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CN-122012640-A - Preparation method of ergothioneine

CN122012640ACN 122012640 ACN122012640 ACN 122012640ACN-122012640-A

Abstract

The invention discloses a preparation method of ergothioneine, which comprises the steps of adding L-histidine and dimethyl carbonate into a solvent, carrying out oil bath condensation reflux reaction under alkaline conditions to obtain L-histidine betaine crude liquid, carrying out vacuum distillation on the L-histidine betaine crude liquid, adding water to a certain volume, adding tNcEgt wet thalli, ncEgt wet thalli, PLP and FeSO 4 ·7H 2 O, cys into the solution with the certain volume, and carrying out reaction to obtain the ergothioneine. In the first step, dimethyl carbonate is used as methyl donor to replace expensive adenosylmethionine SAM, after the first step, the reaction is finished, the enzyme can be directly thrown under the condition of no crystallization, the ergothioneine can be synthesized by catalytic conversion under the condition of high substrate concentration, the ergothioneine synthase Egt1 is truncated and expressed by combining protein engineering, the catalytic efficiency is improved while the expression quantity is improved, and the conversion efficiency of in vitro catalysis of 22.5g/L substrate concentration, 28 ℃ and 24h and more than 99% is realized.

Inventors

  • HE DINGBING
  • ZHANG MENGRU

Assignees

  • 无锡冰河生物科技有限公司

Dates

Publication Date
20260512
Application Date
20260316

Claims (10)

  1. 1. The preparation method of ergothioneine is characterized by comprising the following steps: S1, preparing L-histidine betaine, namely adding L-histidine and dimethyl carbonate into a solvent, and carrying out oil bath condensation reflux reaction under alkaline conditions to obtain crude L-histidine betaine liquid; S2, treating the L-histidine betaine, namely carrying out vacuum distillation on the crude L-histidine betaine liquid, and then adding water to fix the volume; S3, preparing ergothioneine, namely adding tNcEgt wet thalli, ncEgt wet thalli, PLP and FeSO 4 ·7H 2 O, cys into the constant volume solution to react to obtain the ergothioneine.
  2. 2. The method according to claim 1, wherein in the step S1, the solvent comprises methanol and water, and the volume ratio of methanol to water is 1:0.5-1.
  3. 3. A process for the preparation of ergothioneine as claimed in claim 2, characterised in that the volume ratio of methanol to water is 1:1.
  4. 4. The method for preparing ergothioneine according to claim 1, wherein in step S1, the concentration of L-histidine is 20-35 g/L.
  5. 5. The method for preparing ergothioneine according to claim 1, wherein in step S1, the alkaline condition is NH 4 OH, and NH 4 OH is used to adjust the pH to 9-11.
  6. 6. The method for preparing ergothioneine according to claim 1, wherein in step S1, the temperature of the oil bath is 50-200 ℃.
  7. 7. A process for the preparation of ergothioneine according to claim 1, characterized in that in step S2, it is distilled under vacuum at 70 ℃.
  8. 8. The method according to claim 1, wherein in the step S3, the concentration of tNcEgt.about.1 wet cells is 8-20 g/L, the concentration of NcEgt.about.2 wet cells is 12-30 g/L, the concentration of PLP is 0.2-1 g/L, the concentration of FeSO 4 ·7H 2 O is 5-25 g/L, and the concentration of Cys is 15-30 g/L.
  9. 9. The method for preparing ergothioneine according to claim 1, wherein in step S3, the concentration of L-histidine betaine is 10-30 g/L, and the pH of the reaction is 7.5-8.5.
  10. 10. The method for preparing ergothioneine according to claim 1, wherein in step S3, the base sequence of tNcEgt1 is shown in SEQ NO.1, and the base sequence of NcEgt2 is shown in SEQ NO. 2.

Description

Preparation method of ergothioneine Technical Field The invention belongs to the technical fields of biochemistry and protein engineering, and particularly relates to a preparation method of ergothioneine. Background Ergothioneine (Ergothioneine, abbreviated EGT) is a special amino acid of thiohistidine betaine, and its unique redox properties make it one of the best natural antioxidants, with unique physiological effects on plants and animals. Ergothioneine is equivalent to rare vitamins of animals, and has good resistance to oxidative stress. Therefore, the ergothioneine can be used as an antioxidant and a potential nutritional food, and has great application prospect in the industries of food, cosmetics, medicines and the like. Although some fungi, actinomycetes and other microorganisms have the capability of synthesizing ergothioneine, the extremely low yield is insufficient to meet the requirement of industrial production, racemization problems (a mixture of D type and L type) are easy to generate in chemical synthesis, and only L-type has biological activity, and chiral resolution is difficult and high in cost. The natural extraction yield is extremely low, the process is complex, the scale is difficult, and the influence of seasons and production places is great. The synthesis of ergothioneine by biological fermentation is the current mainstream development direction, and the construction of recombinant engineering bacteria synthesized by ergothioneine by taking escherichia coli and saccharomycetes as chassis microorganisms is realized, but the expression of a plurality of exogenous genes disturbs the metabolic balance inside the microorganisms to a certain extent, so that the recombinant engineering bacteria still face a plurality of challenges in the industrialized amplification direction. The patent CN120158408A uses colibacillus as chassis to construct recombinant engineering bacteria, and the yield of ergothioneine after 68h culture in a 5L fermentation tank can reach 8.7g/L. The patent CN120025950A takes streptomyces fradiae as an original strain, and is fermented and cultured for 96 hours, and the fermentation level of ergothioneine can reach 172mg/L. The patent CN120464509A uses Saccharomyces cerevisiae as a chassis to construct recombinant engineering bacteria, and the shake flask fermentation yield reaches 122.5 mg/L, and the 10L fermentation tank yield 2540 mg/L. Patent CN118389557B uses Halomonas sp.LY01 as chassis to construct recombinant engineering bacteria, and the highest fermentation level is 230.5mg/L. Although the yield of the recombinant engineering bacteria is greatly improved compared with that of some ergothioneine-producing microorganisms, the strains are used for producing the ergothioneine at low cost and high efficiency, and a certain gap is still provided. Disclosure of Invention The invention aims to provide a preparation method of ergothioneine, which aims to solve the problems in the background technology. In order to achieve the purpose, the invention provides the following technical scheme that the preparation method of the ergothioneine comprises the following steps: S1, preparing L-histidine betaine, namely adding L-histidine and dimethyl carbonate into a solvent, and carrying out oil bath condensation reflux reaction under alkaline conditions to obtain crude L-histidine betaine liquid; S2, treating the L-histidine betaine, namely carrying out vacuum distillation on the crude L-histidine betaine liquid, and then adding water to fix the volume; S3, preparing ergothioneine, namely adding tNcEgt wet thalli, ncEgt wet thalli, PLP and FeSO 4·7H2 O, cys into the constant volume solution to react to obtain the ergothioneine. Preferably, the solvent comprises methanol and water, and the volume ratio of the methanol to the water is 1:0.5-1, preferably 1:1. In any of the above embodiments, the concentration of L-histidine is preferably 20 to 35g/L, more preferably 30g/L. In any of the above schemes, preferably, the alkaline condition adopts NH 4 OH, and NH 4 OH adjusts the pH to 9-11, preferably ph=10. In any of the above embodiments, the temperature of the oil bath is preferably 50 to 200 ℃, preferably 150 ℃. In any of the above schemes, vacuum distillation at 70 ℃ is preferred. In any of the above embodiments, preferably, the concentration of tNcEgt.about.1 wet cells is 8 to 20g/L, preferably 10g/L, the concentration of NcEgt wet cells is 12 to 30g/L, preferably 15g/L, the concentration of pyridoxal phosphate (PLP) is 0.2 to 1g/L, preferably 0.2g/L, the concentration of FeSO 4·7H2 O is 5 to 25g/L, preferably 8g/L, and the concentration of cysteine (Cys) is 15 to 30g/L, preferably 18g/L. In any of the above embodiments, the concentration of the L-histidine betaine is preferably 10 to 30g/L, preferably 22.5g/L, and the pH of the reaction is preferably 7.5 to 8.5, preferably pH=8.0. In any of the above embodiments, it is preferable that in step S3, the base sequence of tNcEgt