Preparation method of ergothioneine

Abstract

The invention discloses a preparation method of ergothioneine, which comprises the following steps: adding L-histidine and dimethyl carbonate into a solvent, carrying out oil bath condensation reflux reaction under an alkaline condition to obtain an L-histidine betaine crude liquid, and carrying out vacuum distillation on the L-histidine betaine crude liquid to obtain the ergothioneine. And adding water to a constant volume, adding tNcEgt1 wet thallus, NcEgt2 wet thallus, PLP, FeSO4. 7H2O and Cys into the solution with the constant volume, and reacting to obtain ergothioneine. In the first step, dimethyl carbonate is adopted as a methyl donor to replace expensive adenosylmethionine SAM, after the reaction in the first step is finished, the ergothioneine can be synthesized through catalytic conversion under the condition of high substrate concentration by directly adding an enzyme without crystallization, and the ergothioneine synthetase Egt1 is subjected to truncated expression by combining protein engineering, so that the expression quantity is improved, and meanwhile, the yield of the ergothioneine synthetase Egt1 is increased. The catalytic efficiency is improved, and the conversion efficiency of in-vitro catalysis of 22.5 g / L substrate concentration, 28 DEG C and 24 h is 99% or above.

Description

Technical Field

[0001] This invention belongs to the fields of biochemistry and protein engineering technology, and specifically relates to a method for preparing ergothioneine. Background Technology

[0002] Ergothioneine (EGT) is a unique amino acid derived from the thiohistidine betaine. Its unique redox properties make it one of the best natural antioxidants, exhibiting unique physiological effects on both plants and animals. Ergothioneine is equivalent to a rare vitamin in animals, effectively resisting oxidative stress. Therefore, ergothioneine has great potential as an antioxidant and a valuable nutritional food, with significant applications in the food, cosmetics, and pharmaceutical industries. Although some fungi, actinomycetes, and other microorganisms have the ability to synthesize ergothioneine, the extremely low yields are insufficient for industrial-scale production. Chemical synthesis is prone to racemic problems (mixtures of D- and L-forms), and only the L-form possesses biological activity. Chiral resolution is difficult and costly. Natural extraction yields are extremely low, the process is complex, it is difficult to scale up, and it is greatly affected by season and origin.

[0003] Bio-fermentation synthesis of ergothioneine is currently the mainstream development direction. Recombinant engineered bacteria for ergothioneine synthesis, constructed using Escherichia coli and yeast as substrate microorganisms, have been achieved. However, the expression of many exogenous genes has disrupted the metabolic balance within the microorganisms to some extent, which means that recombinant engineered bacteria still face many challenges in terms of industrial scale-up.

[0004] Patent CN120158408A describes the construction of recombinant engineered bacteria using Escherichia coli as a substrate. After culturing in a 5L fermenter for 68 hours, the yield of ergothioneine can reach 8.7g / L.

[0005] Patent CN120025950A uses Streptomyces freundii as the original strain and ferments it for 96 hours, achieving an ergothioneine fermentation level of 172 mg / L.

[0006] Patent CN120464509A describes the construction of recombinant engineered bacteria using brewer's yeast as a substrate, achieving a yield of 122.5 mg / L in shake flask fermentation and 2540 mg / L in a 10 L fermenter.

[0007] Patent CN118389557B describes the construction of recombinant engineered bacteria using Halomonas sp. LY01 as a substrate, achieving a maximum fermentation level of 230.5 mg / L. Although the recombinant engineered bacteria significantly improves yield compared to some ergothioneine-producing microorganisms, there is still a gap in the low-cost and high-efficiency production of ergothioneine using these strains. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing ergothioneine to solve the problems mentioned in the background art.

[0009] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing ergothioneine, comprising the following steps: S1: Preparation of L-histidine betaine: L-histidine and dimethyl carbonate were added to a solvent and reacted under alkaline conditions by reflux in an oil bath to obtain crude L-histidine betaine solution. S2: Treatment of L-histidine betaine: The crude L-histidine betaine solution was vacuum distilled and then water was added to make up to a fixed volume. S3: Preparation of ergothionein: Add tNcEgt1 wet cells, NcEgt2 wet cells, PLP, FeSO4·7H2O, and Cys to the above-mentioned solution to obtain ergothionein.

[0010] Preferably, the solvent comprises methanol and water, and the volume ratio of methanol to water is 1:0.5~1, preferably 1:1.

[0011] In any of the above schemes, it is preferred that the concentration of L-histidine is 20~35g / L, preferably 30g / L.

[0012] In any of the above schemes, it is preferred that the alkaline condition is NH4OH, and the pH is adjusted to 9-11 by NH4OH, preferably pH=10.

[0013] In any of the above embodiments, it is preferred that the temperature of the oil bath is 50~200℃, preferably 150℃.

[0014] Of any of the above methods, vacuum distillation at 70°C is preferred.

[0015] In any of the above embodiments, the concentration of the tNcEgt1 wet bacterial cell is preferably 8~20 g / L, more preferably 10 g / L; the concentration of the NcEgt2 wet bacterial cell is preferably 12~30 g / L, more preferably 15 g / L; the concentration of pyridoxal phosphate (PLP) is preferably 0.2~1 g / L, more preferably 0.2 g / L; the concentration of FeSO4·7H2O is preferably 5~25 g / L, more preferably 8 g / L; and the concentration of cysteine ​​(Cys) is preferably 15~30 g / L, more preferably 18 g / L.

[0016] In any of the above embodiments, the concentration of L-histidine betaine is preferably 10~30 g / L, more preferably 22.5 g / L, and the pH of the reaction is 7.5~8.5, more preferably pH=8.0.

[0017] In any of the above schemes, it is preferred that, in step S3, the base sequence of tNcEgt1 is as shown in SEQ NO.1, and the base sequence of NcEgt2 is as shown in SEQ NO.2.

[0018] The technical effects and advantages of this invention are as follows: In the first step of the preparation method of ergothioneine, dimethyl carbonate is used as a methyl donor to replace the expensive SAM. After the first step reaction is completed, enzyme can be directly added under high substrate concentration conditions to catalyze the conversion of ergothioneine. By combining protein engineering to truncate the expression of ergothioneine synthase Egt1, the expression level is improved and the catalytic efficiency is enhanced, achieving a conversion efficiency of over 99% in vitro at a substrate concentration of 22.5 g / L, 28°C, and 24 h. Attached Figure Description Figure 1 This is a schematic diagram of the structure of the present invention. Detailed Implementation

[0019] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0020] 1. Preparation of L-histidine betaine (see reaction route below): Using L-histidine as the raw material, methanol and water in a 1:1 volume ratio as the solvent, and dimethyl carbonate as the methyl donor, the reaction was carried out under alkaline conditions in an oil bath at 150°C under reflux. The process was monitored by liquid phase. The concentration of L-histidine was 30 g / L, and the pH was adjusted to 10 with NH4OH.

[0021] .

[0022] 2. Enzyme-catalyzed synthesis of ergothioneine (see reaction route below): After the first step of the L-histidine betaine synthesis reaction is completed, methanol is removed by vacuum distillation at 70℃, and then water is added to make up to a final volume to bring the L-histidine betaine concentration to about 22.5 g / L. Add 10 g / L of wet cell tNcEgt1 enzyme; 15 g / L of wet cell NcEgt2 enzyme; 0.2 g / L of PLP; 8 g / L of FeSO4·7H2O; 18 g / L of Cys; control the pH at approximately 8.0; react at 28℃ for 24 h; and perform liquid chromatography.

[0023]

[0024] The base sequence of tNcEgt1 is as follows:

[0025] The base sequence of NcEgt2 is as follows:

[0026] The method for preparing the enzyme protein is as follows: (1) The two genes tNcEgt1 and NcEgt2 were directly synthesized by Wuhan Sangon Biotech Co., Ltd. and inserted into the multiple cloning site NcoI and EcoRI of the vector pET28a. The pET28a vectors containing the tNcEgt1 and NcEgt2 genes were named pET28a-tNcEgt1 and pET28a-NcEgt2, respectively. (2) The vectors pET28a-tNcEgt1 and pET28a-NcEgt2 were transformed into Escherichia coli competent cells BL21(DE3) to prepare expression strains. The competent cells were purchased from Shanghai Weidi Biotechnology, catalog number EC1002. Chemical transformation was carried out according to the instructions provided on the official website. Finally, the cells were plated on solid LB plates containing kanamycin resistance and incubated overnight at 37°C in a constant temperature incubator. (3) Select a single clone and put it into a 50mL small shake flask containing 20mL LB medium. Incubate at 37℃ and 220r for 12~16h. (4) Transfer the culture from the small shake flask to a 500mL shake flask containing 100mL TB medium at an inoculation rate of 1%, and incubate at 37℃ and 220r for 3-4h. Monitor OD600 during the incubation process. When OD600 reaches 0.6-0.8, add IPTG to make the final concentration 1mM and induce in a shaker at 25℃ and 220r for 12-16h. (5) Centrifuge to collect bacterial cells and weigh them to obtain the wet weight of Escherichia coli cells. Add 10 mL of phosphate buffer to resuspend the cells according to 1 g of cell wet weight, and then sonicate to break them up. The parameters for sonication are set as follows: 400 W on ice, 10 s sonication for 10 s interval, 10 min sonication, and finally centrifugation at 4℃ and 12000 r to remove bacterial residue and obtain crude enzyme solution. Experimental example: 1L system of enzyme-catalyzed reaction: The substrate L-histidine betaine was chemically synthesized. Based on the calculated concentration, 22.5 g of the substrate, 10 g of wet tNcEgt1 cells, and 15 g of wet tNcEgt2 cells were mixed and added to 100 ml of Tris-HCl buffer at pH 8.0. The mixture was then subjected to low-temperature sonication and the supernatant was collected and added to the reaction system. The following were added to the reaction system: PLP 0.2 g, FeSO4·7H2O 8 g, and Cys 18 g. The pH was maintained at approximately 8.0, and the reaction was carried out at 28℃ for 24 h. Liquid chromatography analysis showed a conversion rate of 99.6%.

[0027] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing ergothioneine, characterized in that, Includes the following steps: S1: Preparation of L-histidine betaine: L-histidine and dimethyl carbonate were added to a solvent and reacted under alkaline conditions by reflux in an oil bath to obtain crude L-histidine betaine solution. S2: Treatment of L-histidine betaine: The crude L-histidine betaine solution was vacuum distilled and then water was added to make up to a fixed volume. S3: Preparation of ergothionein: Add tNcEgt1 wet cells, NcEgt2 wet cells, PLP, FeSO4·7H2O, and Cys to the above-mentioned solution to obtain ergothionein.

2. The method for preparing ergothioneine according to claim 1, characterized in that, In step S1, the solvent includes methanol and water, and the volume ratio of methanol to water is 1:0.5~1.

3. The method for preparing ergothioneine according to claim 2, characterized in that, The volume ratio of methanol to water is 1:

1.

4. The method for preparing ergothioneine according to claim 1, characterized in that, In step S1, the concentration of L-histidine is 20~35 g / L.

5. The method for preparing ergothioneine according to claim 1, characterized in that, In step S1, the alkaline condition is NH4OH, and the pH is adjusted to 9-11 by NH4OH.

6. The method for preparing ergothioneine according to claim 1, characterized in that, In step S1, the temperature of the oil bath is 50~200℃.

7. The method for preparing ergothioneine according to claim 1, characterized in that, In step S2, vacuum distillation is performed at 70°C.

8. The method for preparing ergothioneine according to claim 1, characterized in that, In step S3, the concentration of tNcEgt1 wet cells is 8~20 g / L, the concentration of NcEgt2 wet cells is 12~30 g / L, the concentration of PLP is 0.2~1 g / L, the concentration of FeSO4·7H2O is 5~25 g / L, and the concentration of Cys is 15~30 g / L.

9. The method for preparing ergothioneine according to claim 1, characterized in that, 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. The method for preparing ergothioneine according to claim 1, characterized in that, 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.

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