A method for biosynthesis of beta-aminoethanethiol compounds using tryptophan synthase

Abstract

The application provides a method for biosynthesizing a beta-aminoethyl mercaptan compound by using tryptophan synthetase, which comprises: an enzymatic reaction of a beta-aminoethanol compound shown in formula I and sodium hydrosulfide in a reaction system containing tryptophan synthetase to generate a beta-aminoethyl mercaptan compound shown in formula II; wherein R1 and R2 are each independently selected from H or an optionally-substituted C1-C5 straight-chain or branched alkyl. The preparation method of the beta-aminoethyl mercaptan compound provided by the application avoids the use of highly toxic raw materials, a large amount of acid and alkali and organic reagents, and the enzyme catalytic reaction process is more moderate, the safety is greatly improved, and the method has high product molar conversion rate and high product purity, and has wide industrial development and application prospect.

Description

Technical Field

[0001] This invention relates to the field of biosynthesis technology, and more specifically to a method for biosynthesizing β-aminoethanethiol compounds using tryptophan synthase. Background Technology

[0002] β-Aminoethanethiols are a class of key chemical raw materials containing both thiol and amino (or amine) functional groups, with important applications in pharmaceuticals, food, pesticides, and new material development. Among them, 2-aminoethanethiol, dimethylaminoethanethiol, and diethylaminoethanethiol are the three most widely used compounds in this class—2-aminoethanethiol is an important active ingredient in pharmaceuticals and biochemistry, diethylaminoethanethiol is mainly used in pharmaceuticals and chemical intermediates and synthesis, while dimethylaminoethanethiol plays a key role in catalysis and material modification.

[0003] Currently, the industrial preparation of β-aminoethanethiol compounds mainly relies on traditional chemical synthesis methods, and there are two main industrial synthesis routes:

[0004] The first method is the isothiourea method (as disclosed in CN101250145B), which involves reacting diethylaminochloroethane with thiourea to obtain isothiourea salt, which is then hydrolyzed under strong alkaline conditions to obtain diethylaminoethyl mercaptan. Although the yield of this method can reach more than 80%, it requires three steps, is cumbersome, and consumes a large amount of acid, alkali and organic solvent.

[0005] The second method is the reaction of cyclothioethane with ammonia (such as those disclosed in CN112661679A and CN102153494A), which utilizes the addition reaction of cyclothioethane with amine compounds to obtain β-aminoethanethiol compounds. Although the process is simple and the yield is high, the raw material cyclothioethane is highly toxic and easily polymerized, posing a high safety risk during storage and use.

[0006] With the development of synthetic biology, its advantages of high efficiency, environmental friendliness, high specificity, and mild reaction have provided new pathways for chemical synthesis. Given the important applications of β-aminoethanethiol compounds, and considering the high consumption of acids, bases, and organic solvents and insufficient safety in existing chemical synthesis methods, developing a method for preparing β-aminoethanethiol compounds using biosynthesis to overcome traditional process bottlenecks has become an urgent technical problem to be solved. Summary of the Invention

[0007] Therefore, in order to solve the problems existing in the preparation of β-aminoethanethiol compounds by chemical synthesis in the prior art, the purpose of this invention is to provide a method for preparing β-aminoethanethiol compounds by enzyme catalysis, which not only improves process safety, but also has high molar conversion rate and high product purity, and has important industrial application value.

[0008] As used in this application, the term "tryptophan synthase (TrpS)" has a conventional definition generally recognized by those skilled in the art. This enzyme is mainly found in bacteria, fungi, plants, and some protozoa, such as Escherichia coli. It is a tetramer (α2β2) composed of an α subunit (encoded by the TrpA gene) and a β subunit (encoded by the TrpB gene). It is a key enzyme that catalyzes the biosynthesis of tryptophan for the industrial production of tryptophan.

[0009] The inventors of this application have discovered through research that this enzyme can be used to catalyze β-aminoethanol compounds, thereby realizing the biosynthesis of β-aminoethanethiol compounds.

[0010] The technical solution for achieving the above-mentioned objectives of this invention is as follows:

[0011] This invention provides a method for preparing β-aminoethanethiol compounds, comprising:

[0012] The β-aminoethanol compounds shown in Formula I and sodium hydrosulfide undergo an enzymatic reaction in a reaction system containing tryptophan synthase to generate the β-aminoethanethiol compounds shown in Formula II.

[0013]

[0014] R1 and R2 are each independently selected from H or optionally substituted C1-C5 straight-chain or branched alkyl groups.

[0015] According to some embodiments of the present invention, R1 and R2 are each independently H, -CH3 or -CH2CH3.

[0016] According to some embodiments of the present invention, the β-aminoethanol compound represented by Formula I is 2-aminoethanol, and the β-aminoethanethiol compound represented by Formula II is 2-aminoethanethiol.

[0017] According to some embodiments of the present invention, the β-aminoethanol compound represented by Formula I is dimethylaminoethanol, and the β-aminoethanethiol compound represented by Formula II is dimethylaminoethanethiol.

[0018] According to some embodiments of the present invention, the β-aminoethanol compound represented by Formula I is diethylaminoethanol, and the β-aminoethanethiol compound represented by Formula II is diethylaminoethanethiol.

[0019] According to some embodiments of the present invention, the amino acid sequence of the α subunit of the tryptophan synthase comprises or is composed of the following amino acid sequence:

[0020] (1) The amino acid sequence shown in SEQ ID NO. 1;

[0021] (2) An amino acid sequence obtained by deleting, substituting, or adding one or more amino acids, such as 1, 2, 3, 4, or 5, from the amino acid sequence shown in SEQ ID NO. 1; or

[0022] (3) An amino acid sequence having more than 90% homology to the amino acid sequence shown in SEQ ID NO. 1, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%;

[0023] The amino acid sequence of the β subunit of the tryptophan synthase contains or is composed of the following amino acid sequences:

[0024] (1) The amino acid sequence shown in SEQ ID NO. 2;

[0025] (2) An amino acid sequence obtained by deleting, substituting, or adding one or more amino acids, such as 1, 2, 3, 4, or 5, from the amino acid sequence shown in SEQ ID NO. 2; or

[0026] (3) An amino acid sequence having more than 90% homology to the amino acid sequence shown in SEQ ID NO. 2, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%.

[0027] According to some preferred embodiments of the present invention, the amino acid sequence of the α subunit of the tryptophan synthase comprises or is composed of the amino acid sequence shown in SEQ ID NO. 1, and the amino acid sequence of the β subunit of the tryptophan synthase comprises or is composed of the amino acid sequence shown in SEQ ID NO. 2.

[0028] According to some embodiments of the present invention, the method for preparing tryptophan synthase includes: cleaving Escherichia coli cells expressing tryptophan synthase to obtain an aqueous solution containing tryptophan synthase. Preferably, the concentration of the aqueous solution containing tryptophan synthase is 10-100 U / mL, more preferably 20-60 U / mL.

[0029] Preferably, the Escherichia coli expressing tryptophan synthase is a recombinant Escherichia coli strain containing a recombinant vector for expressing tryptophan synthase.

[0030] According to some embodiments of the present invention, the recombinant Escherichia coli strain for expressing tryptophan synthase is obtained by cloning the gene encoding the α subunit of the tryptophan synthase and the gene encoding the β subunit of the tryptophan synthase into different recombinant expression vectors, or by tandem cloning them into the same recombinant expression vector, and then introducing the recombinant expression vector into a recombinant Escherichia coli strain.

[0031] According to some specific embodiments of the present invention, the gene encoding the α subunit of tryptophan synthase, the sequence encoding the nucleotide of the conserved ribosome binding site, and the gene encoding the β subunit of tryptophan synthase are sequentially linked together and cloned into the pET28a expression vector. The recombinant pET28a expression vector is then introduced into a recombinant Escherichia coli strain to obtain the recombinant Escherichia coli strain used to express tryptophan synthase.

[0032] According to some embodiments of the present invention, the molar ratio of the β-aminoethanol compound represented by Formula I to sodium hydrosulfide is 1:1-3, preferably 1:1-1.8.

[0033] According to some embodiments of the present invention, the concentration of the β-aminoethanol compound represented by Formula I in the reaction system is 10-200 g / L, preferably 30-60 g / L.

[0034] According to some embodiments of the present invention, the reaction system further comprises pyridoxal phosphate, which acts as a coenzyme to assist the enzymatic reaction.

[0035] According to some embodiments of the present invention, the concentration of pyridoxal phosphate in the reaction system is 10-60 mg / L, preferably 20-40 mg / L.

[0036] According to some embodiments of the present invention, the concentration of the tryptophan synthase in the reaction system is 0.3-3 U / mL, preferably 0.6-1.5 U / mL.

[0037] According to some embodiments of the present invention, the enzymatic reaction is carried out at pH 8-9.

[0038] According to some embodiments of the present invention, the enzymatic reaction is carried out at 30°C to 40°C.

[0039] According to some embodiments of the present invention, the enzymatic reaction takes 6-18 hours, preferably 12 hours.

[0040] According to some embodiments of the present invention, the preparation method further includes: separating and purifying the obtained β-aminoethanethiol compound after the enzymatic reaction is completed. Preferably, the separation and purification includes: extracting the solution after the enzymatic reaction with petroleum ether, drying the organic phase, and then distilling to obtain the β-aminoethanethiol compound represented by Formula II; more preferably, the distillation is atmospheric distillation at 80℃ to 100℃.

[0041] Compared with existing technologies, this invention prepares β-aminoethanethiol compounds by reacting the substrate β-aminoethanol compounds with sodium hydrosulfide catalyzed by tryptophan synthase. This reaction process avoids the use of highly toxic raw materials or large amounts of acids, alkalis and organic reagents, thus improving process safety. Moreover, the product has high molar conversion rate and high purity, and has important industrial application value. Attached Figure Description

[0042] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0043] Figure 1 The present invention provides a reaction flow diagram for preparing β-aminoethanethiol compounds of formula II from β-aminoethanol compounds of formula I. R1 and R2 are as defined herein. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0045] Where specific steps or conditions are not specified in the examples, they shall be performed in accordance with the steps or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0046] In the following examples, the chromatographic conditions for detecting the purity of diethylaminoethanethiol or 2-aminoethanethiol using gas chromatography are as follows:

[0047] Equipment: Shimadzu GC2014C gas chromatograph; Column model: DB-624, dimensions: 30m × 0.53mm × 30μm; Detector: Flame ionization detector; Nitrogen flow rate: 30 mL / min; Hydrogen flow rate: 40 mL / min; Air flow rate: 300 mL / min; Split ratio: 30:1; Column flow rate: 1.0 mL / min; Column temperature: 80℃; Detector temperature: 250℃; Injector temperature: 200℃; Injection volume: 0.5μL;

[0048] Operating procedure: Hold at 80℃ for 4 min, increase the temperature to 160℃ at a rate of 8℃ / min, hold for 2 min, increase the temperature to 220℃ at a rate of 8℃ / min, and hold for 10 min; after the equipment and baseline have stabilized, inject the sample by drawing 0.5 μL of sample into the chromatograph using the injection needle, and collect samples for at least 10 min.

[0049] The conditions for determining the purity of dimethylaminoethanol by high performance liquid chromatography-tandem mass spectrometry are as follows:

[0050] Chromatographic column type: ZORBAX SB Aq, size: 150mm×4.6mm×3.5μm; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: methanol; flow rate: 0.40mL / min; column temperature: 40℃; injection volume: 2μL; isocratic elution, elution program: 0-5min, 20% (v / v) mobile phase B, 80% (v / v) mobile phase A;

[0051] Mass spectrometry scanning method: multiple reaction monitoring (MRM); spray voltage: 3500V; nebulizer gas pressure GS1: 35psi; auxiliary gas temperature: 300℃; gas flow rate: 9L / min.

[0052] Preparation of crude enzyme solution:

[0053] 1) According to publicly available information in the NCBI database, the E. coli-derived tryptophan synthase TrpS consists of an α subunit (TrpA, GenBank: AAA57301.1) and a β subunit (TrpB, GenBank: AAA57300.1), with the amino acid sequence of TrpA shown in SEQ ID NO. 1 and the amino acid sequence of TrpB shown in SEQ ID NO. 2. The coding gene for the α subunit TrpA, the sequence encoding the conserved ribosome binding site, and the coding gene for the β subunit TrpB of tryptophan synthase were tandemly linked, with the sequence encoding the conserved ribosome binding site shown in SEQ ID NO. 3. Shanghai Sangon Biotech Co., Ltd. was commissioned to synthesize this tandem sequence, which was then ligated into the pET28a vector to obtain the recombinant vector pET28a-TrpS.

[0054] SEQ ID NO. 1:

[0055] MERYESLFAQLKERKEGAFVPFVTLGDPGIEQSLKIIDTLIEAGADALELGIPFSDPLADGPTIQNATLRAFAAGVTPAQCFEMLALIRQKHPTIPIGLLMYANLVFNKGIDEFYAQCEKVGVDSVLVADVPVEESAPFRQAALRHNVAPIFICPPNADDDLLRQIASYGRGYTYLLSRAGVTGAENRAALPLNHLVAKLKEYNAAPPLQGFGISAPDQVKAAIDAGAAGAISGSAIVKIIEQHINEPEKMLAALKVFVQPMKAATRS

[0056] SEQ ID NO. 2:

[0057] MTTLLNPYFGEFGGMYVPQILMPALRQLEEAFVSAQKDPEFQAQFNDLLKNYAGRPTALTKCQNITAGTNTTLYLKREDLLHGGAHKTNQVLGQALLAKRMGKTEIIAETGAGQHGVASALASALLGLKCRIYMGAKDVERQSPNVFRMRLMGAEVIPVHSGSATLKDACNEALRDWSGSYETAHYMLGTAAGPHPYPTIVREFQRMIGEETKAQILEREGRLPDAVIACVGGGSNAIGMFADFINETNVGLIGVEPGGHGIETGEHGAPLKHGRVGIYFGMKAPMMQTEDGQIEESYSISAGLDFPSVGPQHAYLNSTGRADYVSITDDEALEAFKTLCLHEGIIPALESSHALAHALKMMRENPDKEQLLVVNLSGRGDKDIFTVHDILKARGEI

[0058] SEQ ID NO. 3: TTTGTTTAACTTTAAGAAGGAGATATACAT

[0059] 2) Introduce the above recombinant vector into Escherichia coli BL21(DE3) competent cells to obtain the recombinant expression strain BL21(DE3) / pET28a-TrpS;

[0060] 3) The glycerol strain of the above recombinant expression strain was inoculated into liquid LB medium containing kanamycin resistance and cultured overnight at 37°C and 250 rpm until OD600 = 1~2; then, it was transferred to liquid TB medium containing kanamycin at a 1% inoculum and cultured at 37°C with shaking until OD600 reached 3~5. IPTG was added to a final concentration of 0.1 mM and induced at 25°C for 8-10 h; finally, the fermentation broth was centrifuged at 4°C and 10000 rpm to collect the bacterial cells.

[0061] 4) Use 20mM, pH7.0 phosphate buffer to disperse and suspend the bacterial cells to a bacterial solution with a mass concentration of 3%-10%, and then perform ultrasonic cell disruption to obtain crude tryptophan synthase solution with enzyme activity >10 U / mL.

[0062] Example 1: Preparation of diethylaminoethanethiol

[0063] Add 100 mL of pure water, 5 g of diethylaminoethanol, and 10.6 g of 35% sodium hydrosulfide aqueous solution to a 500 mL four-necked flask. Start stirring at 200 rpm, heat to 35 °C, adjust the pH to 8.5, then add 3 mg of PLP and 3 mL of the prepared crude enzyme solution to begin the reaction. During the reaction, maintain the pH of the reaction system at 8.5 using 3M sulfuric acid. After 4 hours of reaction, sample the remaining substrate and product amount to detect the reaction. Stop the reaction after 12 hours, yielding 118.24 g of reaction solution, of which 4.635 g was product, with a product content of 3.92%.

[0064] Based on the calculation of the molar conversion rate of diethylaminoethanol: Given that the molecular weight of diethylaminoethanol is 117.19 and the molecular weight of diethylaminoethanethiol is 133.2, the molar conversion rate is calculated to be (4.635 / 133.2) / (5 / 117.19) = 81.5%, that is, the molar conversion rate of this example is 81.5%.

[0065] Example 2 Preparation of diethylaminoethanethiol

[0066] 100 mL of pure water, 5 g of diethylaminoethanol, and 7.51 g of 35% sodium hydrosulfide aqueous solution were added sequentially to a 500 mL four-necked flask. Stirring was started at 200 rpm, and the temperature was raised to 35 °C. The pH was adjusted to 8.5, and then 3 mg of PLP and 5 mL of the prepared crude enzyme solution were added to initiate the reaction. During the reaction, the pH of the reaction system was maintained at 8.5 using 3M sulfuric acid. After 4 hours of reaction, samples were taken to detect the remaining substrate and the amount of product formed. The reaction was stopped after 12 hours, yielding 117.51 ​​g of reaction solution, with a product content of 3.64%. Based on the method in Example 1, the molar conversion rate was calculated to be 75.3%.

[0067] Example 3 Preparation of diethylaminoethanethiol

[0068] 100 mL of pure water, 5 g of diethylaminoethanol, and 12.29 g of 35% sodium hydrosulfide aqueous solution were added sequentially to a 500 mL four-necked flask. Stirring was started at 200 rpm, and the temperature was raised to 35°C. The pH was adjusted to 8.5, and then 3 mg of PLP and 4 mL of the prepared crude enzyme solution were added to initiate the reaction. During the reaction, the pH of the reaction system was maintained at 8.5 using 3M sulfuric acid. After 4 hours of reaction, samples were taken to detect the remaining substrate and the amount of product generated. The reaction was stopped after 12 hours, yielding 121.29 g of reaction solution, with a product content of 4.19%. According to the method in Example 1, the molar conversion rate was calculated to be 89.4%.

[0069] Example 4 Preparation of diethylaminoethanethiol

[0070] 100 mL of pure water, 5 g of diethylaminoethanol, and 10.24 g of 35% sodium hydrosulfide aqueous solution were added sequentially to a 500 mL four-necked flask. Stirring was started at 200 rpm, and the temperature was raised to 30°C. The pH was adjusted to 8, and then 3 mg of PLP and 4 mL of the prepared crude enzyme solution were added to initiate the reaction. During the reaction, the pH of the reaction system was maintained at 8 using 3M sulfuric acid. After 4 hours of reaction, samples were taken to detect the remaining substrate and the amount of product generated. The reaction was stopped after 12 hours, yielding 119.24 g of reaction solution, with a product content of 3.84%. According to the method in Example 1, the molar conversion rate was calculated to be 80.6%.

[0071] Example 5 Preparation of diethylaminoethanethiol

[0072] 100 mL of pure water, 5 g of diethylaminoethanol, and 10.24 g of 35% sodium hydrosulfide aqueous solution were added sequentially to a 500 mL four-necked flask. Stirring was started at 200 rpm, and the temperature was raised to 40 °C. The pH was adjusted to 9, and then 3 mg of PLP and 4 mL of the prepared crude enzyme solution were added to initiate the reaction. During the reaction, the pH of the reaction system was maintained at 9 using 3M sulfuric acid. After 4 hours of reaction, samples were taken to detect the remaining substrate and the amount of product generated. The reaction was stopped after 12 hours, yielding 119.24 g of reaction solution, with a product content of 3.91%. According to the method in Example 1, the molar conversion rate was calculated to be 82.1%.

[0073] Example 6 Preparation of 2-Aminoethanethiol

[0074] 100 mL of pure water, 3 g of 2-aminoethanol, and 11.79 g of a 35% sodium hydrosulfide aqueous solution were added sequentially to a 500 mL four-necked flask. Stirring was started at 200 rpm, and the temperature was raised to 35°C. The pH was adjusted to 8.5, and then 3 mg of PLP and 5 mL of the prepared crude enzyme solution were added to initiate the reaction. During the reaction, the pH of the reaction system was maintained at 8.5 using 3M sulfuric acid. After 4 hours of reaction, samples were taken to detect the remaining substrate and the amount of product generated. The reaction was stopped after 12 hours, yielding 119.79 g of reaction solution, with a product content of 2.61%. According to the method in Example 1, the molar conversion rate was calculated to be 82.5%.

[0075] Example 7 Preparation of dimethylaminoethanethiol

[0076] 100 mL of pure water, 4 g of dimethylaminoethanol, and 10.77 g of 35% sodium hydrosulfide aqueous solution were added sequentially to a 500 mL four-necked flask. Stirring was started at 200 rpm, and the temperature was raised to 35 °C. The pH was adjusted to 8.5, and then 3 mg of PLP and 5 mL of the prepared crude enzyme solution were added to initiate the reaction. During the reaction, the pH of the reaction system was maintained at 8.5 using 3M sulfuric acid. After 4 hours of reaction, samples were taken to detect the remaining substrate and product yield. The reaction was stopped after 12 hours, yielding 119.77 g of reaction solution, with a product content of 3.23%. The calculated molar conversion rate of the substrate was 81.9%.

[0077]

[0078] Table 1 summarizes the main parameters and substrate molar conversion rates of the preparation methods in Examples 1-7. A comparison reveals that:

[0079] (1) The three β-aminoethanol compounds, namely diethylaminoethanol, 2-aminoethanol and dimethylaminoethanol, can achieve high molar conversion rates (≥75.3%, 82.5% and 81.9%, respectively) as substrates, indicating that the preparation method provided by the present invention has good catalytic applicability to these substrates.

[0080] (2) Appropriately increasing the amount of sodium hydrosulfide is beneficial to improving the molar conversion rate of the reaction;

[0081] (3) Comparing Examples 4 and 5, it can be seen that when the amount of sodium hydrosulfide and the amount of crude enzyme solution are the same, the reaction temperature increases from 30℃ to 40℃, and the reaction pH increases from 8 to 9. The molar conversion rate increases from 80.6% to 82.1%. This shows that appropriately increasing the reaction temperature and reaction pH in the range of 30℃-40℃ can promote the enzymatic reaction and increase the molar conversion rate to a certain extent.

[0082] Example 8: Separation and purification of diethylaminoethanethiol

[0083] 220 mL of petroleum ether was added to 118.24 g of the reaction solution obtained in Example 1, and the mixture was thoroughly mixed. The mixture was then placed in a separatory funnel and allowed to stand for separation. The aqueous layer and ether layer were separated. Another 220 mL of petroleum ether was added to the aqueous layer for extraction, and the combined ether layers were collected. 10 g of anhydrous calcium chloride was added to the ether layer as a drying agent. The dried ether layer was then distilled at 90 °C under normal pressure until no fraction was effluent. After distillation, the bottom oily residue was collected as the product diethylaminoethanethiol, weighing 4.36 g. Gas chromatography analysis showed that the content of diethylaminoethanethiol was 99.5%, the molar yield based on diethylaminoethanol was 76.34%, and the yield of the extraction step was 93.66%.

[0084] Given that the molecular weight of diethylaminoethanol is 117.19 and the molecular weight of diethylaminoethanethiol is 133.2, the calculation process for the molar yield and extraction yield is as follows: Purified product = product weight / product content = 4.36 * 99.5% = 4.338 g, therefore the molar yield of the product is 4.338 / 133.2 = 0.03256 mol; With 5 g of diethylaminoethanol added, the molar yield is 5 / 117.19 = 0.04266 mol; therefore, the molar yield is 0.03256 / 0.04266 = 76.34%, and the extraction yield is 4.338 / 4.635 = 93.66%.

Claims

1. A method for preparing a β-aminoethanethiol compound, comprising: The β-aminoethanol compounds shown in Formula I and sodium hydrosulfide undergo an enzymatic reaction in a reaction system containing tryptophan synthase to generate the β-aminoethanethiol compounds shown in Formula II. R1 and R2 are each independently selected from H or optionally substituted C1-C5 straight-chain or branched alkyl groups.

2. The preparation method according to claim 1, wherein, R1 and R2 are each independently H, -CH3, or -CH2CH3; Preferably, the β-aminoethanol compound represented by Formula I is 2-aminoethanol, and the β-aminoethanethiol compound represented by Formula II is 2-aminoethanethiol; Preferably, the β-aminoethanol compound represented by Formula I is dimethylaminoethanol, and the β-aminoethanethiol compound represented by Formula II is dimethylaminoethanethiol; Preferably, the β-aminoethanol compound represented by Formula I is diethylaminoethanol, and the β-aminoethanethiol compound represented by Formula II is diethylaminoethanethiol.

3. The preparation method according to claim 1 or 2, wherein, The amino acid sequence of the α subunit of the tryptophan synthase contains or is composed of the following amino acid sequences: (1) The amino acid sequence shown in SEQ ID NO. 1; (2) An amino acid sequence obtained by deleting, substituting, or adding one or more amino acids, such as 1, 2, 3, 4, or 5, from the amino acid sequence shown in SEQ ID NO. 1; or (3) An amino acid sequence having more than 90% homology to the amino acid sequence shown in SEQ ID NO. 1, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%; The amino acid sequence of the β subunit of the tryptophan synthase contains or is composed of the following amino acid sequences: (1) The amino acid sequence shown in SEQ ID NO. 2; (2) An amino acid sequence obtained by deleting, substituting, or adding one or more amino acids, such as 1, 2, 3, 4, or 5, from the amino acid sequence shown in SEQ ID NO. 2; or (3) An amino acid sequence having more than 90% homology to the amino acid sequence shown in SEQ ID NO. 2, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%.

4. The preparation method according to any one of claims 1-3, wherein, The method for preparing tryptophan synthase includes: breaking down Escherichia coli cells expressing tryptophan synthase to obtain an aqueous solution containing tryptophan synthase; Preferably, the aqueous solution containing tryptophan synthase has an enzyme activity of 10-100 U / mL, more preferably 20-60 U / mL; Preferably, the Escherichia coli expressing tryptophan synthase is a recombinant Escherichia coli strain containing a recombinant vector for expressing tryptophan synthase; Preferably, the recombinant Escherichia coli strain for expressing tryptophan synthase is obtained by cloning the gene encoding the α subunit of the tryptophan synthase and the gene encoding the β subunit of the tryptophan synthase into different recombinant expression vectors, or by tandem cloning them into the same recombinant expression vector, and then introducing the recombinant expression vector into the Escherichia coli recombinant strain.

5. The preparation method according to any one of claims 1-4, wherein, The molar ratio of the β-aminoethanol compound shown in Formula I to sodium hydrosulfide is 1:1-3, preferably 1:1-1.

8.

6. The preparation method according to any one of claims 1-5, wherein, The concentration of the β-aminoethanol compound shown in Formula I in the reaction system is 10-200 g / L, preferably 30-60 g / L; Preferably, the concentration of the tryptophan synthase in the reaction system is 0.3-3 U / mL, more preferably 0.6-1.5 U / mL.

7. The preparation method according to any one of claims 1-6, wherein, The reaction system also contains pyridoxal phosphate; Preferably, the concentration of pyridoxal phosphate in the reaction system is 10-60 mg / L, and more preferably 20-40 mg / L.

8. The preparation method according to any one of claims 1-7, wherein, The enzymatic reaction was carried out at pH 8-9; Preferably, the enzymatic reaction is carried out at 30°C to 40°C; Preferably, the enzymatic reaction takes 6-18 hours, more preferably 12 hours.

9. The preparation method according to any one of claims 1-8, further comprising: The β-aminoethanethiol compounds were separated and purified after the enzymatic reaction was completed.

10. The preparation method according to claim 9, wherein, The separation and purification process includes: extracting the solution after the enzymatic reaction is completed with petroleum ether, and then drying the organic phase and distilling it to obtain the β-aminoethanethiol compound represented by Formula II; Preferably, the distillation is atmospheric distillation at 80°C to 100°C.

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