97A-△32-EanB Using the plasmid as a template, PCR was performed with E277K-F and E277K-R primers. The PCR product was transformed into *E. coli* BL21(DE3) via homologous recombination. After obtaining the transformants, the plasmid pET28a(+)-T387M / F97A / E277K-△32-EanB was correctly obtained through sequencing. Then, using the PCR product as a template, PCR was performed with N354Q-F and N354Q-R primers. PCR was performed using primers 41S-F and F441S-R; then, using the PCR product as a template, PCR was performed again using primers S335K-F and S335K-R to obtain the mutant plasmid. The product was ligated by homologous recombination and transformed into Escherichia coli BL21(DE3). After obtaining the transformant, it was correctly sequenced, and the mutant strain pET28a(+)-△32-EanB-F97A / T387M / E277K / N354Q / F441S / S335K was obtained.
[0093] The recombinant Escherichia coli that was constructed was cultured in shake flasks and its enzyme activity was measured according to the method in Example 1. The results are shown in Table 4. The enzyme activity of each mutant strain in the fermentation broth was significantly improved compared with that of the mutant recombinant Escherichia coli BL21(DE3) / pET28a-Δ32-EanB. Among them, the enzyme activity of mutants F97A / T387M / E277K / N354Q / F441S / S335K was 523.6±4.4% of that of Δ32-EanB, which significantly improved its catalytic level.
[0094] Table 3 Primer sequences for constructing the EanB amino acid mutant
[0095]
[0096]
[0097] Table 4. Relative enzyme activities of the EanB amino acid mutants
[0098]
[0099] Note: The relative enzyme activity of the EanB enzyme mutant is the percentage of the mutant's activity compared to the truncated mutant's activity of Δ32EanB enzyme.
[0100] Example 5: Chemical-enzymatic synthesis of ergothionein
[0101] 1. Screening of methyl donors for the chemical synthesis of histidine betaine
[0102] like Figure 1 As shown, histidine betaine was produced using L-histidine as a substrate in the presence of sodium hydroxide, methanol, and a methyl donor, serving as the substrate for the subsequent synthesis of ergothioneine. In a 1L reactor, 31g of L-histidine and 36g of sodium hydroxide were dissolved in 300mL of water, and 150mL of methanol was added. The reactor was cooled to 0℃, and a methyl donor three times the amount of histidine was added. After the addition, the reactor was shaken at 30℃ for 16 hours to ensure complete consumption of the methyl donor. The concentration of histidine betaine was determined by taking a sample after the reaction, serially diluting the reaction solution, and completely converting it to ergothioneine under EanB enzyme catalysis. The yield of histidine betaine was calculated by detecting the ergothioneine yield using liquid chromatography. Calculations showed that iodomethane, dimethyl carbonate, and dimethyl sulfate all produced high yields, with iodomethane as the substrate yielding a histidine betaine yield of 38g / L. Figure 6 Therefore, iodomethane was chosen as the methyl donor for subsequent use.
[0103] 2. Scale-up synthesis of histidine betaine
[0104] In a 1L reactor, 47g of L-histidine and 72g of sodium hydroxide were dissolved in 300mL of water, followed by the addition of 150mL of methanol. The reactor was then cooled to 0℃, and 85.5g of iodomethane was added. After the addition was complete, the reactor was shaken at 30℃ until the histidine betaine concentration no longer increased (14–16 h). The histidine betaine concentration was determined by taking a sample after the reaction, serially diluting the reaction solution, and completely converting it to ergothioneine under EanB enzyme catalysis. The histidine betaine yield was calculated by detecting the ergothioneine yield using liquid chromatography. The calculated histidine betaine yield was 77g / L, with a conversion rate of 93%. The pH of the reaction solution was adjusted to 7.2 with hydrochloric acid and used directly for subsequent catalytic reactions.
[0105] The original strain pET-28a(+)-EanB and the mutant strain pET28a(+)-△32-EanB-F97A / T387M / E277K / N354Q / F441S / S335K from Example 4 were cultured in shake flasks and the cells were lysed according to the method in Example 1. The crude enzyme solution was collected for the reaction. After pH adjustment of the above reaction solution, a reaction solution with a final histidine betaine concentration of 31 g / L, a final enzyme activity of 25000 U / L for wild-type EanB and △32F97A / T387M / E277K / N354Q / F441S / S335K-EanB enzyme mutants, and a final concentration of 40 g / L of potassium polysulfide was prepared, with a total volume of 0.5 L. The reaction was carried out at 30 °C for 5 h. During the reaction, 1 mL of the reaction solution was taken and serially diluted and measured. The results were obtained from... Figure 7 It can be seen that the conversion rate of ergothionein was 60% and the yield was 21 g / L after 5 h of catalysis by wild-type EnaB; while the conversion rate of ergothionein reached 95% and the yield was 33.8 g / L after 5 h of catalysis by mutant EanB.
[0106] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A method for synthesizing ergothioneine, characterized in that, Ergothionein was synthesized using a combination of chemical and enzymatic methods, starting with histidine as the substrate. The chemical method used L-histidine and a methyl donor as substrates, methanol and water as solvents, and histidine betaine as the substrate in an alkaline environment at 20–35 °C. The enzymatic method used histidine betaine sulfonase as the catalyst and histidine betaine and potassium polysulfide as substrates to catalyze the synthesis of ergothionein.
2. The method according to claim 1, characterized in that, The methyl donor includes one or more of iodomethane, dimethyl carbonate, methyl p-toluenesulfonate, dimethyl sulfate, and betaine; the alkaline environment is an environment containing alkaline substances; the alkaline substances include one or more of ammonia, sodium hydroxide, potassium hydroxide, and potassium carbonate.
3. The method according to claim 2, wherein in the chemical method, the volume ratio of water to methanol is 2:(1-2); the molar ratio of methyl donor to L-histidine is 3:(1-2); and the molar ratio of methyl donor to basic substance is 1:(2-3.5).
4. The method according to claim 3, wherein in the chemical method, the reaction lasts for at least 1 hour.
5. The method according to claim 4, characterized in that, The histidine betaine sulfonase comprises the EanB enzyme or a mutant thereof with the amino acid sequence shown in SEQ ID NO:1; the mutant is: based on the histidine betaine sulfonase shown in SEQ ID NO:1, the N-terminal amino acids 1-32 are truncated, and it has any of the following mutations: (1) Mutate the 97th phenylalanine to alanine; (2) Mutate the threonine at position 387 to methionine; (3) Mutate the glutamic acid at position 277 to lysine; (4) Mutate the asparagine at position 354 to glutamine; (5) Mutate serine at position 335 to lysine; (6) Mutate the phenylalanine at position 441 to serine; (7) Mutate phenylalanine at position 97 to alanine, threonine at position 387 to methionine, and glutamic acid at position 277 to lysine. (8) Mutate phenylalanine at position 97 to alanine, threonine at position 387 to methionine, glutamic acid at position 277 to lysine, asparagine at position 354 to glutamine, phenylalanine at position 441 to serine, and serine at position 335 to lysine.
6. The method according to claim 5, characterized in that, The histidine betaine sulfonase or its mutant is expressed in recombinant microbial cells; the host of the recombinant microbial cells includes Escherichia coli, Pichia pastoris or Saccharomyces cerevisiae, and the expression vector includes pET28a(+), pAO815 or pYC16.
7. The method according to claim 6, characterized in that, The recombinant microbial cells were fermented in a culture medium, and OD... 600 When the concentration reaches 0.6–1.0, add 0.2–0.5 mM IPTG, induce expression at 25–30 °C for 10–15 hours, collect recombinant microbial cells for processing, and obtain the catalyst; The catalyst was added to a reaction system containing histidine betaine and potassium polysulfide, and catalyzed at pH 7.2–7.4 and 20–40°C for at least 1 hour. The catalyst includes the lysate of the recombinant microbial cells, or purified histidine betaine sulfonase or its mutant.
8. The application according to claim 7, characterized in that, In the reaction system, the catalytic activity of the histidine betaine sulfonase or its mutant is 10000-30000 U / L.
9. The application according to claim 8, characterized in that, In the reaction system, the mass ratio of histidine betaine to potassium polysulfide is 1:(0.8-1.5).
10. The use of the method according to any one of claims 1 to 9 in the preparation of products containing ergothioneine.