Multi-enzyme cascade catalytic reaction system and application thereof

Abstract

This invention provides a multi-enzyme cascade catalytic reaction system and its application, belonging to the field of enzyme catalysis production technology. Using glycerol as a raw material, it includes a first module, a second module, and a third module. The first module involves the production of glyceric acid from glycerol under the catalysis of alditol oxidase. The second module contains glyceric acid, ATP, and NAD+. + L-glutamic acid is synthesized into phosphoserine via glycerate kinase, D-3-phosphoglycerate dehydrogenase, and phosphoserine transaminase. The third module involves phosphoserine and nucleophiles being catalyzed by phosphoserine thioylase to synthesize L-cysteine ​​or a non-natural L-α-amino acid. The multi-enzyme cascade catalytic reaction system provided by this invention can catalyze a variety of nucleophiles, yields high products, is low in cost, causes minimal environmental pollution, and is easily industrialized.

Description

Technical Field

[0001] This invention relates to the field of enzyme catalysis production technology, specifically to multi-enzyme cascade catalytic reaction systems and their applications. Background Technology

[0002] L-cysteine ​​is one of the 20 naturally occurring amino acids and plays an important physiological role. In most organisms, it is the only pathway for reduced sulfur to enter cellular metabolism. As an important sulfur-containing amino acid, L-cysteine ​​is widely used in food, medicine, cosmetics, and other fields. Currently, the main methods for producing L-cysteine ​​include hair hydrolysis, enzymatic synthesis, and fermentation. Traditional hair hydrolysis and enzymatic synthesis methods suffer from low yields and severe environmental pollution. While fermentation offers an environmentally friendly and cost-effective approach, the cytotoxicity and strict metabolic regulation of L-cysteine ​​make bacterial fermentation production a significant challenge in the amino acid fermentation industry. Therefore, there is an urgent need to develop a green and efficient method for preparing L-cysteine.

[0003] The 20 naturally occurring amino acids that make up proteins represent only a portion of the biologically important amino acids. Non-natural amino acids, with their diverse functional groups, D-type stereochemistry, and atypical carbon skeletons, enrich the complexity of the amino acid pool. The unique chemical and biological properties of non-natural amino acids have attracted widespread attention in protein engineering and drug discovery. Incorporating non-natural amino acids into proteins provides a rational method for regulating their physical and chemical properties; non-natural amino acids are widely used in antibody-drug conjugates, and due to their unique orthogonal coupling properties, site specificity, and low toxicity, they can lead to better therapeutic approaches; simultaneously, adding non-natural amino acids to antimicrobial peptides can effectively improve their physicochemical and pharmacological diversity. Although non-natural amino acids are valuable biological tools, current production methods such as chemical synthesis, fermentation, and biocatalysis have limitations in terms of cost, yield, and product range. Chemical synthesis uses intermediates such as serine-derived lactones, hydantoin, or aziridine to synthesize non-natural amino acids; this method requires the use of harmful reagents, generates large amounts of waste, or requires further racemization of the products. Fermentation requires a host capable of synthesizing non-natural amino acids, but many non-natural amino acid synthesis pathways are low-yield or even remain unclear. Biocatalysis utilizes enzymes such as ammonia lyases, tyrosine-phenol lyases, and tryptophan synthases to produce optically pure non-natural amino acids from simple and inexpensive substrates. However, thermodynamically controlled reversibility or a limited substrate range remains an obstacle to the synthesis of non-natural amino acids. Summary of the Invention

[0004] The purpose of this invention is to provide a multi-enzyme cascade catalytic reaction system that can catalyze a variety of nucleophiles, has high product yield, low cost, low environmental pollution, and is easy to industrialize, and its application in the preparation of L-cysteine ​​or non-natural L-α-amino acids.

[0005] To achieve the above objectives, the present invention provides a multi-enzyme cascade catalytic reaction system. This system uses glycerol as a raw material and includes a first module, a second module, and a third module. The first module involves the conversion of glycerol to glyceric acid under the catalysis of alditol oxidase. The second module contains glyceric acid, ATP, and NAD+. + L-glutamic acid is synthesized into phosphoserine via glycerokinase, D-3-phosphoglycerate dehydrogenase, and phosphoserine transaminase. The third module is phosphoserine and nucleophilic reagents are synthesized into L-cysteine ​​or non-natural L-α-amino acids via phosphoserine thiolase.

[0006] As a preferred embodiment, catalase is added to the first module; and polyphosphokinase and glutamate dehydrogenase are added to the second module. The addition of catalase facilitates oxygen regeneration and prevents hydrogen peroxide from damaging the reaction system; the addition of polyphosphokinase and glutamate dehydrogenase facilitates the production of cofactors ATP and NAD+. + And the regeneration of L-glutamic acid.

[0007] As a preferred embodiment, in the second module, the ATP concentration is 0.5-5 mM, and the NAD concentration is... + The concentration is 0.5-5 mM, and the L-glutamate concentration is 5-50 mM. The preferred ATP concentrations are 0.5 mM, 1 mM, 2 mM, and 3 mM, and NAD+ concentrations are... + The preferred concentrations are 0.5 mM, 1 mM, 2 mM, and 3 mM, and the preferred concentrations of L-glutamic acid are 20 mM, 30 mM, and 40 mM.

[0008] As a preferred option, 5-50 mM Mg is also added to the second module. 2+ 10-50 mM polyP and 50-200 mM ammonium chloride. Among them, Mg... 2+ The preferred concentrations are 20mM, 30mM, and 40mM, the preferred polyP concentrations are 20mM, 30mM, and 40mM, and the preferred ammonium chloride concentrations are 80mM, 100mM, and 150mM.

[0009] As a preferred embodiment, the amino acid sequence of the phosphoserine thioylase is shown in SEQ ID NO.3, or a mutant thereof, the mutant sequence of which is shown in SEQ ID NO.4. In the mutant, Thr at position 120 of the phosphoserine thioylase amino acid sequence is mutated to Gln, Phe at position 144 to Tyr, Thr at position 178 to Cys, and Ala at position 218 to Arg, while other amino acid residues remain unchanged. Under the same reaction conditions, using the phosphoserine thioylase mutant can significantly improve production efficiency.

[0010] As a preferred embodiment, in the third module, the nucleophile is a nucleophilic compound of general formula (1):

[0011] Thiol R1-SH(1),

[0012] R1 is a monovalent substituted or unsubstituted alkyl, aryl, or heteroaryl group, and the catalytic product is L-cysteine ​​or a non-natural L-α-amino acid.

[0013] As a preferred embodiment, the nucleophile is sodium sulfide, 3-mercapto-1,2-propanediol, 1-naphthiophenol, cyclopentanethiol, mercaptoacetic acid, 2-mercaptoethanol, 2-aminoethanethiol, benzylthiol, sodium thiosulfate, 4-chlorobenzylthiol, thiophenethiol, 4-(trifluoromethyl)-benzylthiol, allylthiol, 4-nitrobenzylthiol, 4-cyanobenzylthiol, triphenylmethanethiol, 2-ethyl-1-hexylthiol, 2-bromobenzylthiol, or 2-pyrazinylethanethiol.

[0014] As a preferred embodiment, in the third module, the nucleophile is a nucleophilic compound of general formula (2):

[0015] Selenol R2-Se-H(2),

[0016] R2 is a monovalent substituted or unsubstituted aryl group, in which case the catalytic product is a non-natural L-α-amino acid.

[0017] As a preferred embodiment, the nucleophile is selenoylbenzene or 3,5-dimethylselenoylbenzene.

[0018] As a preferred embodiment, the nucleophile is an azole of general formula (3) or (4):

[0019]

[0020] Where X and Y are the same or different and represent C or N, and R3, R4, R5, R6, R7, and R8 are the same or different and represent H, COOH, OH, NH2, Br, I, F, Cl, NO2, CN, or alkyl, the catalytic product is a non-natural L-α-amino acid.

[0021] As a preferred embodiment, the nucleophile is pyrazole, 3-methylpyrazole, 3-cyanopyrazole, 5-hydroxypyrazole, 4-aminopyrazole, 5-iodo-1H-pyrazole, 3,5-dimethylpyrazole, 1,2,3-triazole, 1,2,4-triazole, 3-amino-1,2,4-triazole, indazole, indazole-3-carboxylic acid, indazole-3-carboxylic acid, or 5-aminoindazole.

[0022] As a preferred embodiment, the concentration of the nucleophile in the third module is greater than the concentration of phosphoserine generated in the second module.

[0023] As a preferred embodiment, the catalytic reaction of the first module has a pH of 5.0–9.0, a temperature of 20–50°C, and a reaction time of 8–24 hours; the catalytic reaction of the second module has a pH of 5.0–9.0, a temperature of 20–60°C, and a reaction time of 2–10 hours; and the catalytic reaction of the third module has a pH of 5.0–9.0, a temperature of 30–60°C, and a reaction time of 1–6 hours.

[0024] This invention also discloses the application of the above-mentioned multi-enzyme cascade catalytic reaction system in the preparation of L-cysteine ​​or non-natural L-α-amino acids.

[0025] The present invention also discloses a phosphoserine thioylase mutant, the sequence of which is shown in SEQ ID NO.4.

[0026] The reaction pathway for the entire process is shown below:

[0027]

[0028] In the third module of this invention, the nucleophile is sodium sulfide from thiols of general formula (1), and the product is L-cysteine. The nucleophile is thiols of general formula (1) excluding sodium sulfide, selenool of general formula (2), azole of general formula (3) and (4), and the product is non-natural L-α-amino acid. The nucleophile is a nucleophile containing a benzene ring from thiols of general formula (1), a nucleophile containing a benzene ring from selenool of general formula (2), and an azole of general formula (3), and the product is a non-natural L-α-amino acid of aromatic acid.

[0029] The non-natural L-α-amino acid products of this invention refer to non-natural L-α-amino acids with modified side chains, that is, non-natural L-α-amino acids with different side chains (R groups) connected by CS bonds, C-Se bonds, and CN bonds.

[0030] As a preferred embodiment, catalase and D-3-phosphoglycerate dehydrogenase in this invention are derived from *Escherichia coli*, aldose alcohol oxidase (TfAldO) from *Thermopolyspora flexuosa* (GenBank TQM75161.1), glycerate kinase (ScG3K) from *Saccharomyces cerevisiae* (GenBank AAA66317.1), with its amino acid sequence shown in SEQ ID NO.1; phosphoserine transaminase (AbPSAT) from *Acinetobacter baylyi* (GenBank CAG69404.1), with its amino acid sequence shown in SEQ ID NO.2; polyphosphate kinase (SlPPK) from *Sulfurovum lithotrophicum* (GenBank AKF24980.1); and glutamate dehydrogenase (PaGDH) from *Peptoniphilus*. asaccharolyticus (GenBank AAA25611.1); Phosphosserine thiolase (AsOPSS) is derived from Acetobacterium sp (GenBank MBP8031540.1), and its amino acid sequence is shown in SEQ ID NO.3.

[0031] The three modules of the multi-enzyme cascade catalytic reaction system of this invention can be selected from suitable buffer solutions. As a preferred option, the buffer solution in this invention is Tris buffer.

[0032] The sequence of the enzyme in this invention is not specifically limited. Any enzyme with the same function can achieve the purpose of this invention. The vector used in this invention is not specifically limited. Any suitable vector known in the art can be used as long as the vector is reproducible in the host.

[0033] The advantages of this invention are that it uses inexpensive glycerol as a substrate, and converts the substrate into L-cysteine ​​or non-natural L-α-amino acids through in vitro multi-enzyme catalysis. The cofactor regeneration system in this invention effectively reduces NAD5 levels. + The dosage of ATP and glutamate significantly reduces production costs while achieving high yields and conversion rates. Furthermore, this invention modifies the phosphoserine sulfhydrylase to remove product inhibition; under the same reaction conditions, using the phosphoserine sulfhydrylase mutant significantly improves production efficiency. The phosphoserine sulfhydrylase in this invention can accept a wide range of nucleophiles to form CS, CN, and C-Se bonds, providing a green and efficient platform for the production of L-cysteine ​​and non-natural L-α-amino acids. Attached Figure Description

[0034] Figure 1 This is a standard curve for L-cysteine.

[0035] Figure 2 A diagram showing the synthesis of L-cysteine ​​using module three without optimized reaction conditions.

[0036] Figure 3 The diagram shows the optimized reaction conditions for the synthesis of L-cysteine ​​from multiple enzymes. Detailed Implementation

[0037] The present invention will now be described in detail with reference to specific embodiments. It should be understood that the following specific embodiments are only for the purpose of helping those skilled in the art to understand the present invention, and are not intended to limit the present invention.

[0038] Example 1: Enzyme Preparation

[0039] In this invention, catalase and D-3-phosphoglycerate dehydrogenase are derived from Escherichia Coli, and corresponding expression vectors pET28a-EcKatG and pET28a-EcPGDH are constructed.

[0040] Aldolipid oxidase (TfAldO) was derived from *Thermopolyspora flexuosa* (GenBank TQM75161.1); glycerate kinase (ScG3K) was derived from *Saccharomyces cerevisiae* (GenBank AAA66317.1), and its amino acid sequence is shown in SEQ ID NO.1; phosphoserine transaminase (AbPSAT) was derived from *Acinetobacter baylyi* (GenBank CAG69404.1), and its amino acid sequence is shown in SEQ ID NO.2; polyphosphate kinase (SlPPK) was derived from *Sulfurovum lithotrophicum* (GenBank AKF24980.1); glutamate dehydrogenase (PaGDH) was derived from *Peptoniphilus asaccharolyticus* (GenBank AAA25611.1); and phosphoserine thiolase (AsOPSS) was derived from *Acetobacterium sp.* (GenBank). MBP8031540.1), whose amino acid sequence is shown in SEQ ID NO.3.

[0041] TfAldO, ScG3K, AbPSAT, SlPPK, PaGDH, and AsOPSS were synthesized after codon optimization and then ligated into the pET-28a vector using BamHI and XhoI restriction sites to obtain recombinant plasmids pET28a-TfAldO, pET28a-ScG3K, pET28a-AbPSAT, pET28a-SlPPK, pET28a-PaGDH, and pET28a-AsOPSS.

[0042] The recombinant plasmid was transformed into *E. coli* strain BL21(DE3), the expression host strain purchased from Tulu Harbour Biotechnology Co., Ltd. The cells were cultured at 37°C until OD600 = 0.7, then IPTG was added, and the expression of the target protein was induced at 18°C. After 16 h of induction, the cells were collected by centrifugation at 12000 rpm for 10 min. The cells were resuspended in PBS buffer, hyperplatelet-rich, and centrifuged again to collect the supernatant for crude enzyme solution. The crude enzyme solution was purified by nickel affinity chromatography and eluted with phosphate buffer containing 200 mM imidazole to obtain the target protein.

[0043] Example 2: Multi-enzyme-level synthesis of L-cysteine

[0044] Using TfAldO, EcKatG, ScG3K, EcPGDH, AbPSAT, SlPPK, PaGDH, and AsOPSS obtained in Example 1, L-cysteine ​​was synthesized via multi-enzyme catalysis with glycerol and Na2S as substrates according to a modular strategy.

[0045] First module reaction: 1 ml reaction system containing 100 mM pH 7.0 potassium phosphate buffer, 100 mM glycerol, 2 U / ml TfAldO, and 100 U / ml EckatG, catalyzed at 40℃ for 24 hours.

[0046] Second module reaction: After the first module reaction is completed, add the substrate and enzyme for this module. Specifically, add 20mM sodium hexametaphosphate, 10mM ATP, and 10mM NAD. + The following ingredients were added: 30 mM glutamic acid, 200 mM ammonium chloride, 0.1 mM pyridoxal phosphate, 10 mM MgCl2, 1 U / ml ScG3K, 1 U / ml EcPGDH, 1 U / ml AbPSAT, 1 U / ml SlPPK, and 1 U / ml PaGDH. The total volume was controlled at 1.1 ml, and the reaction was carried out at 40 °C for 10 hours.

[0047] The third reaction module: After the second reaction module was completed, the substrate and enzyme for this module were added. Specifically, 200 mM Na2S and 1 U / ml AsOPSS were added, maintaining a total volume of 1.2 ml, and the reaction was continued for 6 hours. HPLC analysis was performed on the reaction solution at different time points.

[0048] The HPLC detection conditions were as follows: detection wavelength: 330 nm; column: WonderalSil C18 column; mobile phase: KH2PO4 solution (50 mM, pH 3.9) / 50% methanol = 90 / 10 (v / v); flow rate: 0.5 ml / min; temperature: 35 ℃; sample loading volume: 10 μl.

[0049] The reaction solution showed a chromatographic peak at 11.5 min, consistent with the retention time of the L-cysteine ​​standard, indicating that this invention can achieve multi-enzyme-level synthesis of L-cysteine. The reaction was then carried out according to the standard curve (…). Figure 1 Calculate the amount of L-cysteine ​​produced in the reaction solution. For example... Figure 2 As shown, 50.2 mM L-cysteine ​​was obtained, with a yield of 50.2%.

[0050] Example 3: Optimization of Multi-enzyme-level Combined System

[0051] The optimal conditions for the synthesis of L-cysteine ​​by multi-enzyme synthesis were obtained through condition optimization.

[0052] The initial reaction system for Module 1 was as described in Example 2. Reaction temperature optimization was performed between 20-50°C, and the results showed that the optimal temperature for Module 1 was 40°C. Figure 3 A). pH optimization was performed at pH 5-9, and the results showed that the optimal pH for Module 2 was 8.0, with Tris buffer being the most suitable buffer. Figure 3 B). Optimizing the reaction time using an 8-24 hour timeframe, the results show that the optimal reaction time for Module 1 is 12 hours. Figure 3 C).

[0053] The initial reaction system for Module 2 was as described in Example 2. Reaction temperature optimization was performed within the range of 20-60℃, and the results showed that the optimal temperature for Module 2 was 45℃. Figure 3 D). pH optimization was performed at pH 5-9, and the results showed that the optimal pH for Module 2 was 8.0, with Tris buffer being the most suitable buffer. Figure 3 E). The optimal amount of sodium hexametaphosphate (SHSP) added was selected from 10-50 mM, and the results showed that the optimal amount of SHSP for the reaction was 30 mM. Figure 3 F). The optimal ATP addition amount was selected from 0.5-5 mM, and the results showed that the optimal ATP addition amount for the reaction was 1 mM. Figure 3 G). Select 5-50mM for Mg.2+ The amount of Mg added was optimized, and the results showed that the optimal Mg content for the reaction was... 2+ The addition amount is 20mM ( Figure 3 H). Select 0.5-5mM for NAD. + The amount added was optimized, and the results showed that the optimal NAD for the reaction was... + The amount added is 2mM ( Figure 3 I). The optimal amount of glutamate added was selected from 5-50 mM, and the results showed that the optimal amount of glutamate added for the reaction was 30 mM. Figure 3 J). The optimal ammonium chloride addition amount was selected from 50-200 mM, and the results showed that the optimal ammonium chloride addition amount for the reaction was 100 mM. Figure 3 K). The reaction time was optimized between 2 and 10 hours, and the results showed that the optimal reaction time for Module 2 was 4 hours. Figure 3 L).

[0054] The initial reaction system for Module 3 was as described in Example 2. Reaction temperature optimization was performed within the range of 30-60℃, and the results showed that the optimal temperature for Module 3 was 50℃. Figure 3 pH optimization was performed at pH 5-9, and the results showed that the optimal pH for module three was 8.0. Figure 3 The optimal Na₂S addition amount was selected from 50-200 mM, and the results showed that the optimal Na₂S addition amount for the reaction was 100 mM. Figure 3 O). The reaction time was optimized by selecting 1-6 hours, and the results showed that the optimal reaction time for Module 3 was 4 hours. Figure 3 P).

[0055] Under the above-mentioned optimal reaction conditions, HPLC analysis was performed after the reaction was completed, and 66.8 mM L-cysteine ​​was synthesized with a yield of 66.8%.

[0056] Example 4: Modification of phosphoserine sulfhydrylase to improve production efficiency

[0057] To alleviate the product inhibition of phosphoserine thiolsase by L-cysteine ​​and non-natural L-α-amino acids and improve production efficiency, phosphoserine thiolsase was subjected to directed evolution to obtain a phosphoserine thiolsase mutant that could relieve product inhibition, and was named AsOPSSmut.

[0058] The AsOPSSmut mutant is obtained by mutating Thr at position 120 to Gln, Phe at position 144 to Tyr, Thr at position 178 to Cys, and Ala at position 218 to Arg in the OPSS amino acid sequence (SEQ ID NO.3), while keeping other amino acid residues unchanged. The resulting amino acid sequence is shown in SEQ ID NO.4.

[0059] AsOPSSmut was used for the production of L-cysteine. The enzyme preparation was the same as in Example 1, and the reaction system was the same as in Example 3. Reaction solutions at different time points were analyzed by HPLC. The results are as follows: Figure 3 As shown, after a total reaction time of 17 hours, i.e. the first hour of the third module reaction, the final concentration of L-cysteine ​​was 80 mM, the yield reached 80% (an increase of 19.7%), and the production rate was 379% higher than that of wild-type OPSS.

[0060] Example 5: Multi-enzyme-level synthesis of non-natural L-α-amino acids

[0061] The reaction system was as described in Example 2, except that the 100mM Na2S in Module 3 was replaced with 100mM of the other nucleophiles. Non-natural L-α-amino acids were synthesized by multi-enzyme catalysis according to the modular strategy. After the reaction was completed, HPLC detection was performed, and the results are shown in Table 1.

[0062] The HPLC detection conditions were as follows: detection wavelength: 210 nm; column: WonderalSil C18 column; mobile phase: 0.1% phosphoric acid / 100% acetonitrile = 95 / 5 (v / v); flow rate: 1.0 ml / min; temperature: 30 ℃; sample loading volume: 10 μl.

[0063] Table 1. Multi-enzyme-level synthesis of non-natural L-α-amino acids and aromatic acids

[0064]

[0065]

[0066] Although the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, these descriptions are intended to provide a clear understanding of the invention and not to limit its implementation. The present invention is not limited to the specific embodiments described, and any person skilled in the art can make various improvements and modifications to the invention without departing from its core spirit. Furthermore, embodiments and their features can be combined and applied without conflict. The scope of protection of the present invention should be determined by the claims, and all changes and improvements made by those skilled in the art within the scope of the invention's concept should be considered within the scope of protection of the present invention.

Claims

1. A multi-enzyme cascade catalytic reaction system, characterized in that, The multi-enzyme cascade catalytic reaction system uses glycerol as a raw material and includes a first module, a second module, and a third module. The first module is for the production of glyceric acid from glycerol under the catalysis of alditol oxidase. The second module contains glyceric acid, ATP, and NAD. + L-glutamic acid is synthesized into phosphoserine via glycerokinase, D-3-phosphoglycerate dehydrogenase, and phosphoserine transaminase. The third module is phosphoserine and nucleophilic reagents are synthesized into L-cysteine ​​or non-natural L-α-amino acids via phosphoserine thiolase.

2. The multi-enzyme cascade catalytic reaction system according to claim 1, characterized in that, The first module also contains catalase; the second module also contains polyphosphate kinase and glutamate dehydrogenase.

3. The multi-enzyme cascade catalytic reaction system according to claim 1, characterized in that, In the second module, the ATP concentration is 0.5-5 mM, and the NAD... + The concentration is 0.5-5 mM, and the concentration of L-glutamic acid is 5-50 mM.

4. The multi-enzyme cascade catalytic reaction system according to claim 3, characterized in that, The second module also contains 5-50 mM Mg 2+ 10-50mM polyP and 50-200mM ammonium chloride.

5. The multi-enzyme cascade catalytic reaction system according to claim 1, characterized in that, The amino acid sequence of the phosphoserine thiolase is shown in SEQ ID NO.3, or is a mutant of it, the mutant sequence of which is shown in SEQ ID NO.

4.

6. The multi-enzyme cascade catalytic reaction system according to claim 1, characterized in that, In the third module, the nucleophilic reagent is a nucleophilic compound of general formula (1): Thiol R1-SH(1), R1 is a monovalent substituted or unsubstituted alkyl, aryl, or heteroaryl group.

7. The multi-enzyme cascade catalytic reaction system according to claim 6, characterized in that, The nucleophiles are sodium sulfide, 3-mercapto-1,2-propanediol, 1-naphthiophenol, cyclopentanethiol, mercaptoacetic acid, 2-mercaptoethanol, 2-aminoethanethiol, benzylthiol, sodium thiosulfate, 4-chlorobenzylthiol, thiophenethiol, 4-(trifluoromethyl)-benzylthiol, allylthiol, 4-nitrobenzylthiol, 4-cyanobenzylthiol, triphenylmethanethiol, 2-ethyl-1-hexylthiol, 2-bromobenzylthiol, and 2-pyrazinylethanethiol.

8. The multi-enzyme cascade catalytic reaction system according to claim 1, characterized in that, In the third module, the nucleophilic reagent is a nucleophilic compound of general formula (2): Selenol R2-Se-H(2), R2 is a monovalent substituted or unsubstituted aryl group.

9. The multi-enzyme cascade catalytic reaction system according to claim 8, characterized in that, The nucleophile is selenobenzyl alcohol or 3,5-dimethylselenobenzyl alcohol.

10. The multi-enzyme cascade catalytic reaction system according to claim 1, characterized in that, In the third module, the nucleophile is an azole of general formula (3) or (4): X and Y may be the same or different and represent C or N, while R3, R4, R5, R6, R7, and R8 may be the same or different and represent H, COOH, OH, NH2, Br, I, F, Cl, NO2, CN, or alkyl.

11. The multi-enzyme cascade catalytic reaction system according to claim 10, characterized in that, The nucleophile is pyrazole, 3-methylpyrazole, 3-cyanopyrazole, 5-hydroxypyrazole, 4-aminopyrazole, 5-iodo-1H-pyrazole, 3,5-dimethylpyrazole, 1,2,3-triazole, 1,2,4-triazole, 3-amino-1,2,4-triazole, indazole, indazole-3-carboxylic acid, indazole-3-carboxylic acid, and 5-aminoindazole.

12. The multi-enzyme cascade catalytic reaction system according to claim 1, characterized in that, The concentration of the nucleophile in the third module is greater than the concentration of phosphoserine generated in the second module.

13. The multi-enzyme cascade catalytic reaction system according to claim 1, characterized in that, The first module's catalytic reaction has a pH of 5.0–9.0, a temperature of 20–50°C, and a reaction time of 8–24 hours; the second module's catalytic reaction has a pH of 5.0–9.0, a temperature of 20–60°C, and a reaction time of 2–10 hours; the third module's catalytic reaction has a pH of 5.0–9.0, a temperature of 30–60°C, and a reaction time of 1–6 hours.

14. The use of the multi-enzyme cascade catalytic reaction system shown in any one of claims 1-13 in the preparation of L-cysteine ​​or non-natural L-α-amino acids.

15. A phosphoserine sulfhydrylase mutant, characterized in that, The mutant sequence is shown in SEQ ID NO.4.

Images