O-succinyl-l-homoserine sulfuryltransferase mutants and uses thereof

Abstract

The application discloses an O-succinyl-L-homoserine thiotransferase mutant and application thereof, wherein the mutant is obtained by single mutation or multiple mutation of positions 59 and 119 of an O-succinyl-L-homoserine thiotransferase amino acid sequence shown in SEQ ID NO. 1. The application improves MetZ by semi-rational design and site-directed mutagenesis technology, and finally obtains a mutant with improved tolerance to high-concentration sodium methanethiol under the condition of not losing enzyme activity. The mutant of the application has greatly improved tolerance to sodium methanethiol, is increased from 5% before mutation to 15%, and has great industrial value. The mutant of the application has significantly improved conversion rate of a substrate, and is beneficial to production and application.

Description

(I) Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to an O-succinyl-L-homoserine thiotransferase mutant with enhanced tolerance to sodium methanethiol and its application. (II) Background Technology

[0002] L-Methionine is the only sulfur-containing amino acid essential for humans and animals, and it has significant applications in medicine, food, and feed. Its green and efficient synthesis has attracted widespread attention. Currently, the mainstream synthesis method for L-methionine is the chemical method, but this involves the use of highly volatile and toxic substrates such as hydrogen cyanide and methanethiol, harsh reaction conditions, and large amounts of waste, which increasingly limits its application. Direct fermentation production via microbial cell factories is also difficult to industrialize due to the complex metabolic pathway and challenges in metabolic regulation. In contrast, the fermentation-enzyme catalysis coupling route is an important method for L-methionine synthesis. Using glucose as a substrate, fermentation produces the precursor O-succinyl-L-homoserine, which further reacts with methanethiol under the action of O-succinyl-L-homoserine thiotransferase (MetZ) to generate L-methionine. This method offers high atom economy, low waste emissions, and has significant application potential.

[0003] MetZ is a pyridoxal-dependent phosphatase type I with a structure that can be divided into dimers and tetramers, requiring pyridoxal 5′-phosphate (PLP) as a cofactor. MetZ is a tetrameric protein with similar subunits. Each monomer consists of three subregions: an N-terminal domain, a PLP-binding domain, and a C-terminal domain. The N-terminal domain has a cyclic structure that protrudes from the main body of the monomer, forming a clamping effect on neighboring monomers. Each monomer's PLP-binding domain has a β-sheet containing an active site. Lysine residues near this active site bind to the PLP cofactor via a Schiff base bond. This active site requires the N-terminal domain of another monomer to be connected to the phosphate group of the PLP via a salt bridge. The C-terminal domain has a slightly twisted antiparallel β-sheet that binds to the PLP-binding domain, contributing to the formation of a dense monomer shape. In recent years, based on structure-function relationship studies of MetZ, mutants with enhanced activity have been obtained by modifying MetZ and applied to the catalytic synthesis of L-methionine from OSH and sodium methanethiol (Adv. Synth. Catal. 2023, 365, 1048-1057). However, studies have found that when sodium methanethiol is used as a thiol donor, increasing its concentration limits the reaction rate, and the poor tolerance of MetZ to high concentrations of sodium methanethiol is the main reason. Therefore, developing MetZ with good tolerance to high concentrations of sodium methanethiol is of great significance to meet the industrial demand for the fermentation-enzymatic synthesis of L-methionine. (III) Summary of the Invention

[0004] The purpose of this invention is to provide an O-succinyl-L-homoserine thiotransferase mutant with enhanced tolerance to sodium methanethiol and its applications.

[0005] The technical solution adopted in this invention is:

[0006] This invention provides an O-succinyl-L-homoserine thiotransferase mutant (denoted as MetZ-M) with enhanced tolerance to sodium methanethiol. The mutant is obtained by single or multiple mutations at positions 59 and 119 of the amino acid sequence of the O-succinyl-L-homoserine thiotransferase from Chromobacterium violaceum, as shown in SEQ ID NO.1.

[0007] Furthermore, the O-succinyl-L-homoserine thiotransferase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1 to one of the following: (1) mutating threonine at position 59 to isoleucine, with the amino acid sequence shown in SEQ ID NO.2, denoted as MetZ-M1; (2) mutating threonine at position 119 to tyrosine, with the amino acid sequence shown in SEQ ID NO.3, denoted as MetZ-M2; (3) mutating threonine at position 59 to isoleucine and threonine at position 119 to tyrosine, with the amino acid sequence shown in SEQ ID NO.4, denoted as MetZ-M3.

[0008] This invention provides the encoding gene of the O-succinyl-L-homoserine thiotransferase mutant, a recombinant plasmid, and a recombinant genetically engineered bacterium. The recombinant plasmid is preferably based on Pet-28b and uses Escherichia coli BL21(DE3) as the host bacterium.

[0009] This invention also provides an application of the O-succinyl-L-homoserine thiotransferase mutant in the catalytic production of L-methionine from sodium methanethiol. The application is as follows: using wet bacterial cells obtained by fermentation culture of engineered bacteria containing the encoding gene of the O-succinyl-L-homoserine thiotransferase mutant, or pure enzyme solution extracted by ultrasonic disruption of wet bacterial cells, as a catalyst; using O-succinyl-L-homoserine (OSH) and sodium methanethiol as substrates; and using a buffer solution with pH 7-9 as the reaction medium to form a reaction system; reacting in a constant temperature shaking metal bath at 30°C and 800 rpm; separating and purifying the reaction solution to obtain L-methionine.

[0010] Furthermore, in the reaction system, the amount of wet bacterial cells added is 5-15 g / L (preferably 10 g / L); the amount of pure enzyme solution added is 50-150 mg / L (preferably 100 mg / L) based on protein concentration; the concentration of O-succinyl-L-homoserine (OSH) added is 20-80 g / L (preferably 50 g / L); and the volume concentration of sodium methanethiol added is 1-15%.

[0011] Furthermore, the reaction system also contains pyridoxal 5′-phosphate (PLP), with a final concentration of 10-50 mM.

[0012] Furthermore, the buffer solution is a 2M Tris-HCl buffer solution with a pH of 8.0.

[0013] Furthermore, the catalyst is prepared by the following method:

[0014] (1) Engineered bacteria containing the gene encoding the O-succinyl-L-homoserine thiotransferase mutant were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37℃ and 180 r / min for 10-12 h to obtain seed culture; the seed culture was inoculated into fresh LB medium containing a final concentration of 50 μg / mL kanamycin at a volume concentration of 1% and cultured at 37℃ and 180 r / min to obtain OD. 600 When the concentration reaches 0.6-0.8, add IPTG to the culture medium to a final concentration of 0.1 mM. Induce expression at 28℃ for 12 h, centrifuge at 4℃ and 8000 r / min for 10 min, discard the supernatant, and collect the wet cells.

[0015] (2) The wet bacterial cells were resuspended in Tris-HCl buffer (100mM, pH 8.0) and disrupted by sonication. The disruption program was: disruption time 3s, interval time 4s, power 40w (Note: the bacterial suspension should be disrupted in an ice bath). After disruption, the suspension was a clear yellowish-brown color. The cell disruption solution was centrifuged at 4℃, 12000rpm for 20min and the supernatant was collected.

[0016] (3) After ultrafiltration through a 0.22 μm filter membrane, the supernatant was loaded onto a Ni-NTA gel column and the flow rate was controlled by gravity. Elution was performed using a pH 8.0 buffer containing 300 mM NaCl and 50 mM imidazole and 50 mM Tris-HCl at a flow rate of 1.0 mL / min to remove some impurities and unbound proteins until the protein purification instrument reached equilibrium under UV detection. Elution was then performed again using a pH 8.0 buffer containing 300 mM NaCl and 50 mM imidazole and 500 mM Tris-HCl at a flow rate of 1.0 mL / min to collect the eluent of the target protein. The eluent was placed in a dialysis bag (MwCO = 100000) and dialyzed overnight at 4°C using pure water as the dialysate. The choked liquid was collected to obtain the pure enzyme solution.

[0017] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0018] (1) This invention modifies MetZ using semi-rational design and site-directed mutagenesis, ultimately obtaining a mutant with improved tolerance to high concentrations of sodium methanethiol without loss of enzyme activity. The mutant of this invention exhibits significantly improved tolerance to sodium methanethiol, increasing from 5% before mutation to 15%, which has great industrial value.

[0019] (2) After incubation at 5%, 10%, and 15% sodium methanethiol, the half-life of mutants T59I, T119Y, and T59I / T119Y was increased by 300%-360% compared to the wild type, with mutant T59I / T119Y showing the highest increase of 360% over the wild type. The residual enzyme activity of mutants T59I, T119Y, and T59I / T119Y after 10 hours was increased by 190%-210% compared to the wild type, with mutant T59I showing the highest increase of 210% over the wild type.

[0020] (3) The mutants of this invention significantly improve the conversion rate of the substrate. In the 10% sodium methanethiol system, the conversion rate of WT no longer increases after 1.5 h of reaction, with a maximum conversion rate of 32%. The conversion rates of mutants T59I and T119Y no longer increase after about 4 h of reaction, with mutant T59I reaching a maximum conversion rate of 60% and mutant T119Y reaching a conversion rate of 52%, both of which are improved compared to WT, which is beneficial for production applications. (IV) Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0022] LB liquid medium composition: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, dissolved in distilled water; LB solid medium is LB liquid medium with 20 g / L agar powder added; autoclave at 121℃ for 20 min; add kanamycin to a final concentration of 100 μg / mL before use.

[0023] Example 1: MetZ enzyme activity assay

[0024] 1. Construction of wild-type recombinant genetically engineered bacteria

[0025] The gene encoding O-succinyl-L-homoserine thiotransferase (MetZ, GeneID: MK948096.1, amino acid sequence as shown in SEQ ID NO.1) from Chromobacterium violaceum was artificially synthesized. The expression vector was Pet-28b, the host cell was E. coli BL21(DE3), and positive clones were screened to obtain wild-type recombinant genetically engineered bacteria E. coli BL21(DE3)-Pet-28b-MetZ, denoted as WT.

[0026] 2. Induction of wild-type recombinant engineered bacteria expression

[0027] The wild-type recombinant genetically engineered bacteria constructed in step 1 were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37℃ and 180 r / min for 10–12 h to obtain seed culture. The seed culture was then inoculated into fresh LB medium containing a final concentration of 50 μg / mL kanamycin at a volume concentration of 1% and cultured at 37℃ and 180 r / min until OD was obtained. 600 To a concentration of 0.6–0.8, add IPTG to the culture medium to a final concentration of 0.1 mM. Induce expression at 28°C for 12 h, then centrifuge at 4°C and 8000 r / min for 10 min. Discard the supernatant, take the wet cells, and resuspend them in Tris-HCl buffer (100 mM, pH 8.0) to a concentration of 0.1 g / L for later use.

[0028] 3. Ultrasonic disruption of wild-type recombinant genetically engineered bacteria

[0029] The bacterial suspension from step 2 was disrupted using ultrasonication. The ice bath disruption program was as follows: disruption time 3 seconds, interval 4 seconds, power 40W. After disruption, the suspension was a clear, light yellowish-brown color. Finally, the cell disruption solution was centrifuged at 4°C, 12,000 rpm for 20 minutes, and the supernatant was collected.

[0030] 4. Enzyme separation and purification

[0031] The supernatant from step 3 was ultrafiltered through a 0.22 μm membrane and loaded onto a Ni-NTA gel column. The flow rate was controlled by gravity. Elution was performed using a pH 8.0 buffer containing 300 mM NaCl and 50 mM imidazole, and 50 mM Tris-HCl buffer (HCl was adjusted to pH 8.0 and then sonicated through the membrane). The flow rate was 1.0 mL / min to elute some impurities and unbound proteins until the protein purification instrument reached equilibrium as detected by UV. Elution was then performed using a pH 8.0 buffer containing 300 mM NaCl and 50 mM imidazole, and 500 mM Tris-HCl buffer (HCl was adjusted to pH 8.0 and then sonicated through the membrane) at a flow rate of 1.0 mL / min. The eluent of the target protein was collected. The eluent was then placed in a dialysis bag (MwCO 100000), and dialyzed overnight at 4°C using pure water as the dialysate. The choked fluid was collected to obtain the pure enzyme solution. SDS gel electrophoresis analysis yielded a high-purity pure enzyme solution with correct bands, which was stored for later use.

[0032] 5. MetZ enzyme activity assay

[0033] (1) Standard enzyme activity detection

[0034] To a 2 mL reaction system, add MetZ enzyme solution with a protein concentration of 0.1 g / L dissolved in Tris-HCl buffer (2 M, pH 8.0), incubate at 30 °C for 5 min, then add OSH to a final concentration of 50 g / L and 1% (v / v) sodium methanethiol, and react in a constant temperature shaking metal bath at 30 °C and 800 rpm for 5 min. After the reaction is complete, take 200 μL of the reaction solution and add 10 μL of 6 M HCl to terminate the reaction. After the catalytic reaction is complete, centrifuge at 12000 rpm for 1 min, collect the supernatant, dilute, derivatize, and determine the amount of L-methionine produced by high performance liquid chromatography (HPLC) to calculate the initial enzyme activity.

[0035] (2) Effect of PLP on enzyme activity

[0036] Under the same conditions in step (1), pyridoxal 5′-phosphate (PLP) was added to the reaction system to a final concentration of 10 mM, and other operations were the same. The enzyme activity of the wild-type pure enzyme solution without PLP in step (1) was 71.30 U / mg, and the enzyme activity of the wild-type pure enzyme solution with PLP in step (2) was 106.02 U / mg.

[0037] (3) Effect of sodium methanethiol concentration on enzyme activity

[0038] In step (1), the volumetric concentration of sodium methanethiol was changed to 1%, 5%, 10%, and 15%, respectively. The reaction time in a constant-temperature shaking metal bath at 30°C and 800 rpm for 5 minutes was changed to 30 minutes, while other operations remained the same. Using the enzyme activity of MetZ in the 1% sodium methanethiol system as 100%, the relative enzyme activity was calculated as follows: 79.3% in the 5% sodium methanethiol system, 53.3% in the 10% sodium methanethiol system, and 39.7% in the 15% sodium methanethiol system.

[0039] Enzyme activity is defined as the amount of enzyme required to produce 1 μmol of L-methionine per minute, which is defined as one unit of enzyme activity (U).

[0040] Enzyme specific activity (U·mg) -1 The enzyme activity unit is defined as the number of enzyme activity units contained in each milligram of enzyme protein.

[0041] The formula for calculating specific enzyme activity is: Specific enzyme activity (U·mg) -1 = Enzyme activity (U) / Protein content (mg).

[0042] HPLC detection method:

[0043] Preparation of derivatization reagent: Weigh 0.27g of 4-chloro-3,5-dinitrotrifluorotoluene (CNBF) and mix with 10mL of acetonitrile to prepare a stock solution, which should be stored away from light.

[0044] Preparation of boric acid-borax buffer solution: Dissolve boric acid and borax separately in sterile water to a final concentration of 0.2 mol / L and a final concentration of borax of 0.05 mol / L, then mix and sonicate to dissolve.

[0045] Sample derivatization: 100 μL of sample, 300 μL of derivatization reagent, and 500 μL of boric acid-borax buffer were mixed and reacted in a constant-temperature shaking metal bath at 60℃ and 400 rpm for 1 h in the dark. The mixture was then filtered through a 0.22 μm organic phase filter. The concentration of the product L-methionine was quantitatively analyzed by HPLC. Mobile phase preparation: Two mobile phases were prepared. Mobile phase A was pure acetonitrile, and mobile phase B was prepared according to a ratio of water:acetonitrile:triethylamine:acetic acid = 850:150:2:7. The mobile phases were then sonicated through a membrane. Chromatographic column: Welchrom C18 column (4.6 mm × 250 mm, 5 μm); operating temperature: 30℃; UV detection wavelength: 260 nm; flow rate: 0.8 mL / min. Elution method: L-methionine was detected using gradient elution, as shown in Table 1.

[0046] Table 1 HPLC detection system

[0047]

[0048] Example 2: Construction and Screening of MetZ Single-Point Mutants

[0049] 1. Selection of mutation sites

[0050] The wild-type MetZ protein was modeled using the Swiss-Model tool, and the active site and substrate channels of the enzyme were predicted using pymol and Caver software. Combined with molecular dynamics simulation and computer virtual screening, the T59 and T119 sites that affect tolerance to sodium methanethiol were predicted and targeted for site-directed mutagenesis.

[0051] 2. Construction of mutants

[0052] Based on the nucleotide sequence of MetZ (GeneID:MK948096.1), mutation primers were designed. Using whole plasmid PCR technology, a single-point mutation was introduced into the amino acid sequence of MetZ shown in SEQ ID NO.1 using the recombinant vector pET28b-MetZ as a template. The primers used are shown in Table 2.

[0053] Table 2 Primer design for mutant construction

[0054]

[0055] PCR reaction system: 25 μL 2×phanta Max buffer, 2 μL forward / reverse primers, 1 μL template DNA, add ddH2O to 50 μL.

[0056] PCR amplification conditions were: 98℃ for 5 min; (98℃ for 30 s, 60℃ for 30 s, 72℃ for 2000 bp / min); 35 cycles; 72℃ for 10 min.

[0057] Add 0.5 μL of DpnI enzyme and 1 μL of duffer to the PCR product, incubate at 37℃ for 1 h and 70℃ for 15 min. Take 10 μL of the PCR product and add it to 100 μL of E. coli BL21(DE3) competent cell suspension in ice bath. Incubate on ice for 30 min, then immediately heat shock at 42℃ for 90 s, incubate on ice for 2-5 min, then add 600 μL of LB medium, incubate at 37℃ for 1 h, centrifuge at 4000 r / min for 1 min, discard part of the supernatant, resuspend the remaining bacterial culture and plate it on LB agar plates (containing kanamycin, final concentration 50 μg / mL), and incubate upside down at 37℃ for 12 h.

[0058] 3. Induced expression of recombinant engineered bacteria

[0059] Single colonies were picked from the plate and placed into 5 mL LB tubes containing a final concentration of 50 μg / mL kanamycin, and cultured at 37°C and 180 rpm for 12 h. Then, at a 2% (v / v) inoculum, they were transferred to 100 mL LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 180 rpm for 2–3 h (OD). 600 =0.6~0.8), add IPTG (final concentration 0.1mM), and induce culture at 28℃ and 180rpm for 12h. After the culture is completed, centrifuge at 4℃ and 8000rpm for 10min, discard the supernatant, and collect the wet bacterial cells.

[0060] 3. Screening for mutants

[0061] Wet bacterial cells were added to Tris-HCl buffer (2M, pH 8.0) at a concentration of 10 g / L and incubated at 30°C for 5 min. Then, 50 g / L OSH and 1% (v / v) sodium methanethiol were added to form a 2 mL reaction system, which was reacted at 30°C in a shaking metal bath at 800 rpm for 5 min. After the reaction, the amount of L-methionine produced was determined using the method in Example 1. The relative enzyme activity was calculated with the enzyme activity of MetZ in the 1% sodium methanethiol system as 100%.

[0062] Under the same conditions, the volume concentration of sodium methanethiol was changed to 5%, 10%, and 15%, and other operations were the same. The relative enzyme activities are shown in Table 3. The residual enzyme activities of T59I and T119Y were significantly increased after incubation in sodium methanethiol of different concentrations.

[0063] Table 3. Tolerance of MetZ and its mutants to sodium methanethiol

[0064]

[0065] Example 3: Construction and Screening of Saturated Mutants

[0066] Based on the single mutant sequence constructed in Example 2, site-directed saturation mutagenesis primers were designed. Using whole plasmid PCR technology, with the recombinant vector pET28b / MetZ as a template, saturation mutagenesis was performed at positions 59 and 119 of the MetZ amino acid sequence. The primer sequences are shown in Table 4.

[0067] Table 4. Primer design for saturation mutations at MetZ sites 59 and 119.

[0068]

[0069] N represents A / T / G / C, and K represents G / T.

[0070] The mutants were constructed using the method in Example 2 and then induced for expression and screened. The results are shown in Table 5.

[0071] Table 5. Screening results of MetZ 59 and 119 site saturation mutants to tolerance to different concentrations of sodium methanethiol.

[0072]

[0073] Example 4: Construction and Screening of Combinatorial Mutants

[0074] Based on the single mutant sequence constructed in Example 2, a combination mutation was designed. Using whole plasmid PCR technology, the 59th and 119th amino acid positions of the MetZ amino acid sequence were combined and mutated using the recombinant vector pET28b / MetZ as a template.

[0075] The mutants were constructed using the method in Example 2 and then induced for expression and screened. The results are shown in Table 6.

[0076] Table 6. Tolerance of MetZ T59I / T119Y combined mutants to different concentrations of sodium methanethiol

[0077]

[0078] Tests showed that the mutant T59I / T119Y exhibited further improved tolerance compared to WT.

[0079] Example 5: Determination of tolerance of MetZ and its mutants to sodium methanethiol

[0080] Wet cells of WT and its mutants T59I, T119Y, and T59I / T119Y were prepared using the method in Example 1. The enzyme activity of the wet cells under 10% sodium methanethiol conditions was detected using the method in Example 1.

[0081] The results showed that the half-life of mutants T59I, T119Y, and T59I / T119Y was increased by 300%–360% compared to WT, with mutant T59I / T119Y showing the highest increase of 360%. The residual enzyme activity of mutants T59I, T119Y, and T59I / T119Y at 10 hours was increased by 190%–210% compared to WT, with mutant T59I showing the highest increase of 210%, indicating a significantly improved tolerance of the mutants to sodium methanethiol.

[0082] Example 6: Determination of transformation efficiency of MetZ and its mutants

[0083] Wet cells of WT and its mutants T59I, T119Y and T59I / T119Y were prepared using the method in Example 1 and used for substrate transformation.

[0084] In a 10 mL reaction system, 10 g / L of the above-mentioned wet bacterial cells dissolved in Tris-HCl buffer (2 M, pH 8.0) were added. After incubation at 30 °C for 5 min, a final concentration of 60 g / L OSH, 10.0% (v / v) sodium methanethiol, and 50 mM PLP were added in a single batch. The reaction was continued in a metal bath at 30 °C and 800 rpm with constant temperature shaking. Samples were taken every 30 min for the first 3 h, and then every 1 h thereafter, until the reaction was completed at 8 h. The residual amount of substrate OSH was determined using an amino acid analyzer, and the conversion rate was calculated.

[0085] After 1.5 hours of reaction, the conversion rate of WT mutants no longer increased, with a maximum conversion rate of 32%. For mutants T59I and T119Y, the conversion rate no longer increased after approximately 4 hours of reaction. The highest conversion rate was 60% for mutant T59I and 52% for mutant T119Y. The highest conversion rate for the T59I / T119Y mutant combination reached 64%, all showing improvements compared to WT, which is beneficial for production applications.

Claims

1. An O-succinyl-L-homoserine thiotransferase mutant with enhanced tolerance to sodium methanethiol, characterized in that, The O-succinyl-L-homoserine thiotransferase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1 to one of the following: (1) mutating threonine at position 59 to isoleucine, with the amino acid sequence shown in SEQ ID NO.2; (2) mutating threonine at position 59 to isoleucine and threonine at position 119 to tyrosine, with the amino acid sequence shown in SEQ ID NO.

4.

2. A recombinant genetically engineered bacterium containing the coding gene of the mutant described in claim 1.

3. The application of the O-succinyl-L-homoserine thiotransferase mutant of claim 1 in the catalytic production of L-methionine from sodium methanethiol.

4. The application as described in claim 3, characterized in that, The application is as follows: using wet bacterial cells obtained by fermentation culture of engineered bacteria containing the encoding gene of the O-succinyl-L-homoserine thiotransferase mutant, or pure enzyme solution extracted by ultrasonic disruption of wet bacterial cells, as a catalyst, O-succinyl-L-homoserine and sodium methanethiol as substrates, and a buffer solution with pH 7-9 as the reaction medium, the reaction is carried out in a constant temperature shaking metal bath at 30°C and 800 rpm. The reaction solution is then separated and purified to obtain L-methionine.

5. The application as described in claim 4, characterized in that, In the reaction system, the amount of wet bacterial cells added is 5-15 g / L; the amount of pure enzyme solution added is 50-150 mg / L based on protein concentration; the concentration of O-succinyl-L-homoserine added is 20-80 g / L; and the volume concentration of sodium methanethiol added is 1-15%.

6. The application as described in claim 5, characterized in that, The reaction system also contains pyridoxal 5′-phosphate, with a final concentration of 10-50 mM.

7. The application as described in claim 4, characterized in that, The buffer solution is a 2M Tris-HCl buffer solution with a pH of 8.

0.

8. The application as described in claim 4, characterized in that, The catalyst is prepared according to the following method: (1) Inoculate the engineered bacteria containing the gene encoding the O-succinyl-L-homoserine thiotransferase mutant into LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and culture at 37℃ and 180 r / min for 10-12 h to obtain seed culture; The seed culture was inoculated at a volume concentration of 1% into fresh LB medium containing a final concentration of 50 μg / mL kanamycin, and cultured at 37℃ and 180 r / min. 600 When the concentration reaches 0.6-0.8, add IPTG to the culture medium to a final concentration of 0.1 mM. Induce expression at 28℃ for 12 h, centrifuge at 4℃ and 8000 r / min for 10 min, discard the supernatant, and collect the wet cells. (2) The wet bacterial cells were resuspended in 100mM, pH 8.0 Tris-HCl buffer and disrupted by sonication. The ice bath disruption program was: disruption time 3s, interval time 4s, power 40W. After disruption, the cell disruption solution was centrifuged at 4℃, 12000rpm for 20min and the supernatant was collected. (3) After ultrafiltration through a 0.22 μm filter membrane, the supernatant was loaded onto a Ni-NTA gel column and the flow rate was controlled by gravity. Elution was performed using a pH 8.0 buffer containing 300 mM NaCl and 50 mM imidazole and 50 mM Tris-HCl at a flow rate of 1.0 mL / min until the protein was equilibrated by UV detection on the protein purifier. Elution was then performed again using a pH 8.0 buffer containing 300 mM NaCl and 50 mM imidazole and 500 mM Tris-HCl at a flow rate of 1.0 mL / min. The eluent of the target protein was collected. The eluent was placed in a dialysis bag and dialyzed overnight at 4°C using pure water as the dialysate. The choked liquid was collected to obtain the pure enzyme solution.

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