A double-enzyme system for catalyzing D-serine to synthesize L-cysteine and application thereof

Abstract

This invention discloses a dual-enzyme system for catalyzing the synthesis of L-cysteine ​​from D-serine and its applications. This dual-enzyme system achieves the asymmetric synthesis of L-cysteine ​​from D-serine with a substrate conversion rate of 99% and a product yield >110%. The catalytic process of this invention is simple, with no byproducts generated and easy subsequent separation. Therefore, it has advantages such as high atom economy, high product optical purity, mild reaction conditions, environmental friendliness, and simple product post-processing. It has great potential for the industrialization of low-cost and controllable multi-enzyme preparation of L-cysteine.

Description

(I) Technical Field

[0001] This invention belongs to the fields of chemical engineering and enzyme engineering, and in particular relates to a dual-enzyme system for catalyzing the synthesis of L-cysteine ​​from D-serine and its application. (II) Background Technology

[0002] L-cysteine, a key sulfur-containing amino acid, plays a crucial role not only in the pharmaceutical industry but also in food additives and animal feed. Currently, industrial production methods for L-cysteine ​​include hair hydrolysis followed by reduction, chemical synthesis, and enzymatic synthesis. While hair hydrolysis is simple, it requires high temperatures and large amounts of acids and alkalis, resulting in high costs, strong odors, and large amounts of high-salt wastewater. Chemical synthesis, on the other hand, easily produces D-isomers, leading to high separation costs. Therefore, neither hair hydrolysis nor chemical synthesis is suitable for the industrial production of L-cysteine. Developing a green and efficient enzymatic production process for L-cysteine ​​is of great significance for further reducing production costs and improving production efficiency.

[0003] Enzymatic synthesis offers advantages such as mild conditions (25–60℃), high selectivity, and environmental friendliness, significantly improving product yield. There are two main enzymatic routes for L-cysteine ​​preparation: 1) The DL-2-amino-Δ2-thiazoline-4-carboxylic acid (DL-ATC) route: Using DL-ATC as a substrate, L-cysteine ​​is prepared by catalysis with ATC racemic enzymes, hydrolases, and amidases from specific microorganisms (such as Pseudomonas). This route boasts advantages such as short cycle time, high stereoselectivity, and mild reaction conditions, and was once considered an ideal industrial alternative. However, this route has the following problems: ① L-ATC hydrolases are unstable, leading to low yield and production efficiency; ② The synthesis cost of the precursor DL-ATC is high; ③ L-cysteine ​​is easily degraded within cells, resulting in a low overall yield. 2) Multi-enzyme cascade route: First, L-serine is synthesized from glycine, formaldehyde, and tetrahydrofolate using L-serine hydroxymethyltransferase. Second, L-serine and sodium hydrosulfide are used as substrates, and tryptophan synthase catalyzes the synthesis of L-cysteine. This route has advantages such as high conversion rate and high yield; however, tetrahydrofolate accounts for a high raw material cost in the L-serine synthesis process, and its price fluctuates significantly, resulting in poor cost controllability. Therefore, developing a cost-controllable, environmentally friendly, and scalable multi-step enzyme-catalyzed synthesis process is of significant practical importance in reducing the production cost of existing L-cysteine ​​enzymatic synthesis methods. (III) Summary of the Invention

[0004] The purpose of this invention is to provide a dual-enzyme system for catalyzing the synthesis of L-cysteine ​​from D-serine and its application in catalyzing the asymmetric synthesis of L-cysteine ​​from D-serine. This dual-enzyme system can effectively catalyze the conversion of D-serine to L-cysteine, the catalytic process is simple, and the reaction produces no byproducts and is environmentally friendly.

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

[0006] This invention provides a dual-enzyme system for catalyzing the synthesis of L-cysteine ​​from D-serine, the dual-enzyme system being composed of alanine racemic enzyme and tryptophan synthase.

[0007] Furthermore, the alanine racemase is derived from Bacillus pseudofirmus The racemic enzyme family protein, denoted as BpALR, has the amino acid sequence shown in SEQ ID No. 2, and the encoding gene nucleotide sequence shown in SEQ ID No. 1.

[0008] Furthermore, the tryptophan synthase is derived from... Escherichia coli The tryptophan synthase family protein, denoted as Trp, has the amino acid sequence shown in SEQ ID No. 4 and the encoding gene nucleotide sequence shown in SEQ ID No. 3.

[0009] In the dual-enzyme system of this invention, D-serine is partially converted to L-serine by racemic alanine enzyme. This reaction involves the formation of a pyridoxal phosphate (PLP)-dependent Schiff base intermediate, which achieves configuration inversion (L→D or D→L) through proton transfer. Then, under the catalysis of tryptophan synthase Trp, at suitable pH and temperature, L-serine binds to the active site of Trp, stabilizes the carbanion intermediate with the assistance of PLP, and undergoes nucleophilic substitution with hydrides (such as sodium hydrosulfide) or thiols to form a CS bond, ultimately generating L-cysteine.

[0010] The present invention also provides an application of the dual-enzyme system in catalyzing the synthesis of L-cysteine ​​from D-serine.

[0011] Furthermore, the application method is as follows: using crude enzyme solution obtained by ultrasonic disruption of wet bacterial cells from recombinant genetically engineered bacteria expressing a dual-enzyme system after induction culture as a catalyst, D-serine and sodium hydrosulfide as substrates, pyridoxal phosphate (PLP) as coenzyme, and a buffer solution with pH 3-11 as the reaction medium to form a reaction system, and reacting completely at 30-40℃ and 100-400rpm (preferably 37℃ and 200rpm) to obtain a reaction solution containing L-cysteine; the catalyst is a mixture of crude enzyme solutions obtained by ultrasonic disruption of wet bacterial cells from recombinant genetically engineered bacteria expressing alanine racemic enzyme BpALR and tryptophan synthase Trp separately, or crude enzyme solution obtained by ultrasonic disruption of wet bacterial cells from recombinant genetically engineered bacteria expressing both alanine racemic enzyme BpALR and tryptophan synthase Trp.

[0012] Furthermore, in the reaction system, D-serine is added to a final concentration of 20-100 g / L (preferably 100 g / L); sodium hydrosulfide is added to a final concentration of 17.5-87.5 g / L (preferably 87.5 g / L); and pyridoxal phosphate is added to a final concentration of 0.1-0.5 g / L (preferably 0.125 g / L).

[0013] Furthermore, in the reaction system, the amount of crude enzyme solution used is 10-150 g / L based on the weight of wet bacterial cells before rupture. More preferably, when the catalyst is a mixture of crude enzyme solutions, the amount of crude enzyme solution used for the recombinant genetically engineered bacteria expressing alanine racemic enzyme BpALR is 75 g / L based on the weight of wet bacterial cells before rupture, and the amount of crude enzyme solution used for the recombinant genetically engineered bacteria expressing tryptophan synthase Trp is 25 g / L based on the weight of wet bacterial cells before rupture. When the catalyst is the crude enzyme solution of recombinant genetically engineered bacteria that co-expresses alanine racemic enzyme BpALR and tryptophan synthase Trp, the amount used is 50 g / L based on the weight of wet bacterial cells before rupture.

[0014] Furthermore, the pH of the buffer solution is preferably 7-8, more preferably PBS buffer (0.01M, pH=7).

[0015] Furthermore, the reaction system contains 1-5 g / L ammonium sulfate (preferably 2.5 g / L).

[0016] Furthermore, the host cells of the recombinant genetically engineered bacteria expressing alanine racemic enzyme and tryptophan synthase can be any conventional host cell in the art, as long as the recombinant expression vector can stably replicate on its own and can effectively express the target protein after induction by an inducer. Escherichia coli is preferred, and Escherichia coli Lemo 21(DE3) is more preferred.

[0017] Furthermore, the recombinant genetically engineered bacteria expressing alanine racemase were constructed according to the following steps: the gene fragment encoding alanine racemase was inserted between the PacⅠ and AvrⅡ restriction sites of the expression plasmid pCDFDuet-1 to obtain the recombinant plasmid; the recombinant plasmid was transformed into the expression host Escherichia coli Lemo 21(DE3) to obtain the recombinant genetically engineered bacteria.

[0018] Furthermore, the recombinant genetically engineered bacteria expressing tryptophan synthase alone are constructed according to the following steps: the gene fragment encoding tryptophan synthase is inserted between the NdeⅠ and HindⅢ restriction sites of the expression plasmid pET28a to obtain the recombinant plasmid; the recombinant plasmid is transformed into the expression host Escherichia coli Lemo 21(DE3) to obtain the recombinant genetically engineered bacteria.

[0019] Furthermore, the recombinant genetically engineered bacteria co-expressing alanine racemic enzyme and tryptophan synthase were constructed according to the following steps: the alanine racemic enzyme encoding gene fragment was inserted between the PacⅠ and AvrⅡ restriction sites of the expression plasmid pCDFDuet to obtain a recombinant plasmid; the tryptophan synthase encoding gene fragment was inserted between the NdeⅠ and HindⅢ restriction sites of the expression plasmid pET28a to obtain a recombinant plasmid; the two recombinant plasmids were co-transformed into the expression host Escherichia coli Lemo BL21(DE3) to obtain the recombinant genetically engineered bacteria.

[0020] Furthermore, the crude enzyme solution of the recombinant genetically engineered bacteria expressing alanine racemase alone was prepared as follows: The recombinant genetically engineered bacteria expressing alanine racemase alone were inoculated into test tubes containing 50 mg / L streptomycin-resistant LB liquid medium and cultured overnight at 37°C and 220 rpm in a constant temperature shaker; the bacterial solution was then inoculated into LB liquid medium containing 50 mg / L streptomycin at a volume concentration of 1% and cultured at 37°C and 220 rpm in a constant temperature shaker until OD... 600 Once the concentration reaches 0.6, add IPTG to a final concentration of 0.25 mM; incubate at 16℃ and 200 rpm for 20 h to induce expression; after induction, centrifuge the bacterial culture at 4℃ and 4000 rpm for 10 min, remove the supernatant, and store the wet bacterial cells in a -20℃ refrigerator for later use; resuspend the wet bacterial cells in PBS buffer (0.01 M, pH=7), sonicate at 300W for 15 min, working for 2 seconds and then 3 seconds, centrifuge at 4℃ and 12000 rpm for 15 min to obtain the crude enzyme solution.

[0021] Furthermore, the crude enzyme solution of the recombinant genetically engineered bacteria expressing tryptophan synthase alone was prepared as follows: the recombinant genetically engineered bacteria expressing tryptophan synthase alone were inoculated into test tubes containing LB liquid medium of 50 mg / L kanamycin and cultured overnight at 37°C and 220 rpm in a constant temperature shaker; the bacterial solution was then inoculated into LB liquid medium containing 50 mg / L kanamycin at a volume concentration of 1% and cultured at 37°C and 220 rpm in a constant temperature shaker until OD... 600 Once the concentration reaches 0.6, add IPTG to a final concentration of 0.25 mM; incubate at 28℃ and 200 rpm for 18 h to induce expression; after induction, centrifuge the bacterial culture at 4℃ and 4000 rpm for 10 min, remove the supernatant, and store the wet bacterial cells in a -20℃ refrigerator for later use; resuspend the wet bacterial cells in PBS buffer (0.01 M, pH=7), sonicate at 300W for 15 min, working for 2 seconds and then 3 seconds, centrifuge at 4℃ and 12000 rpm for 15 min to obtain the crude enzyme solution.

[0022] Further, the crude enzyme solution of the recombinant genetically engineered bacteria co-expressing alanine racemic enzyme and tryptophan synthase was prepared as follows: the recombinant genetically engineered bacteria co-expressing alanine racemic enzyme and tryptophan synthase were inoculated into test tubes containing LB liquid medium of 50 mg / L streptomycin and 50 mg / L kanamycin, and cultured overnight at 37°C and 220 rpm in a constant temperature shaker; the bacterial solution was inoculated into LB liquid medium containing 50 mg / L streptomycin and 50 mg / L kanamycin at a volume concentration of 1%, and cultured at 37°C and 220 rpm in a constant temperature shaker until OD... 600 Once the concentration reaches 0.6, add IPTG to a final concentration of 0.25 mM; incubate at 16℃ and 200 rpm for 20 h to induce expression; after induction, centrifuge the bacterial culture at 4℃ and 4000 rpm for 10 min, remove the supernatant, and store the wet bacterial cells in a -20℃ refrigerator for later use; resuspend the wet bacterial cells in PBS buffer (0.01 M, pH=7), sonicate at 300W for 15 min, working for 2 seconds and then 3 seconds, centrifuge at 4℃ and 12000 rpm for 15 min to obtain the crude enzyme solution.

[0023] The catalyst of this invention can also be crude enzyme powder after drying crude enzyme solution or pure enzyme solution.

[0024] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0025] This invention provides a dual-enzyme system and its application in catalyzing the synthesis of L-cysteine ​​from D-serine. This invention is the first to discover that the coupling or co-expression of alanine racemic enzyme and tryptophan synthase can effectively catalyze the synthesis of L-cysteine ​​from D-serine and sodium hydrosulfide in the presence of the coenzyme pyridoxal phosphate (PLP). This route fully utilizes the advantages of the D-serine enzymatic preparation process, completely avoiding the use of expensive raw materials such as folic acid, laying an important foundation for the industrialization of a low-cost and controllable two-step enzymatic preparation process.

[0026] The present invention provides a dual-enzyme system for the non-synthetic synthesis of L-cysteine ​​from D-serine, achieving a substrate conversion rate of 99% and a product yield of >110%.

[0027] The catalytic process of this invention is simple, and the reaction produces no byproducts. Subsequent separation is also simple. Therefore, it has advantages such as high atom economy, high product optical purity, mild reaction conditions, environmental friendliness, and simple product post-processing. It has great potential for industrializing the low-cost and controllable multi-enzyme preparation of L-cysteine. (iv) Description of the attached drawings

[0028] Figure 1 Agarose gel electrophoresis images of the bacterial culture, supernatant, and precipitate after BpALR engineered bacteria were induced to express BpALR.

[0029] Figure 2 Agarose gel electrophoresis images of the bacterial culture, supernatant, and precipitate after Trp engineered bacteria were induced to express the bacteria.

[0030] Figure 3 Agarose gel electrophoresis images of the bacterial culture, supernatant, and precipitate after Trp-BpALR engineered bacteria were induced to express the bacteria.

[0031] Figure 4 A schematic diagram illustrating the principle of the coupling catalysis of Trp and BpALR to produce L-cysteine ​​from D-serine and sodium hydrosulfide.

[0032] Figure 5 The curve of serine remaining amount in the BpALR and / or Trp coupling reaction; 1 represents the reaction system containing crude Trp enzyme solution, L-serine and sodium hydrosulfide; 2 represents the reaction system containing crude Trp enzyme solution, D-serine and sodium hydrosulfide; 3 represents the reaction system containing both BpALR and crude Trp enzyme solution, D-serine and sodium hydrosulfide.

[0033] Figure 6 Coupling reaction product yield curves under different ratios of BpALR and Trp.

[0034] Figure 7 Substrate conversion and product yield curves for the coupling reaction of BpALR and Trp.

[0035] Figure 8 The substrate conversion and product yield curves for the co-expression reactions of BpALR and Trp are shown. (V) Detailed Implementation Methods

[0036] 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:

[0037] Example 1: Construction of recombinant genetically engineered bacteria

[0038] 1. Lemo 21(DE3)-pET28a-BpALR

[0039] Amino acid racemic enzymes can achieve reversible conversion between L- and D-amino acids and have important physiological functions. However, few serine racemic enzymes with industrial application potential have been reported in the literature. Furthermore, because serine and alanine have similar molecular structures, and various alanine racemic enzymes can catalyze high-concentration substrates and achieve industrial applications, the activity of alanine racemic enzymes in a laboratory bacterial library for D-serine was tested. A highly active alanine racemic enzyme was screened. It was further coupled with tryptophan synthase, using pyridoxal phosphate (PLP) as a coenzyme and D-serine and sodium hydrosulfide as substrates, to explore the feasibility of asymmetric synthesis from D-serine to L-cysteine, as detailed below:

[0040] The laboratory-preserved source Bacillus pseudofirmus A gene fragment (NCBI accession number WP_261514694.1) was amplified by PCR. The gene fragment was designated as alanine racemase BpALR, and its gene sequence is shown in SEQ ID No. 1. The amino acid sequence is shown in SEQ ID No. 2. This fragment was inserted between the PacⅠ and AvrⅡ restriction sites of the expression plasmid pCDFDuet-1 to obtain the recombinant plasmid pCDFDuet-1-BpALR. After sequencing verification, pCDFDuet-1-BpALR was transformed into the expression host *Escherichia coli* Lemo 21(DE3) to obtain the recombinant genetically engineered bacterium Lemo 21(DE3)-pCDFDuet-1-BpALR, which was used for subsequent expression of the recombinase, designated BpALR.

[0041] 2. Lemo 21(DE3)-pET28a-Trp

[0042] Using laboratory-preserved tryptophan synthase as a catalyst, L-serine and sodium hydrosulfide as substrates, and pyridoxal phosphate (PLP) as a coenzyme, L-cysteine ​​synthesis was compared, and a protein with high synthesis capacity was screened for expression, as detailed below:

[0043] Sourced from NCBI Escherichia coli A tryptophan synthase gene fragment (NCBI accession number CP174267.1) was amplified by PCR and designated as tryptophan synthase Trp. The gene sequence is shown in SEQ ID No. 3, and the amino acid sequence is shown in SEQ ID No. 4. This fragment was inserted between the NdeⅠ and HindⅢ restriction sites of the expression plasmid pET28a to obtain the recombinant plasmid pET28a-Trp. After sequencing verification, pET28a-Trp was transformed into the expression host *Escherichia coli* Lemo 21(DE3) to obtain the recombinant genetically engineered bacterium Lemo 21(DE3)-pET28a-Trp, which was used for subsequent expression of the recombinase, designated Trp.

[0044] 3. Lemo 21(DE3)-pET28a-BpALR-Trp

[0045] A recombinant plasmid was obtained by inserting the alanine racemic enzyme encoding gene fragment between the PacⅠ and AvrⅡ restriction sites of the expression plasmid pCDFDuet-1; a recombinant plasmid was obtained by inserting the tryptophan synthase encoding gene fragment between the NdeⅠ and HindⅢ restriction sites of the expression plasmid pET28a; the two recombinant plasmids were then co-transformed into the expression host Escherichia coli Lemo 21(DE3) to obtain the recombinant genetically engineered bacterium Lemo 21(DE3)-BpALR-Trp, denoted as BpALR-Trp.

[0046] Example 2: Cultivation of engineered bacteria

[0047] 1. Culture of recombinant genetically engineered bacteria expressing alanine racemase alone

[0048] The recombinant genetically engineered bacterium Lemo21(DE3)-pCDFDuet-1-BpALR, which expresses alanine racemase alone and was constructed in Example 1, was inoculated into a test tube containing 5 mL of LB liquid medium containing 50 mg / L streptomycin and cultured overnight at 37°C and 220 rpm. Then, 1 mL of the overnight culture was added to a 250 mL shake flask containing 100 mL of LB liquid medium containing 50 mg / L streptomycin and cultured at 37°C and 220 rpm for approximately 2.5 h. OD 600 Once the concentration reaches 0.6, add IPTG to a final concentration of 0.1 mM. Induce expression for 20 h at 16℃ and 200 rpm using a constant temperature shaker. After induction, transfer the bacterial culture to a 50 mL centrifuge tube and centrifuge at 4℃ and 4000 rpm for 10 min. Obtain the supernatant and wet cell pellet. Discard the supernatant and store the wet cell pellet in a -20℃ refrigerator for later use.

[0049] Take the wet bacterial cells collected after induction expression, add 1 g of wet bacterial cells to 10 mL of PBS buffer (0.1M, pH=7) to resuspend, sonicate at 300W for 15 min, working for 2 s and then resting for 3 s to obtain crude enzyme solution, thus obtaining BpALR crude enzyme solution.

[0050] 2. Culture of recombinant genetically engineered bacteria expressing tryptophan synthase alone

[0051] The recombinant genetically engineered bacterium Lemo 21(DE3)-pET28a-Trp, which expresses tryptophan synthase independently and was constructed in Example 1, was inoculated into a test tube containing 5 mL of LB liquid medium containing 50 mg / L kanamycin and cultured overnight at 37°C and 220 rpm in a shaker. Then, 1 mL of the overnight culture was added to a 250 mL shake flask containing 100 mL of LB liquid medium containing the corresponding antibiotic and cultured at 37°C and 220 rpm in a shaker for approximately 2.5 h. OD 600 Once the concentration reaches 0.6, add IPTG to a final concentration of 0.1 mM. Induce expression for 18 h at 28℃ and 200 rpm in a constant temperature shaker. After induction, transfer the bacterial culture to a 50 mL centrifuge tube and centrifuge at 4℃ and 4000 rpm for 10 min. Obtain the supernatant and wet cell pellet. Discard the supernatant and store the wet cell pellet in a -20℃ refrigerator for later use.

[0052] Take the wet bacterial cells collected after induction expression, add 1 g of wet bacterial cells to 10 mL of PBS buffer (0.1M, pH=7) to resuspend, sonicate at 300W for 15 min, working for 2 s and then resting for 3 s to obtain crude enzyme solution, thus obtaining Trp crude enzyme solution.

[0053] 3. Culture of recombinant genetically engineered bacteria co-expressing alanine racemic enzyme and tryptophan synthase

[0054] The recombinant genetically engineered bacteria co-expressing alanine racemic enzyme and tryptophan synthase constructed in Example 1 were inoculated into a test tube containing 5 mL of LB liquid medium containing 50 mg / L streptomycin and 50 mg / L kanamycin, and cultured overnight at 37°C and 220 rpm in a shaker. Then, 1 mL of the overnight culture was added to a 250 mL shake flask containing 100 mL of LB liquid medium containing 50 mg / L streptomycin and 50 mg / L kanamycin, and cultured at 37°C and 220 rpm in a shaker for approximately 2.5 h. OD 600 Once the concentration reaches 0.6, add IPTG to a final concentration of 0.1 mM. Induce expression for 20 h at 16℃ and 200 rpm in a constant temperature shaker. After induction, transfer the bacterial culture to a 50 mL centrifuge tube and centrifuge at 4℃ and 4000 rpm for 10 min. Obtain the supernatant and wet cell pellet. Discard the supernatant and store the wet cell pellet in a -20℃ refrigerator for later use.

[0055] Take the wet bacterial cells collected after induction expression, add 1 g of wet bacterial cells to 10 mL of PBS buffer (0.1M, pH=7) to resuspend, sonicate at 300W for 15 min, working for 2 s and then resting for 3 s to obtain crude enzyme solution, which is BpALR-Trp crude enzyme solution.

[0056] The crude enzyme solution was poured into 50 mL centrifuge tubes and centrifuged at 6000 rpm for 10 min at 4 °C. The induced bacterial culture (whole bacteria), the supernatant after disruption and centrifugation, and the precipitate after disruption and centrifugation were analyzed by agarose gel electrophoresis. The results are shown below. Figure 1 , Figure 2 and Figure 3 As shown, this indicates that engineered bacteria containing the target gene were successfully obtained.

[0057] Example 3: BpALR coupled with Trp catalyzes the synthesis of L-cysteine ​​from D-serine and sodium hydrosulfide.

[0058] Reference Figure 4 BpALR and / or Trp catalytic systems were constructed, respectively.

[0059] 1. Trp catalyzes the conversion of L-serine to L-cysteine.

[0060] The 10 mL reaction system consisted of: using the crude Trp enzyme solution prepared by the method in Example 2 as a catalyst, adding 25 g / L based on the weight of the wet cells before lysis, then adding L-serine at a final concentration of 20 g / L, sodium hydrosulfide at a final concentration of 17.5 g / L, PLP at a final concentration of 0.125 g / L, and PBS buffer (0.01 M, pH=7) to make up to 10 mL.

[0061] 2. Trp-catalyzed reaction system for the conversion of D-serine to L-cysteine

[0062] The 10 mL reaction system consisted of: using the crude Trp enzyme solution prepared by the method in Example 2 as a catalyst, adding 25 g / L based on the weight of the wet cells before lysis, then adding D-serine at a final concentration of 20 g / L, sodium hydrosulfide at a final concentration of 17.5 g / L, PLP at a final concentration of 0.125 g / L, and then adding PBS buffer (0.01 M, pH=7) and deionized water to make up to 10 mL.

[0063] 3. BpALR-Trp coupled catalytic reaction system for the conversion of D-serine to L-serine

[0064] The 10 mL co-expression catalytic system consisted of: crude BpALR enzyme solution and crude Trp enzyme solution prepared by the method in Example 2 as catalysts, each added at a concentration of 25 g / L based on the weight of the wet cells before lysis; D-serine at a final concentration of 20 g / L; sodium hydrosulfide at a final concentration of 17.5 g / L; PLP at a final concentration of 0.125 g / L; and PBS buffer (0.01 M, pH=7) to a final volume of 10 mL.

[0065] The above reaction systems were reacted at 37℃ and 200 rpm. Samples were taken every half hour, derivatized with DNFB (2,4-dinitrofluorobenzene), and the peak area of ​​serine was detected by HPLC. The residual amount of serine was calculated based on the standard curve of serine standard concentration versus peak area. The results are shown in [Figure number missing]. Figure 5 The results showed that: the tryptophan synthase in curve 1 could utilize L-serine to generate L-cysteine; the BpALR and tryptophan synthase in curve 3 could utilize D-serine to generate L-cysteine, and the D-serine was almost completely consumed after 3 hours; the Trp in curve 2 could not catalyze the synthesis of L-cysteine ​​from D-serine and sodium hydrosulfide, but the miscellaneous enzymes in the crude enzyme would also slowly consume D-serine. The conversion rate was 19% after 3 hours of reaction, but no L-cysteine ​​was detected.

[0066] HPLC detection conditions: Thermo Fisher Scientific liquid chromatograph, using a C18 column (4.6). The column temperature was 30℃, and the UV detection wavelength was 260 nm. The injection volume was 10 μL, the flow rate was 0.8 mL / min, and the column temperature was 350 mm (5 μm). Gradient elution was used. Mobile phase A consisted of pure acetonitrile; mobile phase B consisted of 826 mL ddH₂O + 170 mL acetonitrile + 2 mL triethylamine + 2 mL acetic acid. The elution times for D-serine and L-serine were both 8.8 min, and the elution time for L-cysteine ​​was 16.7 min.

[0067] Table 1. Final composition of the 10mL reaction system:

[0068]

[0069] Table 2. Gradient elution program for liquid chromatography:

[0070]

[0071] Example 4: Optimization of the enzyme dosage for the reaction of D-serine and sodium hydrosulfide to produce L-cysteine ​​using BpALR-coupled Trp.

[0072] The amount of enzyme added has a significant impact on the enzyme catalytic process. The catalytic conditions were optimized by adjusting the amount (ratio) of BpALR and Trp. Since the expression level of BpALR protein is lower than that of Trp, a larger amount of BpALR added to the catalytic system is beneficial to improving the conversion rate of D-serine to L-serine.

[0073] Composition of the 10 mL reaction system: The crude BpALR enzyme solution and crude Trp enzyme solution prepared by the method in Example 2 were added to a final concentration of (12.5 g / L Trp + 50 g / L BpALR, 25 g / L Trp + 50 g / L BpALR, 50 g / L Trp + 50 g / L BpALR, 25 g / L Trp + 25 g / L BpALR, 25 g / L Trp + 75 g / L BpALR), 87.5 g / L sodium hydrosulfide, 100 g / L D-serine, 0.125 g / L PLP, and PBS buffer (0.01 M, pH=7) to a final volume of 10 mL, based on the weight of the wet cells before lysis. The reaction was carried out at 37 °C and 200 rpm for 5 h. Samples were taken at 1, 2, 3, 4, and 5 hours. The production of L-cysteine ​​and the reduction of D-serine were detected using the method described in Example 3. The yield of L-cysteine ​​was calculated, and the results are shown in [Figure 3]. Figure 6 The results showed that when the amounts of Trp and BpALR added were 25 g / L and 75 g / L, respectively, the highest L-cysteine ​​yield (59.9%) was achieved after 3 hours of reaction. Decreasing BpALR dosage led to a decrease in L-cysteine ​​yield, with the highest yield (33.71%) corresponding to a dosage of 25 g / L. Changes in Trp dosage did not significantly alter the L-cysteine ​​yield. These results indicate that BpALR is the rate-determining step in the BpALR-Trp coupling reaction system, suggesting that the influence on L-cysteine ​​yield may be caused by the degradation of D-serine by intracellular enzymes in the host cell.

[0074] Yield (%) = L-cysteine ​​production / serine reduction (1g serine can be completely converted to produce 1.15g L-cysteine)

[0075] Example 5: Ammonium sulfate-enhanced BpALR-Trp coupling catalyzes the reaction of D-serine and sodium hydrosulfide to produce L-cysteine.

[0076] Literature reports that the addition of ammonium sulfate helps inhibit the degradation of D-serine by miscellaneous enzymes in the expression host, thereby improving the overall conversion yield. Therefore, under the optimal enzyme addition conditions, the effects of ammonium sulfate addition on the conversion of D-serine and the mass yield of L-cysteine ​​were determined.

[0077] The 10 mL reaction system consisted of: crude BpALR and crude Trp enzyme solutions prepared according to the method in Example 2, added to a final concentration of 75 g / L and 25 g / L respectively, based on the weight of the wet cells before lysis; 87.5 g / L sodium hydrosulfide; 100 g / L D-serine; 0.125 g / L PLP; 2.5 g / L ammonium sulfate; and PBS buffer (0.01 M, pH=7) to a final volume of 10 mL. The reaction was carried out at 37°C and 200 rpm for 5 h. Samples were taken at 1, 2, 3, 4, and 5 h, and the production of L-cysteine ​​and the reduction of D-serine were determined using the method in Example 3. The yield of L-cysteine ​​and the molar conversion rate of D-serine were calculated. The results are shown in [Figure 1]. Figure 7 The results showed that D-serine gradually decreased during the reaction, reaching a molar conversion of 100% after 5 hours; the product L-cysteine ​​gradually accumulated, with a mass yield of 108.51% after 5 hours, close to the theoretical value (115%). These results indicate that, under optimal enzyme addition conditions, adding ammonium sulfate to inhibit the degradation of D-serine by other enzymes can effectively improve the overall yield of D-serine to L-cysteine, demonstrating promising industrial application potential.

[0078] Molar conversion rate (%) = Reduction in D-serine / Initial amount of D-serine

[0079] Example 6: The BpALR-Trp co-expression system catalyzes the reaction of D-serine and sodium hydrosulfide to generate L-cysteine.

[0080] To reduce the amount of enzyme required and the cost of enzyme production through fermentation, BpALR and Trp were co-expressed. The co-expressing bacteria were then induced to express the enzymes and subjected to catalytic tests to assess their potential for industrial application.

[0081] The 10 mL reaction system consisted of: crude enzyme solution (BpALR-Trp) prepared according to the method in Example 2, with a final concentration of 50 g / L sodium hydrosulfide, 87.5 g / L sodium hydrosulfide, 100 g / L D-serine, and 0.125 g / L PLP added based on the weight of the wet cells before lysis. PBS buffer (0.01 M, pH=7) was then added to bring the total volume to 10 mL. The reaction was carried out at 37°C and 200 rpm for 4 hours.

[0082] The 10 mL reaction system consisted of: crude enzyme solution (BpALR-Trp) prepared according to the method in Example 2, with a final concentration of 50 g / L sodium hydrosulfide, 87.5 g / L sodium hydrosulfide, 100 g / L D-serine, 0.125 g / L PLP, and 2.5 g / L ammonium sulfate added based on the weight of the wet cells before lysis. PBS buffer (0.01 M, pH=7) was then added to bring the total volume to 10 mL. The reaction was carried out at 37°C and 200 rpm for 4 hours.

[0083] Samples were taken at 1, 2, 3, and 4 hours, and the production of L-cysteine ​​and the reduction of D-serine were detected using the method described in Example 3. The yield of L-cysteine ​​and the molar conversion rate of D-serine were calculated. The results are shown in [Figure 3]. Figure 8 When ammonium sulfate was not added to reaction system 1, D-serine was completely converted after 4 hours, but the mass yield of L-cysteine ​​was only 79.4%. When ammonium sulfate was added, although the catalytic efficiency decreased, D-serine was almost completely converted after 4 hours, and the mass yield of L-cysteine ​​reached 98.5%. These results are consistent with those of Example 5, indicating that D-serine degradation is a key factor affecting the mass yield, and the addition of ammonium sulfate can effectively inhibit D-serine degradation. However, compared with the case of expressing the two enzymes alone in Example 5, the product yield catalyzed by the co-expression strain decreased by nearly 10%, which may be due to the low expression ratio of BpALR. It is necessary to further increase its expression ratio to achieve a higher L-cysteine ​​yield.

Claims

1. A two-enzyme system for catalyzing the synthesis of L-cysteine ​​from D-serine, characterized in that, The dual-enzyme system consists of alanine racemic enzyme and tryptophan synthase.

2. The dual-enzyme system as described in claim 1, characterized in that, The alanine racemase is derived from Bacillus pseudofirmus A racemic enzyme family protein, designated BpALR, has the amino acid sequence shown in SEQ ID No. 2; the tryptophan synthase is derived from... Escherichia coli The tryptophan synthase family protein, denoted as Trp, has the amino acid sequence shown in SEQ ID No.

4.

3. The application of the dual-enzyme system of claim 1 in the catalytic synthesis of L-cysteine ​​from D-serine.

4. The application as described in claim 3, characterized in that, The application method is as follows: using crude enzyme solution obtained by ultrasonic disruption of wet bacterial cells from recombinant genetically engineered bacteria expressing a dual-enzyme system after induction culture as a catalyst, D-serine and sodium hydrosulfide as substrates, pyridoxal phosphate as coenzyme, and a buffer solution with pH 3-11 as the reaction medium to form a reaction system, and reacting completely at 30-40℃ and 100-400rpm to obtain a reaction solution containing L-cysteine; the catalyst is a mixture of crude enzyme solutions obtained by ultrasonic disruption of wet bacterial cells from recombinant genetically engineered bacteria expressing alanine racemic enzyme BpALR and tryptophan synthase Trp separately, or crude enzyme solution obtained by ultrasonic disruption of wet bacterial cells from recombinant genetically engineered bacteria expressing both alanine racemic enzyme BpALR and tryptophan synthase Trp.

5. The application as described in claim 4, characterized in that, In the reaction system, D-serine is added to a final concentration of 20-100 g / L; sodium hydrosulfide is added to a final concentration of 17.5-87.5 g / L; and pyridoxal phosphate is added to a final concentration of 0.1-0.5 g / L.

6. The application as described in claim 4, characterized in that, In the reaction system, the amount of crude enzyme solution used is 10-150 g / L based on the weight of the wet bacterial cells before crushing.

7. The application as described in claim 6, characterized in that, When the catalyst is a mixture of crude enzyme solutions, the amount of crude enzyme solution used for recombinant genetically engineered bacteria expressing alanine racemic enzyme BpALR is 75 g / L based on the weight of wet bacterial cells before lysis, and the amount of crude enzyme solution used for recombinant genetically engineered bacteria expressing tryptophan synthase Trp is 25 g / L based on the weight of wet bacterial cells before lysis; when the catalyst is a crude enzyme solution of recombinant genetically engineered bacteria that co-expresses alanine racemic enzyme BpALR and tryptophan synthase Trp, the amount used is 50 g / L based on the weight of wet bacterial cells before lysis.

8. The application as described in claim 4, characterized in that, The reaction system contains 1-5 g / L of ammonium sulfate.

9. The application as described in claim 4, characterized in that, The recombinant genetically engineered bacteria expressing alanine racemase were constructed according to the following steps: the gene fragment encoding alanine racemase was inserted between the PacⅠ and AvrⅡ restriction sites of the expression plasmid pCDFDuet-1 to obtain a recombinant plasmid; the recombinant plasmid was then transformed into the expression host *Escherichia coli* Lemo 21(DE3) to obtain the recombinant genetically engineered bacteria. The recombinant genetically engineered bacteria expressing tryptophan synthase were constructed according to the following steps: the gene fragment encoding tryptophan synthase was inserted between the NdeⅠ and HindⅢ restriction sites of the expression plasmid pET28a to obtain a recombinant plasmid; the recombinant plasmid was then transformed into the expression host *Escherichia coli* Lemo 21(DE3) to obtain the recombinant genetically engineered bacteria.

10. The application as described in claim 4, characterized in that, The recombinant genetically engineered bacteria co-expressing alanine racemic enzyme and tryptophan synthase were constructed according to the following steps: the alanine racemic enzyme encoding gene fragment was inserted between the PacⅠ and AvrⅡ restriction sites of the expression plasmid pCDFDuet to obtain a recombinant plasmid; the tryptophan synthase encoding gene fragment was inserted between the NdeⅠ and HindⅢ restriction sites of the expression plasmid pET28a to obtain a recombinant plasmid; the two recombinant plasmids were co-transformed into the expression host Escherichia coli Lemo BL21(DE3) to obtain the recombinant genetically engineered bacteria.

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