Process for the preparation of ergothioneine and use of mercapto compounds therein

Abstract

The application provides a preparation method of ergothioneine and application of a mercapto compound in the method. The method comprises the following steps: in the presence of a catalyst system, a sulfur oxide is reacted to generate ergothioneine. The catalyst system comprises a first catalyst and a second catalyst. The first catalyst is ergothioneine synthase, and the second catalyst is one or more of a mercapto-containing amino acid and a mercapto-containing polypeptide. The preparation method of ergothioneine provided by the application does not need to use a large amount of a chemical mercaptan reagent with a foul smell, has the advantages of high efficiency, green environmental protection, and friendly production scene, and is beneficial to industrial production of ergothioneine.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology. Specifically, this invention relates to a method for preparing ergothioneine and the application of thiol compounds therein. Background Technology

[0002] Ergothioneine is an amino acid containing a thiol group, isolated from ergot by Tanret in 1909. As a highly bioactive thiol reducing agent, ergothioneine effectively captures high-energy oxidative intermediates within cells, preventing cellular damage from oxidants. Its unique redox properties make it one of the best natural antioxidants. Human cells have identified a transport protein (OrganicCation / Ergothioneine Transporter 1, OCTN1) that specifically absorbs ergothioneine, and ergothioneine has been found to accumulate to varying degrees (100 μM to 2 mM) in different tissues and cells, indicating that ergothioneine likely plays an important physiological role in the human body. Currently, ergothioneine is approved for use in cosmetics and food, and has broad potential for future growth.

[0003] Currently, ergothioneine synthesis mainly involves chemical synthesis, natural extraction (from fungal mycelium), fermentation using genetically engineered strains, and enzymatic catalysis. Chemical synthesis suffers from drawbacks such as long pathways, low conversion rates, and significant pollution. Natural extraction is limited by the low ergothioneine content in mycelium. Introducing the ergothioneine biosynthetic pathway into model strains (such as *Escherichia coli*, *Yarrowia lipolytica*, or *Saccharomyces cerevisiae*) and then using bio-fermentation to synthesize ergothioneine using the modified strains is an ideal method. However, due to limitations imposed by the host cell's regulatory system, the yield of fermented ergothioneine is limited to the gram level, making large-scale production difficult. In vitro enzymatic catalysis can overcome the limitations of the host cell's regulatory system and synthesize high concentrations of the target product. However, due to limited understanding of the enzymatic catalytic mechanisms involved in ergothioneine biosynthesis, no high-conversion-rate enzymatic catalytic methods have been reported to date.

[0004] Previous research has found that, such as Figure 1As shown, the CS bond in sulfur oxide 2 is cleaved by EgtE (or Egt2) to generate pyruvate and ergothioneine. In verifying the enzyme catalytic mechanism of EgtE or Egt2 (Sci. Rep. 2015, 5, 11870 and Cell Chemical Biology 2018, 25, 519), it was found that the presence of dithiothreitol (DTT) in the reaction system promotes the synthesis of ergothioneine. Based on this, it is speculated that DTT participates in the synthesis of ergothioneine as a reducing agent, possibly responsible for reducing intermediate 6 after the CS bond cleavage to ergothioneine, that is, reducing the 0-valent sulfur atom in 6 to the -2 valent in ergothioneine. In the above reaction process, to ensure the complete conversion of sulfur oxide to ergothioneine, an equivalent level of DTT needs to be added, generating the corresponding oxidized DTT as a byproduct of the reaction. Since the market price of DTT is high, the large-scale addition of DTT will increase the production cost of ergothioneine. In addition, if mercaptoethanol is used to replace DTT in the production of ergothioneine, the volatile odor of mercaptoethanol requires additional environmental protection equipment to control odor leakage and treat reaction wastewater, which will increase the company's equipment investment. Summary of the Invention

[0005] The purpose of this invention is to provide a simple reaction system, highly efficient catalytic and environmentally friendly method for preparing ergothioneine, as well as the application of thiol compounds therein.

[0006] In a first aspect of the present invention, a method for preparing ergothioneine is provided, the method comprising the following steps:

[0007] In the presence of a catalyst system, sulfur oxides are reacted to yield ergothioneine;

[0008] The catalyst system includes a first catalyst and a second catalyst, wherein the first catalyst is ergothionein synthase and the second catalyst is one or more of a thiol-containing amino acid and a thiol-containing polypeptide.

[0009] The structural formula of the sulfur oxide is shown in Formula I:

[0010]

[0011] The structural formula of the ergothionein is shown in Formula II or Formula III:

[0012]

[0013] In another preferred embodiment, the thiol-containing amino acid is selected from any one or a combination of the following groups: L-cysteine, homocysteine.

[0014] In another preferred embodiment, the thiol-containing polypeptide is reduced glutathione.

[0015] In another preferred embodiment, the concentration of the sulfur oxide is 1-100 mM, preferably 9-75 mM.

[0016] In another preferred embodiment, the concentration of the ergothioneine synthase is 1 × 10⁻⁶. -3 -1mM, preferably 0.02-0.2mM.

[0017] In another preferred embodiment, the concentration of the second catalyst is 1-100 mM, preferably 1-5 mM or 50-100 mM.

[0018] In another preferred embodiment, the molar ratio of the second catalyst to the sulfur oxide is 1:1-200, preferably 1:1-100, for example 1:1-5 or 1:50-100.

[0019] In another preferred embodiment, the reaction is carried out at a pH of 7.0-9.0, preferably at pH 8.0.

[0020] In another preferred embodiment, the reaction is carried out in a KPi buffer solution with a pH of 7.0-9.0, preferably with a pH of 8.0.

[0021] In another preferred embodiment, the reaction is carried out at 25-37°C, preferably 30°C.

[0022] In another preferred embodiment, the reaction time is 2-24 hours, preferably 2-16 hours.

[0023] In another preferred embodiment, the ergothioneine synthase is Egt2 and / or EgtE.

[0024] In another preferred embodiment, the ergothioneine synthase is selected from EgtE derived from Mycobacterium smegmatis or trEgt2 derived from Trichoderma reesei.

[0025] In another preferred embodiment, the Egt2 is derived from Trichoderma reesei, with GeneBank accession number XP_006968735.1.

[0026] In another preferred embodiment, the reaction is carried out in the presence of a regenerated system of a second catalyst.

[0027] In another preferred embodiment, when the reaction is carried out in the presence of a regenerated system of a second catalyst, the molar ratio of the second catalyst to the sulfur oxide is 1:50-200, preferably 1:50-100, for example 1:75.

[0028] In another preferred embodiment, when the reaction is carried out in the presence of a regeneration system without a second catalyst, the molar ratio of the second catalyst to the sulfur oxide is 1:1-5, preferably 1:1-2, for example 1:1.

[0029] In another preferred embodiment, the second catalyst regeneration system includes: a reductase for regenerating the second catalyst, and a cofactor for enabling the reductase to exert its catalytic activity.

[0030] In another preferred embodiment, the reductase is selected from any one or a combination of the following groups: glutathione reductase, cystine reductase.

[0031] In another preferred embodiment, the glutathione reductase (Gsr) is a glutathione reductase (EC 1.8.1.7 or EC 1.6.4.2) obtained by PCR using the genome of Escherichia coli as a template.

[0032] In another preferred embodiment, the cystine reductase is designated as EC 1.6.4.1 or EC 1.8.1.6.

[0033] In another preferred embodiment, the cofactor is selected from any one or a combination of the following groups: NADPH, NADH.

[0034] In another preferred embodiment, the concentration of the reductase is 1 × 10⁻⁶. -3 -1mM, preferably 0.01-0.1mM.

[0035] In another preferred embodiment, the concentration of the cofactor is 0.5-2 mM, preferably 1 mM.

[0036] In another preferred embodiment, the reaction is carried out in the presence of a cofactor regeneration system.

[0037] In another preferred embodiment, the cofactor regeneration system includes: a hydrogen-donating substrate and a dehydrogenase that uses the hydrogen-donating substrate to regenerate the cofactor.

[0038] In another preferred embodiment, the hydrogen-donating substrate is glucose, and the dehydrogenase is glucose dehydrogenase.

[0039] In another preferred embodiment, the hydrogen-donating substrate is isopropanol, and the dehydrogenase is isopropanol dehydrogenase.

[0040] In another preferred embodiment, the hydrogen-donating substrate is D-glucose-6-phosphate, and the dehydrogenase is glucose-6-phosphate dehydrogenase.

[0041] In another preferred embodiment, the hydrogen-donating substrate is formic acid, and the dehydrogenase is formic acid dehydrogenase.

[0042] In another preferred embodiment, the concentration of the sulfur oxide is 1-100 mM, preferably 50-100 mM, for example 75 mM.

[0043] In another preferred embodiment, the concentration of the dehydrogenase is 1 × 10⁻⁶. -3 -1mM, preferably 0.01-0.1mM.

[0044] In another preferred embodiment, the concentration of the hydrogen-donating substrate is 100-200 mM, preferably 150 mM.

[0045] In another preferred embodiment, the second catalyst is reduced glutathione, the reductase is glutathione reductase, and the cofactor is NADPH or NADH.

[0046] In another preferred embodiment, the concentration of the glutathione reductase is 1 × 10⁻⁶. -3 -1mM, preferably 0.01-0.1mM.

[0047] In another preferred embodiment, the concentration of the reduced glutathione is 0.5-2.5 mM, preferably 1 mM.

[0048] In another preferred embodiment, the second catalyst is L-cysteine, the reductase is cystine reductase, and the cofactor is NADH.

[0049] In another preferred embodiment, the concentration of L-cysteine ​​is 0.5-2.5 mM, preferably 1 mM.

[0050] In another preferred embodiment, the concentration of the cystine reductase is 1 × 10⁻⁶. -3 -1mM, preferably 0.01-0.1mM.

[0051] In another preferred embodiment, when the second catalyst is reduced glutathione, the second catalyst regeneration system includes glutathione reductase and NADPH, and the cofactor regeneration system includes glucose and glucose dehydrogenase.

[0052] In another preferred embodiment, the glutathione reductase (Gsr) is a glutathione reductase (EC 1.8.1.7 or EC 1.6.4.2) obtained by PCR using the genome of *E. coli* as a template. The glucose dehydrogenase (Gdh) is derived from *Bacillus subtilis* 168, NCBI accession number NP_388275.1.

[0053] In another preferred embodiment, the reaction system comprises: 50-100 mM sulfur oxides, 0.02-0.2 mM ergothioneine synthase, 0.5-2 mM reduced glutathione, 0.01-0.1 mM glutathione reductase, and 0.5-2 mM NADP. + 100-200mM glucose and 0.01-0.1mM glucose dehydrogenase.

[0054] In another preferred embodiment, when the second catalyst is L-cysteine, the second catalyst regeneration system includes cysteine ​​reductase and NADH, and the cofactor regeneration system includes glucose and glucose dehydrogenase. In another preferred embodiment, the reaction system includes: 50-100 mM sulfur oxides, 0.02-0.2 mM ergothioneine synthase, 0.5-2 mM L-cysteine, 0.01-0.1 mM cysteine ​​reductase, and 0.5-2 mM NAD+. + 100-200 mM glucose and 0.01-0.1 mM glucose dehydrogenase. The cysteine ​​reductase is designated as EC 1.6.4.1 or EC 1.8.1.6.

[0055] In a second aspect of the invention, there is provided the use of a thiol compound in the catalytic generation of ergothionein from sulfur oxides by ergothionein synthase, wherein the thiol compound is a thiol-containing amino acid or a thiol-containing polypeptide.

[0056] In another preferred embodiment, the thiol-containing amino acid is selected from any one or a combination of the following groups: L-cysteine, homocysteine.

[0057] In another preferred embodiment, the thiol-containing polypeptide is reduced glutathione;

[0058] In another preferred embodiment, the catalysis is carried out at a pH of 7.0-9.0, preferably at a pH of 8.0.

[0059] In another preferred embodiment, the catalysis is carried out at 25-37°C, preferably 30°C.

[0060] In another preferred embodiment, the thiol compound is the same as the second catalyst described in the first aspect of the invention, the second catalyst being as described in the first aspect of the invention.

[0061] In another preferred embodiment, the ergothionein synthase is the same as the first catalyst described in one aspect of the present invention, the first catalyst being as described in the first aspect of the present invention.

[0062] In another preferred embodiment, the catalysis is the same reaction as described in the first aspect of the invention, the reaction being as described in the first aspect of the invention.

[0063] The main advantages of this invention include:

[0064] (1) The preparation method of ergothionein of the present invention uses amino acids or polypeptides containing thiol groups, which avoids the use of thiol compounds with volatile odors, reduces the impact of odor on the environment, and eliminates the need to add deodorization equipment, thus reducing the investment cost of equipment.

[0065] (2) After the introduction of the second catalyst regeneration system, only a very small amount (catalytic amount) of the second catalyst is needed to meet the requirements of sulfur oxide to ergothioneine, without the need to add the second catalyst and sulfur oxide equivalents; in addition, after the introduction of the cofactor regeneration system, the continuous addition of inexpensive hydrogen-donating substrates can complete the continuous conversion. The reaction has the advantages of high substrate concentration, high conversion efficiency and low cost, and can be used for the industrial production of ergothioneine.

[0066] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0067] Figure 1 The biosynthetic pathway of ergothionein using DTT catalysis in the prior art is shown.

[0068] Figure 2 The normalized HPLC chromatograms of Examples 2, 3, 4, 5, Comparative Example 1, and Comparative Example 2 are shown. Figure 2 In the middle, 2 (i.e.) Figure 1 In the figure, sulfur oxide 2) represents the peak formed by the substrate sulfur oxide, and Ergothioneine represents the peak formed by the ergothioneine product.

[0069] Figure 3 The normalized HPLC chromatograms of Examples 6 and 7 are shown. Figure 3 In the figure, 2 represents the peak formed by the substrate sulfur oxide, and Ergothioneine represents the peak formed by the ergothioneine product.

[0070] Figure 4This diagram shows the cyclic regeneration pathway of L-cysteine ​​in a reaction system containing Egt2 and cystine reductase.

[0071] Figure 5 This diagram illustrates the process of ergothioneine synthesis involving reduced glutathione (GSH) in a reaction system containing Egt2 and glutathione reductase (Gsr), as well as a schematic diagram of the GSH recycling process. Figure 5 It is known that sulfur oxides undergo continuous carbon-sulfur bond breaking and reduction reactions, ultimately producing ergothioneine.

[0072] Figure 6 This diagram illustrates the cyclic regeneration pathway of reduced glutathione in a reaction system containing Egt2 and glutathione reductase. Detailed Implementation

[0073] Through extensive and in-depth research, the inventors have discovered for the first time a method for preparing ergothioneine products using thiol-containing amino acids and / or thiol-containing polypeptides as substrates with sulfur oxides. This method does not use small molecule thiol compounds (also known as mercaptans) such as DTT or β-mercaptoethanol, significantly reducing the volatile odor of small molecule thiol compounds and helping to reduce environmental pollution.

[0074] On the other hand, to ensure the complete conversion of sulfur oxides to ergothioneine, small-molecule thiol compounds need to be added in an equimolar ratio with the sulfur oxide substrate (also known as equivalent addition), and byproducts that are difficult to recycle are generated. Therefore, the amount of small-molecule thiol compounds added is large, resulting in high production costs. Furthermore, additional environmental protection equipment is required to control odor emissions and the treatment of wastewater, which increases the company's equipment investment. In contrast, the thiol-containing amino acids and / or thiol-containing peptides of this invention are easily recyclable at room temperature and can be added in catalytic amounts. Therefore, the amount added is small, the production cost is low, and there is no odor, eliminating the need for additional investment in environmental protection equipment.

[0075] Furthermore, the thiol-containing amino acids and / or thiol-containing polypeptides of the present invention can be regenerated at room temperature using enzymatic methods with hydrogen-donating substrates such as small-molecule sugars, small-molecule alcohols, or small-molecule acids. The reaction conditions are mild and controllable, reducing the probability of enzyme inactivation and byproduct generation, thus contributing to improved conversion rates of sulfur oxide substrates. Moreover, the continuous regeneration of thiol-containing amino acids and / or thiol-containing polypeptides can also drive the reaction towards the formation of ergothioneine products from sulfur oxide substrates, thereby further improving the conversion rate of sulfur oxide substrates.

[0076] In addition, when thiol-containing amino acids and / or thiol-containing polypeptides are regenerated at room temperature via enzymatic methods, the final consumable is an inexpensive hydrogen-donating substrate. Therefore, the preparation method of the present invention has a low total cost and good prospects for industrialization.

[0077] Therefore, the ergothioneine preparation method of this application has the advantages of being odorless, low-cost, having high substrate concentration, and high substrate conversion rate, and can be used for the industrial production of ergothioneine. Based on this, the present invention was completed.

[0078] the term

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0080] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0081] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0082] The technical problem this invention aims to solve is that existing methods for preparing ergothioneine involve high costs and significant volatile odor from the addition of small-molecule thiols, leading to environmental pollution and unsuitability for industrial production. Therefore, this invention provides a method for preparing ergothioneine. This method avoids the use of large quantities of volatile odorous thiols, making it more economical and environmentally friendly, and suitable for the industrial production of ergothioneine. This invention primarily solves the above-mentioned technical problems through the following technical solutions:

[0083] This invention optimizes the codons of ergothioneine synthase Egt2 and replaces DTT or β-mercaptoethanol with peptides or amino acids containing sulfhydryl groups, such as L-cysteine, homocysteine, and reduced glutathione (GSH). This achieves a high substrate conversion rate while avoiding the use of expensive and volatile odorous DTT or β-mercaptoethanol.

[0084] Furthermore, by introducing a second catalyst regeneration system, only a catalytic amount, rather than an equimolar amount, of thiol amino acids or peptides is needed to meet the requirements for the conversion of sulfur oxides to ergothioneine, thus avoiding the need to add an equimolar amount of DTT or β-mercaptoethanol to the sulfur oxides. For example, in the reaction system (5 ml) of this application, after introducing the second catalyst regeneration system, the amount of the second catalyst added to the reaction system is only 1 / 75 of the molar amount of sulfur oxides, which is sufficient to meet the requirements for the conversion of sulfur oxides to ergothioneine.

[0085] In some embodiments of the present invention, the regeneration process of GSH is as follows: Figure 5 and Figure 6 As shown, GSH acts as a reducing agent to reduce intermediate 6 to ergothioneine and oxidized glutathione (GSSG). Subsequently, NADPH-dependent glutathione reductase Gsr (EC 1.8.1.7) reduces GSSG to regenerate GSH. The newly formed GSH participates in the next round of conversion. GSH is one of the raw materials for the cyclic reaction in the reaction with intermediate 6. Glucose, as the final consumed substrate, completes NADP under the action of Gdh. + The cycle to NADPH cofactor.

[0086] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0087] Ion-pair analysis method for product detection:

[0088] Instrument used: High-performance liquid chromatograph equipped with a UV detector

[0089] Column: Welch Ultimate AQ-C18 (5μm, 4.6×250mm); Buffer: 0.05mol / L diammonium hydrogen phosphate: tetrabutylammonium hydroxide aqueous solution = 91:1, pH = 3.6; Mobile phase: acetonitrile: buffer = 8:92; Detection wavelength: 210nm; Column temperature: 30℃; Flow rate: 1.0ml / min; Injection volume: 10μl; Isocratic run time: 15min.

[0090] The conversion rate of sulfur oxide substrates was calculated using the area normalization method based on HPLC chromatographic data. The conversion rate is calculated as: (Amount of substrate converted / Total amount of substrate added) × 100%.

[0091] Sulfur oxides were prepared according to the literature Organic Letters, 16(20), 5382-5385;

[0092] pET28a was purchased from Novagen.

[0093] BL21 competent cells were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0094] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0095] TB medium: 2% tryptone, 2.4% yeast extract, 72mM K2HPO4, 17mM KH2PO4, 0.4% glycerol.

[0096] The relevant enzymes involved in the embodiments of this invention are as follows:

[0097] Glutathione reductase, with NAD(P)H as its cofactor, is designated as EC1.8.1.7 or EC1.6.4.2.

[0098] Cystine reductase, with NADH as a cofactor, is designated EC1.6.4.1 or EC1.8.1.6. Its source is Maresca, B., Jacobson, E., Medoff, G. and Kobayashi, G. Cystine reductase in the dimorphic fungus Histoplasmacapsulatum. J. Bacteriol. 135 (1978) 987-992.

[0099] Glucose dehydrogenase (Gdh) is also known as glucose dehydrogenase EC 1.1.1.118, in which case the cofactor is NAD+. + And / or NADH. Glucose dehydrogenase can also be glucose dehydrogenase EC 1.1.1.119, in which case the cofactor is NADP. + And / or NADPH. The glucose dehydrogenase in this invention is derived from Bacillus subtilis.

[0100] Glucose-6-phosphate dehydrogenase, with the enzyme designation EC 1.1.1.49.

[0101] Ergothioneine synthase, also known as hercynylcysteine ​​S-oxide lyase, abbreviated as Egt2 (from fungi) or EgtE (from prokaryotes), is designated EC 4.4.1.36.

[0102] Unless otherwise specified, the experimental methods used in this invention are conventional methods. Unless otherwise specified, the experimental reagents used in this invention are all commercially available.

[0103] Example 1: Preparation of crude enzyme solutions of ergothioneine synthase (trEgt2), glucose dehydrogenase (Gdh), and glutathione reductase (Gsr).

[0104] The gene sequence of trEgt2 from Trichoderma reesei (GeneBank accession number XP_006968735.1) and the gene sequence of glucose dehydrogenase (Gdh) from Bacillus subtilis 168 (NCBI accession number NP_388275.1) were codon-optimized and synthesized by Sangon Biotech (Shanghai) Co., Ltd. (698 Xiangmin Road, Songjiang District, Shanghai) and constructed into the pET28a vector, respectively, to obtain the pET28a-trEgt2 recombinant plasmid and the pET28a-Gdh recombinant plasmid.

[0105] Using the genome of Escherichia coli as a template, the gene sequence of glutathione reductase Gsr (EC 1.8.1.7, also known as EC1.6.4.2) was obtained by PCR and constructed into the pET28a vector to obtain the pET28a-Gsr recombinant plasmid.

[0106] The three recombinant plasmids were transformed into Ecoli BL21(DE3) host cells and plated on LB agar plates containing 50 μg / mL kanamycin. The plates were incubated upside down at 37°C for 16 h. Single colonies were picked and inoculated into LB agar containing 50 μg / mL kanamycin. The colonies were incubated at 37°C for 5 h, and then transferred at a 1% (v / v) inoculation rate to TB agar containing 50 μg / mL kanamycin. The cells were incubated at 37°C and 220 rpm until OD500 reached. 600 The concentration was approximately 0.4-0.6. After inducing expression with 0.5 mM IPTG at 25°C for 16 h, the bacterial cells were collected. The cells were resuspended in Tris-HCl buffer (50 mM Tris, 100 mM NaCl) at a ratio of 1 g: 5 mL and sonicated to disrupt the cells. After centrifugation, the supernatant was treated with 0.5‰ polyethyleneimine (PEI) to obtain crude enzyme solutions of ergothioneine synthase (trEgt2), glucose dehydrogenase (Gdh), and glutathione reductase (Gsr), respectively.

[0107] Example 2: The reaction of sulfur oxides to ergothionein in the presence of GSH

[0108] 2.1 A 5.0 mL reaction system was constructed. This reaction system included: KPi buffer solution with a final concentration of 100 mM and pH 8.0, sulfur oxides with a final concentration of 9 mM (approximately 15 g / L), a first catalyst (crude enzyme solution of ergothioneine synthase trEgt2) with a final concentration of 0.02 mM, and a second catalyst GSH with a final concentration of 9.0 mM. The reaction system was incubated at 30 °C and 200 rpm for 2 h.

[0109] 2.2 Take 20 μL of the reaction solution after 2 h of reaction and add it to 480 μL of deionized water. Then add an equal volume of methanol (500 μL) and treat it. Then use ion pair analysis to determine the conversion rate of sulfur oxides.

[0110] like Figure 2 As shown in Figure a, the conversion rate of sulfur oxides in this embodiment is 99.7%. In this reaction system (5 ml), the equivalent conversion of low-concentration sulfur oxides (approximately 15 g / L) to ergothioneine can be achieved within 2 hours.

[0111] Example 3: The reaction of sulfur oxides to ergothionein in the presence of GSH

[0112] The difference between Example 2 and Example 3 is that the concentration of the sulfur oxide substrate was increased.

[0113] 3.1 A 5.0 mL reaction system was constructed. This reaction system included: KPi buffer with a final concentration of 100 mM and a pH of 8.0, sulfur oxides with a final concentration of 75.0 mM, a first catalyst (crude enzyme solution of ergothioneine synthase trEgt2) with a final concentration of 0.02 mM, and a second catalyst GSH with a final concentration of 75.0 mM. The reaction system was incubated at 30 °C and 200 rpm for 16 h.

[0114] 3.2 Take 20 μL of the reaction solution after 16 h of reaction and add it to 480 μL of deionized water. Then add an equal volume of methanol (500 μL) and treat it. Then use ion pair analysis to determine the conversion rate of sulfur oxides.

[0115] like Figure 2 The experimental results shown in b indicate that the conversion rate of sulfur oxides in this embodiment is 99.5%, which means that when the concentration of sulfur oxide substrate is increased, the second catalyst GSH can still achieve an equivalent reaction with sulfur oxides under the action of ergothionein synthase.

[0116] Example 4: The reaction of sulfur oxides to ergothionein in the presence of L-cysteine.

[0117] Unlike Example 3, this example uses cysteine ​​instead of GSH in Example 3.

[0118] 4.1 A 5.0 mL reaction system was constructed, comprising: a KPi buffer solution with a final concentration of 100 mM and a pH of 8.0; sulfur oxides with a final concentration of 75.0 mM; a first catalyst (crude ergothioneine synthase solution, also known as trEgt2 crude enzyme solution) with a final concentration of 0.02 mM; and a second catalyst, L-cysteine, with a final concentration of 75.0 mM. The reaction system was incubated at 30 °C and 200 rpm for 16 h.

[0119] 4.2 Take 20 μL of the reaction solution after 16 h of reaction and add it to 480 μL of deionized water. Then add an equal volume of methanol (500 μL) and treat it. Then use ion pair analysis to determine the conversion rate of sulfur oxides.

[0120] like Figure 2 The experimental results shown in c indicate that the conversion rate of ergothionein in this embodiment is 43.5%. Figure 2 In c, the peak on the left (marked as 2 in the figure) represents the peak formed by the sulfur oxide substrate, and the peak on the right represents the peak formed by the ergothioneine product.

[0121] Example 5: The reaction of sulfur oxides to ergothionein in the presence of homocysteine.

[0122] Unlike Example 3, this example uses homocysteine ​​instead of GSH in Example 3.

[0123] 5.1 A 5.0 mL reaction system was constructed. This reaction system included: a KPi buffer solution with a final concentration of 100 mM and a pH of 8.0; sulfur oxides with a final concentration of 75.0 mM; a first catalyst (crude ergothioneine synthase solution, also known as trEgt2 crude enzyme solution) with a final concentration of 0.02 mM; and a second catalyst, homocysteine, with a final concentration of 75.0 mM. The reaction system was incubated at 30 °C and 200 rpm for 16 h.

[0124] 5.2 Take 20 μL of the reaction solution after 16 h of reaction and add it to 480 μL of deionized water. Then add an equal volume of methanol (500 μL) and treat it. Then use ion pair analysis to determine the conversion rate of sulfur oxides.

[0125] like Figure 2 The experimental results shown in d indicate that the conversion rate of sulfur oxides in this embodiment is 44.1%.

[0126] The experimental results of Examples 3, 4, and 5 show that the conversion rate of sulfur oxides in the reaction system containing GSH is higher than that in the reaction system containing cysteine ​​and the reaction system containing homocysteine.

[0127] Comparative Example 1: The reaction of sulfur oxides to ergothionein in the presence of DTT

[0128] (1) Construct a 5.0 mL reaction system. This reaction system includes: KPi buffer with a final concentration of 100 mM and pH 8.0, sulfur oxides with a final concentration of 75.0 mM, a first catalyst (crude enzyme solution of ergothioneine synthase) with a final concentration of 0.02 mM, and DTT with a final concentration of 75.0 mM. The reaction system was subjected to reaction at 30 °C, pH 8.0, and 200 rpm for 16 h.

[0129] (2) Take 20 μL of the reaction solution after 16 h of reaction and put it into 480 μL of deionized water. Then add an equal volume of methanol and use ion pair analysis to determine the conversion rate of sulfur oxides.

[0130] like Figure 2 The experimental results shown in Figure e indicate that the conversion rate of sulfur oxides in this embodiment is 99.7%, indicating that DTT can react with sulfur oxides in stoichiometric amounts under the action of ergothioneine synthase, and the conversion rate of sulfur oxides is relatively high. However, due to the large amount of DTT added in this reaction, the raw material cost is high. In addition, the reaction system has a strong odor, which is not conducive to environmental protection.

[0131] Comparative Example 2: The reaction of sulfur oxides to ergothionein in the presence of β-mercaptoethanol

[0132] (1) A 5.0 mL reaction system was constructed. This reaction system included: KPi buffer solution with a final concentration of 100 mM and pH 8.0, sulfur oxides with a final concentration of 75.0 mM, a first catalyst (crude ergothioneine synthase solution) with a final concentration of 0.02 mM, and β-mercaptoethanol with a final concentration of 75.0 mM. The reaction system was carried out at 30 °C, pH 8.0, and 200 rpm for 16 h.

[0133] (2) Take 20 μL of the reaction solution after 16 h of reaction and put it into 480 μL of deionized water. Then add an equal volume of methanol and use ion pair analysis to determine the conversion rate of sulfur oxides.

[0134] like Figure 2 The experimental results shown in f indicate that the conversion rate of sulfur oxides in this embodiment is 93.6%, and a small portion of the sulfur oxide substrate was not converted into ergothioneine products.

[0135] The experimental results from Examples 1, 1, and 2 show that in the reaction process of generating ergothioneine using sulfur oxides as substrates, although DTT was used as a catalyst, the conversion rate of ergothioneine was the highest. However, DTT, as a small molecule thiol, is highly volatile and has a strong odor, causing environmental pollution. While the conversion rate of ergothioneine using GSH as a catalyst is similar to that of DTT, it is odorless because it is non-volatile. Therefore, GSH can replace DTT and β-mercaptoethanol.

[0136] Example 6: The reaction of sulfur oxides to ergothionein in the presence of L-cysteine, with the addition of a second catalyst regeneration system and a cofactor regeneration system.

[0137] 6.1 Construct a 5.0 mL reaction system. This system included: a KPi buffer solution with a final concentration of 100 mM and a pH of 8.0; sulfur oxides with a final concentration of 75.0 mM; a first catalyst (ergothioneine synthase crude enzyme solution, also known as trEgt2 crude enzyme solution) with a final concentration of 0.02 mM; a second catalyst, L-cysteine, with a final concentration of 1.0 mM; cysteine ​​reductase enzyme solution with a final concentration of 0.01 mM (commercially available as a reductase); and NAD+ with a final concentration of 1.0 mM. + The reaction system consisted of glucose (as an oxidizing cofactor), glucose at a final concentration of 150.0 mM (as a hydrogen-donating substrate), and crude glucose dehydrogenase at a final concentration of 0.01 mM (also known as crude Gdh enzyme solution). The reaction system was incubated at 30°C and 200 rpm for 16 h. The reaction principle diagram is shown below. Figure 4 As shown.

[0138] 6.2 Take 20 μL of the reaction solution after 16 h of reaction and add it to 480 μL of deionized water. Then add an equal volume of methanol and treat it. Analyze the conversion rate of sulfur oxides using ion-pair analysis.

[0139] like Figure 3 The experimental results shown in i indicate that the conversion rate of sulfur oxides in this example is 58.3%. Compared with the results of Example 4 (43.5%), the conversion rate of sulfur oxides is improved in the presence of L-cysteine ​​and after the addition of a regeneration system.

[0140] Example 7: The reaction of sulfur oxides to ergothionein in the presence of GSH, with the addition of a second catalyst regeneration system and a cofactor regeneration system.

[0141] 7.1 Construct a 5.0 mL reaction system. This reaction system includes: a KPi buffer solution with a final concentration of 100 mM and a pH of 8.0; sulfur oxides with a final concentration of 75.0 mM; a first catalyst (ergothioneine synthase crude enzyme solution, also known as trEgt2 crude enzyme solution) with a final concentration of 0.02 mM; a second catalyst GSH with a final concentration of 1.0 mM; a glutathione reductase crude enzyme solution (as a reductase, also known as Gsr crude enzyme solution, obtained from Example 1) with a final concentration of 0.01 mM; and NADP with a final concentration of 1.0 mM. + The reaction system consisted of glucose (as an oxidizing cofactor), glucose at a final concentration of 150.0 mM (as a hydrogen-donating substrate), and crude glucose dehydrogenase at a final concentration of 0.01 mM (also known as crude Gdh enzyme solution). The reaction system was incubated at 30°C and 200 rpm for 16 h. The specific reaction schematic is shown below. Figure 5 and Figure 6 As shown.

[0142] 7.2 Take 20 μL of the reaction solution after 16 h of reaction and add it to 480 μL of deionized water. Then add an equal volume of methanol and treat it. Analyze the conversion rate of sulfur oxides using ion-pair analysis.

[0143] like Figure 3 The experimental results shown in ii indicate that the conversion rate of sulfur oxides in this embodiment is 99.5%.

[0144] The experimental results from Examples 2 and 7 show that, without the introduction of a second catalyst (GSH) for regeneration, GSH reacts with sulfur oxides in an equimolar ratio (equivalent reaction), requiring a relatively large amount of GSH. However, after introducing the regeneration system, although the amount of GSH added decreases, the conversion rate of sulfur oxides does not decrease, thus reducing the amount of GSH used and consequently lowering raw material costs.

[0145] Furthermore, DTT and β-mercaptoethanol cannot be regenerated using enzymatic methods; their reaction with sulfur oxides can only occur in an equimolar ratio, resulting in large addition amounts. This not only increases raw material costs but also produces a strong odor, which is detrimental to environmental protection and increases investment in deodorization equipment and other environmental protection facilities. In contrast, this embodiment overcomes the shortcomings of DTT or β-mercaptoethanol as reducing agents by constructing an enzymatic regeneration system for GSH, which results in the inability to regenerate and the strong odor.

[0146] The parameters and conversion rates of sulfur oxides for the above embodiments and comparative examples are shown in Table 1 below.

[0147] Table 1. Parameters and conversion rates for different embodiments.

[0148]

[0149]

[0150] The results showed that thiol-containing amino acids or peptides, when providing thiol groups alone, can convert sulfur oxides into ergothioneine in essentially equivalent quantities. When thiol-containing amino acids or peptides can be regenerated, only about 1 / 75 of the previous amount is needed to convert the same amount of sulfur oxides into ergothioneine. This obviously reduces the amount of thiol-containing amino acids or peptides used and avoids the odor of small molecule thiols, thereby reducing the raw material cost of ergothioneine production and reducing environmental pollution.

[0151] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A process for the preparation of ergothioneine, characterized in that, The method comprises the following steps: The sulfur oxide is reacted in the presence of a catalyst system to generate ergothioneine; The catalyst system comprises a first catalyst and a second catalyst, the first catalyst is ergothioneine synthase, and the second catalyst is a polypeptide containing a mercapto group; The ergothioneine synthase is Egt2, and the ergothioneine synthase is derived from *Reeseidon* (Egt2). Trichoderma reesei trEgt2, which is derived from Reesei mold ( Trichoderma reesei The GeneBank accession number for trEgt2 is XP_006968735.1; the thiol-containing polypeptide is reduced glutathione, and the molar ratio of the second catalyst to the sulfur oxide is 1:1-200. The structural formula of the sulfur oxide is shown in Formula I: Formula I; The structural formula of the ergothioneine is shown in Formula II or Formula III: Formula II; Formula III.

2. The method of claim 1, wherein, The concentration of the sulfur oxide is 1-100 mM.

3. The method of claim 1, wherein, The concentration of the sulfur oxide is 9-75 mM.

4. The method of claim 1, wherein, The concentration of said ergothioneine synthase is 1 x 10 -3 -1 mM.

5. The method of claim 1, wherein, The concentration of the ergothioneine synthase is 0.02-0.2 mM.

6. The method of claim 1, wherein, The reaction is performed at a pH of 7.0-9.

0.

7. The method of claim 1, wherein, The reaction is performed at a pH of 8.

0.

8. The method of claim 1, wherein, The reaction is performed in a KPi buffer at a pH of 7.0-9.

0.

9. The method of claim 8, wherein, The pH of the KPi buffer is 8.

0.

10. The method of claim 1, wherein, The reaction is performed at 25-37 DEG C.

11. The method of claim 1, wherein, The reaction is performed at 30 DEG C.

12. The method of claim 1, wherein, The duration of the reaction is 2-24 h.

13. The method of claim 1, wherein, The duration of the reaction is 2-16 h.

14. The method of claim 1, wherein, The concentration of the second catalyst is 1-100 mM.

15. The method of claim 1, wherein, The concentration of the second catalyst is 1-5 mM or 50-100 mM.

16. The method of claim 1, wherein, The molar ratio of the second catalyst to the sulfur oxide is 1:1-5 or 1:50-100.

17. The method of claim 1, wherein, The reaction is performed in the presence of a second catalyst regeneration system, and the second catalyst regeneration system comprises a reductase for regenerating the second catalyst and a co-factor for catalytic activity of the reductase.

18. The method of claim 17, wherein, The reductase is glutathione reductase.

19. The method of claim 17, wherein, The co-factor is selected from any one or a combination of NADPH and NADH.

20. The method of claim 17, wherein, The molar ratio of the second catalyst to the sulfur oxide is 1:50-200.

21. The method of claim 17, wherein, The molar ratio of the second catalyst to the sulfur oxide is 1:75-100.

22. The method of claim 17, wherein, The concentration of the reductase is 1 x 10 -3 -1 mM.

23. The method of claim 17, wherein, The concentration of the reductase is 0.01-0.1 mM.

24. The method of claim 17, wherein, The concentration of the co-factor is 0.5-2 mM.

25. The method of claim 17, wherein, The concentration of the co-factor is 1 mM.

26. The method of claim 17, wherein, The reaction is performed in the presence of a co-factor regeneration system, and the co-factor regeneration system comprises a hydrogen donor and a dehydrogenase for regenerating the co-factor by using the hydrogen donor.

27. The method of claim 26, wherein, The hydrogen donor is glucose, and the dehydrogenase is glucose dehydrogenase; and / or, The hydrogen donor is isopropanol, and the dehydrogenase is isopropanol dehydrogenase; and / or, The hydrogen donor is D-glucose-6-phosphate, and the dehydrogenase is glucose-6-phosphate dehydrogenase; and / or, The hydrogen donor is formic acid, and the dehydrogenase is formic acid dehydrogenase.

28. The method of claim 26, wherein, The concentration of the sulfur oxide is 1-100 mM; and / or, the concentration of said dehydrogenase is 1 x 10 -3 -1 mM; and / or, The concentration of the hydrogen donor is 100-200 mM.

29. The method of claim 26, wherein, The concentration of the sulfur oxide is 75 mM; and / or, The concentration of the dehydrogenase is 0.01-0.1 mM; and / or, The concentration of the hydrogen donor is 150 mM.

30. The method of claim 17, wherein, The reductase is glutathione reductase, and the co-factor is NADPH or NADH.

31. The method of claim 30, wherein, The concentration of the glutathione reductase is 1 x 10 -3 -1 mM; and / or the concentration of reduced glutathione is 0.5-2.5 mM.

32. The method of claim 30, wherein, the concentration of glutathione reductase is 0.01-0.1 mM; and / or the concentration of reduced glutathione is 1 mM.

33. Use of a thiol compound in the synthesis of ergothioneine by an ergothioneine synthase catalyzed oxidation of a sulfur oxide, the thiol compound being a polypeptide containing a thiol group; the structure of the sulfur oxide is shown in Formula I: Formula I; the structure of the ergothioneine is shown in Formula II or Formula III: Formula II; Formula III; wherein The ergothioneine synthase is Egt2, the ergothioneine synthase is trEgt2 derived from Trichoderma reesei Rasamsonia emersonii, the GeneBank accession number of the trEgt2 derived from Trichoderma reesei Rasamsonia emersonii is XP_006968735.1; the thiol-containing polypeptide is reduced glutathione; and the molar ratio of the thiol-containing polypeptide to the sulfur oxide is 1:1-200.

34. The use of claim 33, wherein, the catalysis is carried out at a pH of 7.0-9.0; and / or the catalysis is carried out at 25-37 °C.

35. The use of claim 33, wherein the compound is ###00023### 34. the catalysis is carried out at a pH of 8.0; and / or the catalysis is carried out at 30 °C.

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