Preparation method of ergothioneine and application of sulfhydryl compound in ergothioneine

Abstract

The invention provides a preparation method of ergothioneine and application of a sulfhydryl compound in the ergothioneine. The method comprises the following step: in the presence of a catalyst system, sulfur oxides react to generate ergothioneine. The catalyst system includes a first catalyst and a second catalyst. The first catalyst is ergothioneine synthetase, and the second catalyst is one or more of sulfydryl-containing amino acid and sulfydryl-containing polypeptide. The preparation method of the ergothioneine does not need to use a large amount of stink chemical mercaptan reagent, has the remarkable advantages of high production efficiency, environmental protection, friendly production scene and the like, and is beneficial to industrial production of the ergothioneine.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and in particular, relates to a method for preparing ergothioneine and application of a thiol compound therein. Background Art

[0002] Ergothioneine is an amino acid containing a thiol group that was isolated from ergot by Tanret in 1909. As a highly biologically active thiol reducing agent, ergothioneine can effectively capture high-energy oxidative intermediates inside cells and prevent cells from being damaged by oxides. Its unique redox properties make it one of the best natural antioxidants. Transporters (Organic Cation / Ergothioneine Transporter 1, OCTN1) that specifically absorb ergothioneine have been identified in human cells, and it has been found that ergothioneine accumulates to varying degrees in different tissue cells (100μM to 2mM), indicating that ergothioneine should play an important physiological function in the human body. Currently, ergothioneine has been approved for use in cosmetics and food, and there is broad room for growth in the future.

[0003] At present, the synthesis of ergothioneine mainly includes chemical synthesis, natural extraction (extracted from fungal mycelium), genetically engineered strain fermentation and enzyme catalysis. Chemical synthesis method has defects such as long path, low conversion rate and large pollution. Natural extraction method is due to the low content of ergothioneine in mycelium, which limits the efficiency of extraction method. The biosynthetic path of ergothioneine is imported into model strains (such as Escherichia coli, Yarrowia lipolytica or Saccharomyces cerevisiae), and then synthesizing ergothioneine by transforming strains through biological fermentation is an ideal synthesis method. However, it may be subject to the regulatory system of the host cell itself, and the output of fermented ergothioneine can only reach gram level, which is difficult to be applied to large-scale production preparation. The method of in vitro enzyme catalysis can get rid of the limitation of the host cell regulatory system and synthesize high-concentration target products. It may be due to the limited understanding of the enzyme catalysis mechanism related to ergothioneine biosynthesis, and the report of high conversion rate enzyme catalysis method is not found at present.

[0004] Previous studies have found that Figure 1As shown, the CS bond in sulfur oxide 2 (Sulfoxide 2) is catalytically cracked by EgtE (or Egt2) to generate pyruvic acid and thioneine. In verifying the catalytic mechanism of EgtE or Egt2 enzymes, it is found that (Sci.Rep.2015,5,11870 and Cell Chemical Biology 2018,25,519), the presence of dithiothreitol (Dithiothreitol, DTT) in the reaction system can promote the synthesis of thioneine, and it is inferred that DTT participates in the synthesis process of thioneine as a reducing agent, and may be responsible for reducing the intermediate 6 after the CS bond is broken to form thioneine, that is, the sulfur atom in the 0 valence state in 6 is reduced to -2 valence in thioneine. In the process of the above reaction, in order to ensure the full conversion of sulfur oxide to thioneine, it is necessary to add DTT of equivalent level (Equivalent level), and generate corresponding oxidized DTT as a by-product of the reaction. Due to the high market price of DTT, the large amount of addition of DTT will increase the production cost of thioneine. In addition, if mercaptoethanol is used to replace DTT in the production of ergothioneine, since mercaptoethanol has the volatile odor of thiol, it is necessary to add environmental protection equipment to control odor overflow and treat reaction wastewater, which will increase the company's equipment investment. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing ergothioneine with simple reaction system, high catalytic efficiency and environmental friendliness, and 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 steps of:

[0007] In the presence of a catalyst system, the sulfur oxide is reacted to obtain ergothioneine;

[0008] Wherein 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 sulfhydryl-containing amino acid and a sulfhydryl-containing polypeptide;

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

[0010]

[0011] The structural formula of the ergothioneine is as 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 -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, such as 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 a pH of 8.0.

[0020] In another preferred embodiment, the reaction is carried out in a KPi buffer having a pH of 7.0-9.0, preferably 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 of the reaction is 2-24 h, preferably 2-16 h.

[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, and the GeneBank accession number is XP_006968735.1.

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

[0027] In another preferred embodiment, when the reaction is carried out in the presence of a regeneration system for the 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 absence of a regeneration system for the 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 comprises: a reductase for regenerating the second catalyst, and a cofactor for the reductase to exert catalytic activity.

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

[0031] In another preferred embodiment, the glutathione reductase (Gsr) is a glutathione reductase (EC 1.8.1.7 or EC1.6.4.2) obtained by PCR using the genome of Escherichia coli as a template.

[0032] In another preferred embodiment, the cystine reductase is numbered 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 -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 comprises: a hydrogen-donating substrate, and a dehydrogenase that utilizes 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 formate, and the dehydrogenase is formate dehydrogenase.

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

[0043] In another preferred embodiment, the concentration of the dehydrogenase is 1×10 -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 glutathione reductase is 1×10 -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 cystine reductase is 1×10 -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 EC1.6.4.2) obtained by PCR using the genome of Escherichia coli as a template. The glucose dehydrogenase (Gdh) is derived from Bacillus subtilis 168, and the NCBI accession number is NP_388275.1.

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

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

[0055] In a second aspect of the present invention, there is provided a use of a thiol compound in ergothioneine synthase catalyzing sulfur oxide to generate ergothioneine, 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 present invention, and the second catalyst is as described in the first aspect of the present invention.

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

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

[0063] The main advantages of the present invention include:

[0064] (1) the preparation method of thioneine of the present invention uses sulfhydryl-containing amino acid or polypeptide, avoids the use of thiol compounds with volatile odor, reduces the impact of odor on the environment, does not need to increase deodorizing equipment, and reduces the investment cost of equipment.

[0065] (2) after introducing the second catalyst regeneration system, only the second catalyst of a very small amount (catalytic amount) needs to be added to satisfy the demand for sulfur oxides to be converted into thioneine, without the need to add the second catalyst and sulfur oxide equivalent; in addition, after introducing the cofactor regeneration system, continuously adding cheap hydrogen supply substrate can complete continuous conversion, and the reaction has the advantages of high substrate concentration, high conversion efficiency and low cost, and can be used for the industrialized production of thioneine.

[0066] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0068] Figure 2 The normalized HPLC spectra of Example 2, Example 3, Example 4, Example 5, Comparative Example 1 and Comparative Example 2 are shown. Figure 2 In, 2 (i.e. Figure 1 The sulfur oxide 2) in the figure 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 profiles of Example 6 and Example 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 4Schematic diagram showing the cyclic regeneration pathway of L-cysteine ​​in a reaction system containing Egt2 and cystine reductase.

[0071] Figure 5 The schematic diagram shows the process of synthesizing ergothioneine with the participation of reduced glutathione (GSH) in a reaction system containing Egt2 and glutathione reductase (Gsr) and the cyclic regeneration of GSH. Figure 5 It is known that sulfur oxides continuously undergo carbon-sulfur bond cleavage and reduction reactions, ultimately producing ergothioneine.

[0072] Figure 6 Schematic diagram showing the cyclic regeneration pathway of reduced glutathione in a reaction system containing Egt2 and glutathione reductase. DETAILED DESCRIPTION

[0073] The inventors have, through extensive and in-depth research, discovered for the first time a method for preparing ergothioneine products using sulfhydryl-containing amino acids and / or sulfhydryl-containing polypeptides using sulfur oxides as substrates, which does not use small molecule sulfhydryl compounds (also known as mercaptans) such as DTT or β-mercaptoethanol, greatly reduces the volatility of small molecule sulfhydryl compounds. The odor contributes to reducing environmental pollution.

[0074] On the other hand, to ensure the full conversion of sulfur oxides to thioneine, small molecule thiol compounds need to be added with sulfoxide substrates in an equimolar ratio (also known as equivalent addition), and produce a by-product that is difficult to recycle, therefore, the addition of small molecule thiol compounds is large, and the production cost is relatively high. And it is necessary to add an environmental protection device for controlling odor overflow and being in reaction wastewater, which will increase the equipment investment of enterprises. And the sulfhydryl-containing amino acid of the present invention and / or the sulfhydryl-containing polypeptide are easily recycled at room temperature, can be added with a catalytic amount, and therefore the addition is small, the production cost is relatively low, and there is no odor, and there is no need to increase the investment cost of environmental protection equipment.

[0075] On the other hand, the sulfhydryl-containing amino acid and / or sulfhydryl-containing polypeptide of the present invention can utilize hydrogen supply substrates such as small molecule sugars, small molecule alcohols or small molecule acids to realize room temperature regeneration by enzymatic method, and reaction conditions are gentle and controllable, and the probability of enzyme inactivation and the generation probability of by-product are reduced, which contributes to improving the conversion rate of sulfoxide substrate. Further, by the continuous regeneration of sulfhydryl-containing amino acid and / or sulfhydryl-containing polypeptide, the reaction can also be promoted to continue in the direction of generating thioneine product by sulfoxide substrate, thus the conversion rate of sulfoxide substrate can be further improved.

[0076] In addition, when the thiol-containing amino acid and / or thiol-containing polypeptide is regenerated at room temperature by an enzymatic method, the final consumption is a low-cost hydrogen supply substrate. Therefore, the preparation method of the present invention has a low total cost and a good industrial prospect.

[0077] Therefore, the preparation method of thioneine of the application has the advantages of no odor, low cost, high substrate concentration and high substrate conversion rate, and can be used for the industrialized production of thioneine.Completed the present invention on this basis.

[0078] the term

[0079] Unless defined otherwise, 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 belongs.

[0080] As used herein, the terms "comprise", "include", and "contain" are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0081] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. 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 to be solved by the present invention is that the small molecule thiol reducing agent used in the existing preparation method of thioneine has high addition cost and obvious volatile odor, thereby causing environmental pollution, being unsuitable for the problems of industrial production, therefore, the present invention provides a preparation method of thioneine. The preparation method of the present invention avoids the use of a large amount of thiols with volatile odor, is more economical and environmentally friendly, and can be used for the industrial production of thioneine. The present invention mainly solves the above-mentioned technical problems through the following technical solutions:

[0083] The invention performs codon optimization on ergothioneine synthase Egt2, and uses polypeptides or amino acids containing thiol groups such as L-cysteine, homocysteine, reduced glutathione (GSH) to replace DTT or beta-mercaptoethanol, thereby achieving a higher substrate conversion rate and avoiding the use of DTT or beta-mercaptoethanol which are expensive and have a volatile odor.

[0084] Moreover, after introducing the second catalyst regeneration system, only need to add catalytic amount and not sulfhydrylamino acid or polypeptide of equimolar amount in reaction system just can meet the demand that sulfur oxide is converted into thioneine, can avoid inputting DTT or beta-mercaptoethanol of equimolar amount with sulfur oxide.For example, in the reaction system (5ml) of the present application, after introducing the second catalyst regeneration system, when the amount of adding the second catalyst in reaction system is only sulfur oxide 1 / 75 molar amount, just can meet the demand that sulfur oxide is converted into thioneine.

[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 generate ergothioneine and oxidized glutathione (GSSG), and then NADPH-dependent glutathione reductase Gsr (EC 1.8.1.7) reduces GSSG to achieve GSH regeneration. The newly formed GSH participates in the next round of conversion process. GSH is one of the raw materials of the cyclic reaction and participates in the reaction with intermediate 6. Glucose, as the final consumption substrate, completes the conversion of NADP under the action of Gdh. + to the NADPH cofactor.

[0086] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0087] Ion pair condition analysis product detection method:

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

[0089] Chromatographic 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 running time: 15min.

[0090] Calculation method of sulfur oxide substrate conversion rate: Calculate based on HPLC spectrum data using area normalization method. Substrate conversion rate = amount of converted substrate / total amount of added substrate × 100%.

[0091] Sulfur oxides were prepared according to 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, 72 mM K2HPO4, 17 mM KH2PO4, 0.4% glycerol.

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

[0097] Glutathione reductase, whose full English name is gluathione reductase, has a cofactor of NAD(P)H and an enzyme number of EC1.8.1.7 or EC1.6.4.2.

[0098] Cystine reductase, whose full English name is cystine reductase, has a cofactor of NADH and an enzyme number of EC1.6.4.1 or EC 1.8.1.6. The source of the enzyme is Maresca, B., Jacobson, E., Medoff, G. and Kobayashi, G. Cystine reductase in the dimorphic fungus Histoplasma capsulatum. J. Bacteriol. 135 (1978) 987-992.

[0099] Glucose dehydrogenase (Gdh) is also called glucose dehydrogenase. Glucose dehydrogenase can also be 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 the present invention is derived from Bacillus subtilis.

[0100] Glucose-6-phosphate dehydrogenase, the full English name is glucose-6-phosphate dehydrogenase, and the enzyme number is 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 numbered EC 4.4.1.36.

[0102] The experimental methods in the present invention are conventional methods unless otherwise specified. The experimental reagents in the present invention are conventionally commercially available unless otherwise specified.

[0103] Example 1 Preparation of crude enzyme solution of ergothioneine synthase (trEgt2), crude enzyme solution of glucose dehydrogenase (Gdh) and crude enzyme solution of 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 synthesized by Sangon Biotech (Shanghai) Co., Ltd. (No. 698, Xiangmin Road, Songjiang District, Shanghai) after codon optimization 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 a pET28a vector to obtain a pET28a-Gsr recombinant plasmid.

[0106] The three recombinant plasmids were transferred into Ecoli BL21 (DE3) host cells, spread on resistant LB plates containing 50 μg / mL kanamycin, and inverted at 37°C for 16 h. A single colony was picked and inoculated into resistant LB medium containing 50 μg / mL kanamycin, incubated at 37°C for 5 h, and then transferred to TB medium containing 50 μg / mL kanamycin at a 1% (v / v) inoculation amount, and incubated at 37°C and 220 rpm until OD 600 Around 0.4-0.6. At 25°C, after inducing expression with a final concentration of 0.5mM IPTG for 16 hours, the bacteria were collected. The bacteria were resuspended in Tris-HCl buffer (50mM Tris, 100mMNaCl) at a ratio of 1g:5mL and ultrasonically broken. 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 Reaction of sulfur oxides into ergothioneine in the presence of GSH

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

[0109] 2.2 Take 20 μL of the reaction solution after 2 hours of reaction and add it to 480 μL of deionized water. Then add an equal volume of methanol (500 μL) for treatment, and analyze the conversion rate of sulfur oxides using ion pair conditions.

[0110] like Figure 2 The experimental result shown in a shows that the conversion rate of sulfur oxide in the present embodiment is 99.7%. In this reaction system (5ml), the equivalent conversion of sulfur oxide (about 15g / L) of low concentration to thioneine can be achieved in 2h.

[0111] Example 3 Reaction of sulfur oxides into ergothioneine in the presence of GSH

[0112] The difference from Example 2 is that Example 3 increases the concentration of the sulfur oxide substrate.

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

[0114] 3.2 Take 20 μL of the reaction solution after 16 hours of reaction and add it to 480 μL of deionized water. Then add an equal volume of methanol (500 μL) for treatment, and analyze the conversion rate of sulfur oxides using ion pair conditions.

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

[0116] Example 4: Reaction of sulfur oxides into ergothioneine in the presence of L-cysteine

[0117] Different from 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 having a final concentration of 100 mM and a pH of 8.0, a sulfur oxide having a final concentration of 75.0 mM, a first catalyst having a final concentration of 0.02 mM (ergothioneine synthase crude enzyme solution, also known as trEgt2 crude enzyme solution), and a second catalyst L-cysteine ​​having a final concentration of 75.0 mM. The reaction system was reacted at 30° C. and 200 rpm for 16 h.

[0119] 4.2 Take 20 μL of the reaction solution after 16 hours of reaction and add it to 480 μL of deionized water. Then add an equal volume of methanol (500 μL) for treatment, and analyze the conversion rate of sulfur oxides using ion pair conditions.

[0120] like Figure 2 The experimental result shown in c shows that the transformation efficiency of thioneine in the present embodiment is 43.5%. Figure 2 In c, the peak on the left (labeled as 2 in the figure) represents the peak formed by the sulfoxide substrate, and the peak on the right represents the peak formed by the thioneine product.

[0121] Example 5 Reaction of sulfur oxides into ergothioneine in the presence of homocysteine

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

[0123] 5.1 Construct a 5.0 mL reaction system. The reaction system includes: a KPi buffer solution with a final concentration of 100 mM and a pH of 8.0, a sulfur oxide 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, and a second catalyst homocysteine ​​with a final concentration of 75.0 mM. The reaction system is reacted at 30° C. and 200 rpm for 16 h.

[0124] 5.2 Take 20 μL of the reaction solution after 16 hours of reaction and add it to 480 μL of deionized water. Then add an equal volume of methanol (500 μL) for treatment, and analyze the conversion rate of sulfur oxides using ion pair conditions.

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

[0126] From the experimental results of Examples 3, 4 and 5, it can be seen 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: Reaction of sulfur oxides converted into ergothioneine in the presence of DTT

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

[0129] (2) After 16 h of reaction, 20 μL of the reaction solution was added to 480 μL of deionized water, and then an equal volume of methanol was added for treatment. The conversion rate of sulfur oxides was analyzed using ion pair conditions.

[0130] like Figure 2 The experimental result shown in e shows that the conversion rate of sulfur oxides in the present embodiment is 99.7%, indicating that under the action of ergothioneine synthetase, DTT can achieve equivalent reaction with sulfur oxides, and the conversion rate of sulfur oxides is higher. However, due to the reaction, the addition amount of DTT is larger, so the raw material cost of the reaction is higher, in addition, the reaction system has a very large odor, which is unfavorable for environmental protection.

[0131] Comparative Example 2: The reaction of converting sulfur oxide into ergothioneine in the presence of β-mercaptoethanol

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

[0133] (2) After 16 h of reaction, 20 μL of the reaction solution was added to 480 μL of deionized water, and then an equal volume of methanol was added for treatment. The conversion rate of sulfur oxides was analyzed using ion pair conditions.

[0134] like Figure 2 The experimental result shown in f shows that the conversion rate of sulfur oxides in the present embodiment is 93.6%, and a small part of sulfur oxide substrate is not converted into thioneine product.

[0135] As can be seen from the test results of Example 1, Comparative Example 1 and Comparative Example 2, in the reaction process of generating thioneine using sulfur oxide as substrate, although the conversion rate of thioneine is the highest when using DTT as catalyst, DTT as small molecule mercaptan has a larger stink due to easy volatility, brings environmental pollution. And the conversion rate of thioneine is almost the same as DTT when using GSH as catalyst, but there is no stink due to not volatilizing, therefore, GSH can replace DTT and beta-mercaptoethanol.

[0136] Example 6 Conversion of Sulfur Oxide to Ergothioneine in the Presence of L-Cysteine ​​and Addition of a Second Catalyst Regeneration System and a Cofactor Regeneration System

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

[0138] 6.2 Take 20 μL of the reaction solution after 16 hours of reaction and add it to 480 μL of deionized water. Then add an equal volume of methanol for treatment and analyze the conversion rate of sulfur oxides using ion pair conditions.

[0139] like Figure 3 The experimental results shown in Figure 1 show that the conversion rate of sulfur oxides in this example is 58.3%. Compared with the result of Example 4 (43.5%), the conversion rate of sulfur oxides is improved in the presence of L-cysteine ​​and after the addition of the regeneration system.

[0140] Example 7 Conversion of sulfur oxides to ergothioneine in the presence of GSH and addition of a second catalyst regeneration system and a cofactor regeneration system

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

[0142] 7.2 Take 20 μL of the reaction solution after 16 hours of reaction and add it to 480 μL of deionized water. Then add an equal volume of methanol for treatment and analyze the conversion rate of sulfur oxides using ion pair conditions.

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

[0144] According to the experimental results of Example 2 and Example 7, when GSH is not introduced for regeneration of the second catalyst, GSH and sulfur oxide react in an equimolar ratio (equivalent reaction), and the amount of GSH added is relatively large. After the regeneration system is introduced, although the amount of GSH added is reduced, the conversion rate of sulfur oxide does not decrease, which reduces the amount of GSH used, thereby reducing the cost of raw materials.

[0145] In addition, since DTT and β-mercaptoethanol cannot be regenerated by enzymatic method, they can only react with sulfur oxides in an equimolar ratio, so the amount added is large, which not only increases the cost of raw materials, but also has a strong odor, which is not conducive to environmental protection and will increase the investment in environmental protection equipment such as deodorization equipment. In contrast, this embodiment overcomes the defects of being unable to regenerate and having a strong odor when DTT or β-mercaptoethanol is used as a reducing agent by constructing an enzymatic regeneration system for GSH.

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

[0147] Table 1 Parameters and conversion rates of different embodiments

[0148]

[0149]

[0150] The result analysis shows that the sulfhydryl-containing amino acid or polypeptide can convert sulfur oxides into the product ergothioneine substantially in equivalent when the sulfhydryl-containing amino acid or polypeptide provides a sulfhydryl group alone, and when the sulfhydryl-containing amino acid or polypeptide can be regenerated, only about 1 / 75 of the previous amount needs to be added to convert the same amount of sulfur oxides into ergothioneine, which obviously reduces the usage of the sulfhydryl-containing amino acid or polypeptide, and avoids the odor of small molecule thiol compounds, thereby reducing the raw material cost when producing ergothioneine, and reducing environmental pollution.

[0151] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for preparing ergothioneine, characterized in that, The method comprises the following steps: In the presence of the catalyst system, the sulfur oxides react to form ergothioneine; Wherein, 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 sulfhydryl-containing amino acid and a sulfhydryl-containing polypeptide; The structural formula of the sulfur oxide is shown in Formula I: The structural formula of the ergothioneine is as shown in Formula II or Formula III:

2. The method according to claim 1, characterized in that The sulfhydryl-containing amino acid is selected from any one or a combination of the following groups: L-cysteine, homocysteine; the sulfhydryl-containing polypeptide is reduced glutathione; and / or The concentration of the sulfur oxide is 1-100 mM, preferably 9-75 mM; and / or, The concentration of the ergothioneine synthase is 1×10 -3 -1 mM, preferably 0.02-0.2 mM; and / or, The molar ratio of the second catalyst to the sulfur oxide is 1:1-200; and / or, The reaction is carried out at a pH of 7.0-9.0, preferably at a pH of 8.0; and / or, The reaction is carried out in a KPi buffer having a pH of 7.0-9.0, preferably a KPi buffer having a pH of 8.0; and / or, The reaction is carried out at 25-37°C, preferably 30°C; and / or, The duration of the reaction is 2-24 hours, preferably 2-16 hours; and / or, The ergothioneine synthase is selected from EgtE derived from Mycobacterium smegmatis or trEgt2 derived from Trichoderma reesei.

3. The method according to claim 1 or 2, characterized in that The reaction is carried out in the presence of a second catalyst regeneration system, wherein the second catalyst regeneration system comprises: a reductase for regenerating the second catalyst, and a cofactor for the reductase to exert catalytic activity.

4. The method according to claim 3, characterized in that The reductase is selected from any one or a combination of the following groups: glutathione reductase, cystine reductase; and / or, The cofactor is selected from any one or a combination of the following groups: NADPH, NADH; and / or, The molar ratio of the second catalyst to the sulfur oxide is 1:50-200, preferably 1:75-100; and / or, The concentration of the reductase was 1×10 -3 -1 mM, preferably 0.01-0.1 mM; and / or, The concentration of the cofactor is 0.5-2 mM, preferably 1 mM.

5. The method according to claim 3, characterized in that The reaction is carried out in the presence of a cofactor regeneration system, wherein the cofactor regeneration system comprises: a hydrogen-donating substrate, and a dehydrogenase that regenerates the cofactor using the hydrogen-donating substrate; Preferably, the hydrogen-donating substrate is glucose, and the dehydrogenase is glucose dehydrogenase; and / or, The hydrogen-donating substrate is isopropanol, and the dehydrogenase is isopropanol dehydrogenase; and / or, The hydrogen supply substrate is D-glucose-6-phosphate, and the dehydrogenase is glucose-6-phosphate dehydrogenase; and / or, The hydrogen supply substrate is formate, and the dehydrogenase is formate dehydrogenase.

6. The method according to claim 5, characterized in that The concentration of the sulfur oxide is 1-100 mM, preferably 75 mM; and / or, The concentration of the dehydrogenase was 1×10 -3 -1 mM, preferably 0.01-0.1 mM; and / or, The concentration of the hydrogen-donating substrate is 100-200 mM, preferably 150 mM.

7. The method according to claim 3, characterized in that The second catalyst is reduced glutathione; the reductase is glutathione reductase; and the cofactor is NADPH or NADH.

8. The method according to claim 7, characterized in that The concentration of glutathione reductase was 1×10 -3 -1 mM, preferably 0.01-0.1 mM; and / or The concentration of the reduced glutathione is 0.5-2.5 mM, preferably 1 mM.

9. The method according to claim 3, characterized in that The second catalyst is L-cysteine; the reductase is cystine reductase; and the cofactor is NADH; Preferably, the concentration of L-cysteine ​​is 0.5-2.5 mM, preferably 1 mM; and / or, The concentration of the cystine reductase is 1×10-3-1 mM, preferably 0.01-0.1 mM.

10. The use of a sulfhydryl compound in the production of ergothioneine by catalyzing sulfur oxides by ergothioneine synthase, wherein the sulfhydryl compound is a sulfhydryl-containing amino acid or a sulfhydryl-containing polypeptide; The structural formula of the sulfur oxide is shown in Formula I: The structural formula of the ergothioneine is as shown in Formula II or Formula III: Further, the thiol-containing amino acid is selected from any one or a combination of the following groups: L-cysteine, homocysteine; the thiol-containing polypeptide is reduced glutathione; Furthermore, the catalysis is carried out at a pH of 7.0-9.0, preferably at a pH of 8.0; and / or The catalysis is carried out at 25-37°C, preferably 30°C.

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