Method for preparing taurine through multi-enzyme catalytic synthesis

Abstract

The invention belongs to the technical field of gene and enzyme engineering, and discloses a method for preparing taurine through multi-enzyme catalytic synthesis, which comprises the following steps: respectively cloning genes for coding cysteine dioxygenase, cysteine sulfinic acid decarboxylase and flavin-containing monooxygenase 1 into a vector pET-28a to obtain recombinant plasmids, and transforming the recombinant plasmids into host cells to obtain the taurine. A recombinant strain corresponding to each enzyme is obtained; the method comprises the following steps: performing propagation culture on a recombinant strain, adding IPTG (isopropyl-beta-d-thiogalactoside) to perform induction culture, centrifuging and collecting thalli, adding a buffer solution to resuspend the thalli, performing ultrasonic cell disruption, centrifuging and purifying to respectively obtain recombinant cysteine dioxygenase, recombinant cysteine sulfinate decarboxylase and recombinant flavin-containing monooxygenase 1; taking cysteine as a substrate, and carrying out combined catalytic reaction on the three enzymes to prepare the taurine. The preparation method is green, environment-friendly, mild in condition, low in energy consumption, high in product purity and suitable for industrial production of taurine.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene and enzyme engineering, and particularly relates to a method for synthesizing taurine by multi-enzyme catalysis. Background Art

[0002] Taurine is an important β-sulfur-containing amino acid, which is widely present in the tissues and cells of mammals and is a conditional essential amino acid for humans and animals. Taurine has various physiological functions in organisms, including bile salt synthesis, calcium regulation, neural inhibition in the central nervous system, etc., and is also crucial for vision, platelet aggregation, and the action of insulin. The synthesis of taurine mainly comes from the sulfur-containing amino acid - cystine, and its biosynthetic pathway has been recorded in multiple tissues such as the liver, kidney, white and brown adipose tissues, pineal gland, and retina.

[0003] The synthesis of taurine mainly occurs through two pathways: the taurine pathway and the hypotaurine pathway. In the taurine pathway, cysteine is first oxidized by cysteine dioxygenase (or cysteine oxidase, hereinafter referred to as "CDO") to form cysteine sulfinic acid (hereinafter referred to as "CSA"), CSA is further oxidized by CDO to form taurine, and finally decarboxylated by cysteine sulfinic acid decarboxylase (hereinafter referred to as "CSD") to form taurine. In the hypotaurine pathway, CSA is decarboxylated by CSD to form hypotaurine, and hypotaurine is then oxidized by NAD+-dependent flavin-containing monooxygenase 1 to form taurine. In the liver cytosol, cystine (CYS) can be rapidly oxidized to 3-sulfonylalanine, which is catalyzed by a specific enzyme. 3-Sulfonylalanine is a metabolic branch point, which can be non-enzymatically oxidized to L-cysteine, or can enter the mitochondria to be converted into 3-sulfonyl-pyruvate, or can be decarboxylated in the cytoplasm to form hypotaurine. Hypotaurine exists in low concentrations in the liver and brain, and at least 90% of hypotaurine can be further oxidized to taurine.

[0004] Currently, the production processes of taurine mainly include chemical synthesis method and enzymatic synthesis method. The chemical synthesis method usually includes two types: ① Using ethanolamine as the raw material, through sulfuric acid esterification and sodium sulfite reduction to obtain taurine, but there are disadvantages such as incomplete esterification and difficult separation of sodium sulfate, which affect the product yield and quality; ② Using ethylene oxide as the raw material, first undergoing ring-opening addition with sodium sulfite, and then reacting with ammonia under heating and pressurization conditions to synthesize taurine, but the ammoniation reaction requires high temperature and high pressure, resulting in large equipment investment and low ammoniation yield, and multiple cycles of ammoniation are required, increasing the cost. In recent years, the enzymatic synthesis of taurine has gradually attracted attention, which has the advantages of mild reaction conditions and environmental friendliness. However, the existing enzymatic synthesis processes still have problems such as low efficiency and high cost. Summary of the Invention

[0005] (I) Object of the Invention

[0006] The object of the present invention is to provide a method for preparing taurine by multi-enzyme catalytic synthesis, which provides a green and efficient in vitro preparation route for taurine, with high reaction efficiency, short reaction time and low cost.

[0007] (II) Technical solution

[0008] To solve the above problems, the present invention provides a method for preparing taurine by multi-enzyme catalytic synthesis, including:

[0009] S1, cloning the genes encoding cysteine dioxygenase, cysteine sulfinic acid decarboxylase and flavin-containing monooxygenase 1 into the vector pET-28a respectively to obtain recombinant plasmids corresponding to each enzyme. It includes: respectively optimizing the codons of the genes encoding cysteine dioxygenase, cysteine sulfinic acid decarboxylase and flavin-containing monooxygenase 1, and cloning the optimized target genes into the vector pET-28a respectively to obtain recombinant plasmids corresponding to each enzyme.

[0010] S2, transforming the recombinant plasmids into host cells to obtain recombinant strains corresponding to each enzyme;

[0011] S3, culturing the recombinant strains until their OD600 values are 0.6 - 0.8, then adding IPTG for induction culture, then centrifuging to collect the thalli, adding buffer solution to resuspend the thalli, ultrasonically disrupting the cells, and centrifuging and purifying to obtain recombinant cysteine dioxygenase, recombinant cysteine sulfinic acid decarboxylase and recombinant flavin-containing monooxygenase 1 respectively;

[0012] S4, using cysteine as a substrate, and jointly catalyzing the reaction with recombinant cysteine dioxygenase, recombinant cysteine sulfinic acid decarboxylase and recombinant flavin-containing monooxygenase 1 to prepare taurine.

[0013] Further, the gene encoding cysteine dioxygenase is derived from human, Cupriavidus, Bacillus or mouse, and its corresponding amino acid sequences are respectively shown in SEQ ID NO.1 - 4.

[0014] Further, the gene encoding cysteine sulfinic acid decarboxylase is derived from Rattus norvegicus, and its corresponding amino acid sequence is shown in SEQ ID NO.5.

[0015] Further, the gene encoding flavin-containing monooxygenase 1 is derived from Homo sapiens, and its corresponding amino acid sequence is shown in SEQ ID NO.6.

[0016] Further, in the step S3, the IPTG concentration is 0.2 - 0.6 mM, preferably 0.2 mM, the induction culture temperature is 22 - 27 °C, preferably 25 °C, and the culture time is 8 - 16 hours, preferably 12 hours.

[0017] Further, the recombinant cysteine dioxygenase is 19 - 22 kDa, the recombinant cysteine sulfinic acid decarboxylase is 55 kDa, and the recombinant flavin-containing monooxygenase 1 is 60 kDa.

[0018] Further, in the step S4, the addition order of each enzyme is: recombinant cysteine dioxygenase → recombinant cysteine sulfinic acid decarboxylase → recombinant flavin-containing monooxygenase 1.

[0019] Further, the catalytic reaction system in the step S4 includes 0.5 - 1 mL of a buffer solution with a pH of 7.0 - 9.0, 0.1 - 1 mg / mL of recombinant cysteine dioxygenase, 0.1 - 1 mg / mL of recombinant cysteine sulfinic acid decarboxylase, 0.1 - 1 mg / mL of recombinant flavin-containing monooxygenase 1, and 1 - 50 mM of cysteine, and the buffer solution with a pH of 7.0 - 9.0 is preferably 1 mL.

[0020] Further, the catalytic reaction system in the step S4 further includes: 1 - 5 mM Fe 2+ and 0.01 - 0.1 mM of nicotinamide adenine dinucleotide.

[0021] Further, in the catalytic reaction, the reaction temperature is controlled to be 10 - 50 °C, and the reaction time is 0.5 - 10 hours.

[0022] Further, the host cells include Escherichia coli, Lactobacillus, and Bacillus subtilis, and preferably Escherichia coli.

[0023] (III) Beneficial effects

[0024] The above technical solution of the present invention has the following beneficial technical effects: The present invention provides a method for preparing taurine by multi-enzyme catalytic synthesis, which is a new method for efficiently, environmentally friendly, highly safe, highly selective and highly specific in vitro preparation of taurine. This method uses cysteine dioxygenase, cysteine sulfinic acid decarboxylase and flavin-containing monooxygenase 1 to react jointly to efficiently prepare taurine. First, a recombinant plasmid is constructed, the genes encoding cysteine dioxygenase, cysteine sulfinic acid decarboxylase and flavin-containing monooxygenase 1 are codon-optimized, the optimized target genes are recombined, and then the recombinant plasmid is transformed into host cells for propagation, induction expression, separation and purification to obtain recombinant cysteine dioxygenase, recombinant cysteine sulfinic acid decarboxylase and recombinant flavin-containing monooxygenase 1 respectively. Finally, using cysteine as a substrate, the recombinant cysteine dioxygenase, recombinant cysteine sulfinic acid decarboxylase and recombinant flavin-containing monooxygenase 1 are used jointly for catalytic reaction to obtain taurine. The production rate of taurine by the preparation method of the present invention is 50%-80%. The present invention provides a green, efficient, environmentally friendly and mild in vitro preparation method for taurine. Compared with the chemical synthesis method, the multi-enzyme synthesis method has the following advantages: ① Environmentally friendly: The reaction conditions are mild, without strong acids or alkalis, reducing wastewater and pollutants. ② High product purity: The reaction has strong selectivity, few impurities, and no complex purification is required. ③ Low energy consumption: The reaction is carried out at normal temperature and pressure, without high temperature and high pressure, reducing energy consumption. ④ High safety: The reaction process does not involve toxic chemicals, avoiding safety risks. ⑤ Low cost: The enzyme can be recycled, reducing the cost of by-product treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a schematic diagram of the synthesis pathway for preparing taurine by multi-enzyme catalytic synthesis of the present invention;

[0026] Figure 2 is a graph of the production amount of taurine by multi-enzyme joint catalytic synthesis in vitro of the present invention;

[0027] Figure 3 is an SDS-PAGE electrophoresis diagram of recombinant cysteine dioxygenase from different sources of the present invention;

[0028] Figure 4 is a standard curve graph of the taurine standard product of the present invention;

[0029] Figure 5 is an SDS-PAGE electrophoresis diagram of the recombinant cysteine sulfinic acid decarboxylase and flavin-containing monooxygenase 1 of the present invention;

[0030] Figure 6 is a liquid chromatogram of taurine detection of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0031] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are exemplary and are not intended to limit the scope of the present invention. In addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present invention.

[0032] The present invention provides a method for the preparation of taurine by multi-enzyme catalytic synthesis, comprising the following steps:

[0033] S1, respectively codon-optimize the genes encoding cysteine dioxygenase, cysteine sulfinic acid decarboxylase, and flavin-containing monooxygenase 1, and clone the optimized target genes into the vector pET-28a respectively to obtain the recombinant plasmids corresponding to each enzyme. The gene encoding cysteine dioxygenase is derived from Human, Cupriavidus, Bacillus, or Mause, and its amino acid sequences are shown in SEQ ID NO.1-4 respectively. SEQ ID NO.1 is CDO-Human (the amino acid sequence of cysteine dioxygenase derived from Human), SEQ ID NO.2 is CDO-Cupriavidus (the amino acid gene sequence of cysteine dioxygenase derived from Cupriavidus), SEQ ID NO.3 is CDO-Bacillus (the amino acid gene sequence of cysteine dioxygenase derived from Bacillus), and SEQ ID NO.4 is CDO-Mause (the amino acid sequence of cysteine dioxygenase derived from Mause). The gene encoding cysteine sulfinic acid decarboxylase is derived from Rattus norvegicus, and its amino acid sequence is shown in SEQ ID NO.5. SEQ ID NO.5 is CSAD-Rattus norvegicus (the amino acid sequence of cysteine sulfinic acid decarboxylase derived from Rattus norvegicus). The gene encoding flavin-containing monooxygenase 1 is derived from Homo sapiens, and the amino acid sequence of flavin-containing monooxygenase 1 is shown in SEQ ID NO.6. SEQ ID NO.6 is FMO-Homo sapiens (the amino acid sequence of flavin-containing monooxygenase 1 derived from Homo sapiens). Insert the target genes corresponding to each enzyme into the corresponding sites of pET-28a through HindIII and NdeI, and identify by restriction enzyme digestion analysis to obtain the recombinant plasmids corresponding to each enzyme.

[0034] S2. Transform the recombinant plasmid into a host cell to obtain recombinant strains corresponding to each enzyme. The host cells include Escherichia coli, Lactobacillus, and Bacillus subtilis, preferably Escherichia coli. This step is specifically as follows: Use the chemical method to introduce the recombinant plasmid into Escherichia coli BL21(DE3) competent cells. After resuscitation, coat them on a solid LB medium containing 1‰ Kana (kanamycin), invert it in an incubator, and culture overnight at 37°C.

[0035] S3. After culturing the recombinant strains until their OD600 value reaches 0.6 - 0.8, add IPTG for induction culture, then centrifuge to collect the bacterial cells, and then resuspend the bacterial cells in a buffer solution. Ultrasonically break the cells, and after centrifugation and purification, obtain recombinant cysteine dioxygenase, recombinant cysteine sulfinic acid decarboxylase, and recombinant flavin-containing monooxygenase 1 respectively. In step S3, the IPTG concentration is 0.2 mM, the induction culture temperature is 25°C, and the culture time is 12 hours. This step is specifically as follows: Select well-growing monoclonal strains corresponding to each enzyme, and inoculate them into 4 mL of LB liquid medium containing 1‰ Kana respectively. After culturing at 37°C for 6 - 7 hours, transfer 1% of the liquid strain to 200 mL of LB liquid medium. When OD600 is 0.6 - 0.8, add IPTG with a final concentration of 0.2 mM, and continue to induce culture at 25°C for 12 hours. Centrifuge to collect the bacterial cells (set the centrifugation parameters as 8000 rpm, centrifuge for 6 min), resuspend the bacterial cells with 20 mL of buffer solution with a pH of 7.0 - 9.0, intermittently ultrasonically treat (ultrasonic at 200 W for 3 s, intermittent for 2 s) to break the cells, and centrifuge (8000 rpm, 45 min) to collect the soluble target protein supernatant expression components (each crude enzyme sample). The volume ratio of the buffer solution to the fermentation broth is 1:10, that is, the bacterial cells collected from 200 mL of the bacterial liquid are resuspended with 20 mL of the buffer solution. Use SDS-PAGE protein electrophoresis to verify whether the induction expression of cysteine dioxygenase, cysteine sulfinic acid decarboxylase, and flavin-containing monooxygenase 1 is achieved. Then use Ni column affinity chromatography to purify and separate the above-mentioned target protein supernatant expression components to obtain recombinant cysteine dioxygenase, recombinant cysteine sulfinic acid decarboxylase, and recombinant flavin-containing monooxygenase 1 respectively. The recombinant cysteine dioxygenase is 19 - 22 kDa, the recombinant cysteine sulfinic acid decarboxylase is 55 kDa, and the recombinant flavin-containing monooxygenase 1 is 60 kDa.

[0036] S4. Using cysteine as a substrate, use recombinant cysteine dioxygenase, recombinant cysteine sulfinic acid decarboxylase, and recombinant flavin-containing monooxygenase 1 to carry out a catalytic reaction jointly to prepare taurine. In step S4, the synthesis pathway is as Figure 1As shown in the figure, the addition order of each enzyme is: recombinant cysteine dioxygenase → recombinant cysteine sulfinic acid decarboxylase → recombinant flavin-containing monooxygenase 1. That is, first add recombinant cysteine dioxygenase to cysteine to generate cysteine sulfinic acid, then mix the generated cysteine sulfinic acid with cysteine sulfinic acid decarboxylase to generate hypotaurine, and finally mix hypotaurine with flavin-containing monooxygenase 1 to react to generate taurine. In the catalytic reaction system of step S4, the experimental conditions are set as follows: add recombinant cysteine dioxygenase with a final concentration of 0.1-1 mg / mL, recombinant flavin-containing monooxygenase 1 with a final concentration of 0.1-1 mg / mL, recombinant cysteine sulfinic acid decarboxylase with a final concentration of 0.1-1 mg / mL, 1-50 mM (millimoles per liter) cysteine, 1-5 mM Fe 2+ 、0.01-0.1 mM NAD+ (nicotinamide adenine dinucleotide) into 1 mL of Tris-Hcl buffer with a pH of 7.0-9.0, and react at 10-50 °C for 0.5-10 hours to obtain 0.67-40.5 mM taurine, and the production rate of taurine is 50%-80%. The following combines Figures 2-6 to specifically describe the above steps:

[0037] Before separating and purifying the soluble target protein supernatant expression component (crude enzyme sample) collected in step S3, first use SDS-PAGE protein electrophoresis to verify whether the induced expression of cysteine dioxygenase, cysteine sulfinic acid decarboxylase, and flavin-containing monooxygenase 1 has been achieved. The specific process is as follows:

[0038] 1. Verification of induced expression results by SDS-PAGE protein electrophoresis

[0039] (1) Prepare the sample: The ratio of protein electrophoresis loading buffer to the crude enzyme sample is 1:3;

[0040] (2) Binding: Boil the sample obtained in step (1) in boiling water for 10 min to fully denature the protein, and then quickly cool it to room temperature;

[0041] (3) Electrophoresis: Add 3 μL of the corresponding protein marker (protein molecular marker), add 15 μL of the sample to different lanes, the voltage is 120 U, the current is 400 mA, and wait for 1 h;

[0042] (4) Color development: Import an appropriate amount of Fast-blue and soak the electrophoresis gel for about 30 min. Use a gel imaging system to analyze and observe. Figure 3 Figure 24 is the SDS-PAGE electrophoresis diagram of recombinant cysteine dioxygenase from different sources. It can be known that cysteine dioxygenase is successfully expressed, and the recombinant cysteine dioxygenase is approximately 20 kDa. Figure 5 Figure 26 is the SDS-PAGE electrophoresis diagram of cysteine sulfinic acid decarboxylase and flavin-containing monooxygenase 1 (Figure 5 From left to right are Maker (standard product), FMO (flavin-containing monooxygenase 1), and CSAD (cysteine sulfinic acid decarboxylase). It can be known that both cysteine sulfinic acid decarboxylase and flavin-containing monooxygenase 1 are successfully expressed. The recombinant cysteine sulfinic acid decarboxylase is approximately 55 kDa, and the recombinant flavin-containing monooxygenase 1 is about 60 kDa.

[0043] 2. Protein purification of cysteine dioxygenase, cysteine sulfinic acid decarboxylase, and flavin-containing monooxygenase 1

[0044] The above supernatant part (crude enzyme sample) was purified by Ni column affinity chromatography to obtain recombinant cysteine dioxygenase, recombinant cysteine sulfinic acid decarboxylase, and recombinant flavin-containing monooxygenase 1. The specific process is as follows:

[0045] (1) Filter the supernatant with a 0.45 μm diameter filter;

[0046] (2) Wash the Ni column: Wash the resin with 6 - 12 column volumes of ultrapure water at a flow rate of 100 cm / h to remove ethanol;

[0047] (3) Equilibrate the Ni column: Equilibrate the medium with 6 - 12 column volumes of equilibration buffer (20 mM Tirs-HCl pH 7.5, 400 mM NaCl, 20 mM imidazole) at a flow rate of 120 cm / h to ensure that the solution components and pH of the medium are consistent with the sample;

[0048] (4) Load the sample: Centrifuge and filter the sample, and then slowly load the sample at a flow rate of 120 cm / h;

[0049] (5) Wash away impurities: Elute non-specific proteins with 12 - 24 column volumes of buffer (20 mM Tirs-HCl pH 7.5, 400 mM NaCl, 20 mM imidazole) at a flow rate of 120 cm / h, and collect the flow-through for subsequent analysis;

[0050] (6) Elute: Elute the protein with 6 - 12 column volumes of elution buffer (20 mM Tirs-HCl pH 7.5, 200 mM NaCl, 20 mM imidazole) at a low flow rate, and collect the eluate for subsequent analysis;

[0051] (7) Ultrafiltration concentration: Transfer the collected eluate to an ultrafiltration tube, ultrafilter at 4000 rpm for 1 hour, and then collect the concentrated protein (target protein) retained;

[0052] (8) Storage: Add an appropriate amount of glycerol to the above target proteins (recombinant cysteine dioxygenase, recombinant cysteine sulfinic acid decarboxylase, and recombinant flavin-containing monooxygenase 1) and store at -80 °C.

[0053] 3. Multi-Enzyme Catalytic Synthesis of Taurine

[0054] Using the recombinant cysteine dioxygenase, recombinant cysteine sulfinic acid decarboxylase, and recombinant flavin-containing monooxygenase 1 obtained in the above step 2-(8) for combined catalytic and highly efficient preparation of taurine. Taurine with a concentration of 0.67 - 33.5 mM can be obtained after reacting for 0.5 - 10 hours. The specific preparation process is as follows in the examples.

[0055] During the experiment, the preparation process of the taurine standard curve solution is as follows:

[0056] Accurately weigh 0.1000 g of taurine standard product, dilute it with water and make the volume up to 100 mL to obtain a 1 mg / mL taurine standard stock solution. Dilute it with water to prepare a series of standard solutions. Take 20 μL of taurine standard solutions with concentrations of 0, 2, 5, 10, 20, and 25 μg / mL respectively, derivatize them, and perform liquid chromatography analysis and fluorescence detector determination. Chromatographic conditions: Chromatographic column Agilent Eclipse plus C18 (4.6 mm × 250 mm, 5 μm); Mobile phase: methanol + acetonitrile: water (25:75, volume ratio), flow rate 0.8 mL / min; Column temperature 30 °C; Fluorescence detector: Excitation wavelength 330 nm, emission wavelength 530 nm; Injection volume 20 μL. The liquid chromatogram of taurine is as Figure 6 shown, and the peak emergence time is at 6.067 min. Calculate the content of taurine according to the method of the national standard (GB5009.169 - 2016). Taking the standard solution concentration as the ordinate and the corresponding peak color (chromatographic peak area) as the abscissa for linear regression, the standard curve of taurine is as Figure 4 shown.

[0057] Example 1

[0058] Place 1 mM cysteine, 0.2 mg / mL recombinant cysteine dioxygenase (parallel tests are carried out with those from Cupriavidus sp. and from humans, each at 0.2 mg / mL, and the results are shown in Figure 2 ), 0.2 mg / mL recombinant flavin-containing monooxygenase 1, 0.2 mg / mL recombinant cysteine sulfinic acid decarboxylase, 2 mM Fe 2+ , 0.1 mM nicotinamide adenine dinucleotide in 1 mL of buffer solution with pH 7.5 for multi-enzyme combined catalytic reaction. React at 37 °C for 1.5 hours to obtain 0.67 mM taurine. The production rate of taurine is 67%. The results are as Figure 2 shown. Figure 2In the case of CDO-CP, it represents the production amount of taurine catalyzed by the multi-enzyme cascade method when using recombinant cysteine dioxygenase derived from Cupriavidus. For CDO-H, it represents the production amount of taurine catalyzed by the multi-enzyme cascade method when using recombinant cysteine dioxygenase derived from humans. CDO-H performed better at 150 minutes, and recombinant CDO-H was used in the subsequent Examples 2-8.

[0059] Example 2

[0060] 1 mM cysteine, enzymes with different concentrations: 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL (recombinant cysteine dioxygenase, recombinant flavin-containing monooxygenase 1, recombinant cysteine sulfinic acid decarboxylase), 2 mM Fe 2+ , 0.1 mM nicotinamide adenine dinucleotide were placed in 1 mL of buffer at pH 7.5 for multi-enzyme combined catalytic reaction. The parallel tests corresponded to Table 1, and the reaction was carried out at 37 °C for 1.5 hours, obtaining 0.53 mM, 0.79 mM, and 0.77 mM of taurine respectively, and the production rates of taurine were 53%, 79%, and 77% respectively.

[0061] Table 1 Different addition amounts of each recombinant enzyme and detection results

[0062]

[0063]

[0064] Example 3

[0065] 1 mM cysteine, 0.5 mg / mL of enzymes (recombinant cysteine dioxygenase, recombinant flavin-containing monooxygenase 1, recombinant cysteine sulfinic acid decarboxylase), 2 mM Fe 2+ , 0.01, 0.05, 0.1 mM of nicotinamide adenine dinucleotide were added respectively (0.5 mg / mL of glucose dehydrogenase and 100 mM of glucose were added) and placed in 1 mL of buffer at pH 7.5 for multi-enzyme combined catalytic reaction. The parallel tests corresponded to Table 2, and the reaction was carried out at 37 °C for 1.5 hours, obtaining 0.76 mM, 0.81 mM, and 0.82 mM of taurine respectively, and the production rates of taurine were 76%, 82%, and 81% respectively.

[0066] Table 2 Different addition amounts of NAD+ and detection results

[0067] Example 3 NAD+ addition amount (mM) Taurine production rate / % 3-a 0.01 76 3-b 0.05 82 3-c 0.1 81

[0068] Example 4 (Determination of cysteine substrate concentration)

[0069] 10, 20, 30, 40, and 50 mM of cysteine, 0.5 mg / mL of enzymes (recombinant cysteine dioxygenase, recombinant flavin-containing monooxygenase 1, recombinant cysteine sulfinic acid decarboxylase), 2 mM of Fe 2+ , 0.05 mM of nicotinamide adenine dinucleotide (with 0.5 mg / mL of glucose dehydrogenase and 100 mM of glucose) were placed in 1 mL of a buffer solution with a pH of 7.5 for a multi-enzyme catalyzed reaction. The reaction was carried out at 37 °C for 5 hours, and 8.47 mM, 15.31 mM, 22.58 mM, 24.61 mM, and 25.89 mM of taurine were obtained respectively. The results showed that when cysteine was at 50 mM, the production rates of taurine were 51.8% respectively. This concentration was used as the optimal example of Example 4, and cysteine at this concentration was used as the substrate concentration for Examples 5 - 8.

[0070] Example 5 (Optimizing Fe 2+ )

[0071] 50 mM of cysteine, 0.5 mg / mL of enzymes (recombinant cysteine dioxygenase, recombinant flavin-containing monooxygenase 1, recombinant cysteine sulfinic acid decarboxylase) were respectively added with 1 mM, 3 mM, and 5 mM of Fe 2+ , 0.05 mM of nicotinamide adenine dinucleotide (with 0.5 mg / mL of glucose dehydrogenase and 100 mM of glucose) and placed in 10 mL of a buffer solution with a pH of 7.5 for a multi-enzyme catalyzed reaction. The reaction was carried out at 30 °C for 10 hours, and 20.35 mM, 23.34 mM, and 28.79 mM of taurine were obtained respectively. The results showed that when Fe 2+ was at 5 mM, the production rates of taurine were 57.6% respectively. This concentration was used as the optimal example of Example 5.

[0072] Example 6 (Optimizing the pH value of the buffer solution)

[0073] 50 mM of cysteine, 0.5 mg / mL of enzymes (recombinant cysteine dioxygenase, recombinant flavin-containing monooxygenase 1, recombinant cysteine sulfinic acid decarboxylase), 5 mM of Fe 2+ , 0.05 mM of nicotinamide adenine dinucleotide (with 0.5 mg / mL of glucose dehydrogenase and 100 mM of glucose) were respectively placed in 10 mL of buffer solutions with pH values of 7.0, 7.5, 8.5, and 9.0 for a multi-enzyme catalyzed reaction. The reaction was carried out at 30 °C for 10 hours, and 29.42 mM, 30.38 mM, 29.63 mM, and 28.55 mM of taurine were obtained respectively. The results showed that when the pH was 7.5, the production rates of taurine were 60.8% respectively. This concentration was used as the optimal example of Example 6.

[0074] Example 7 (Optimizing the reaction temperature)

[0075] 50 mM of cysteine, 0.5 mg / mL of enzymes (recombinant cysteine dioxygenase, recombinant flavin-containing monooxygenase 1, recombinant cysteine sulfinic acid decarboxylase), 5 mM of Fe 2+ , 0.05 mM of nicotinamide adenine dinucleotide (added with 0.5 mg / mL of glucose dehydrogenase and 100 mM of glucose) were placed in 10 mL of buffer solution with pH 7.5 for multi-enzyme combined catalytic reaction. The reaction was carried out at 10, 30, 37, and 50 °C for 10 hours respectively, and 9.42 mM, 35.38 mM, 29.63 mM, and 8.55 mM of taurine were obtained respectively. The results showed that when the catalytic reaction temperature was 30 °C, the production rate of taurine was 70.8%. This temperature was used as the optimal example of Example 7.

[0076] Example 8 (optimizing reaction time)

[0077] 50 mM of cysteine, 0.5 mg / mL of enzymes (recombinant cysteine dioxygenase, recombinant flavin-containing monooxygenase 1, recombinant cysteine sulfinic acid decarboxylase), 5 mM of Fe 2+ , 0.05 mM of nicotinamide adenine dinucleotide (added with 0.5 mg / mL of glucose dehydrogenase and 100 mM of glucose) were placed in 10 mL of buffer solution with pH 7.5 for multi-enzyme combined catalytic reaction. The reaction was carried out at 30 °C for 6, 10, and 16 hours respectively, and 25.41, 36.25, and 40.5 mM of taurine were obtained. The enzymes were removed by filtration through a membrane with a molecular weight of 10K, and ethanol was added to precipitate taurine. The purity detected by liquid phase was 98.5%. The results showed that when the catalytic reaction time was 16 hours, the production rate of taurine was 81%, and the purity of taurine was 98.5%. This duration was used as the optimal example of Example 8. The detection conditions and results of each optimal example are shown in Table 3.

[0078] Table 3 Summary chart of the results of each group's optimal examples

[0079]

[0080] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. Persons familiar with the art of this technology can easily make various modifications to these embodiments and apply the general principles described again to other embodiments without creative labor. The present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art without departing from the scope of the present invention as disclosed should be within the protection scope of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative labor fall within the protection scope of the present invention. In the description of the present invention, it should be noted that the terms "first", "second", and "third" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance. In addition, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. It should be understood that the above specific embodiments of the present invention are only for illustrative or explanatory purposes of the principles of the present invention and do not constitute a limitation to the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modifications that fall within the scope and boundaries of the appended claims or equivalent forms of such scope and boundaries. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is not necessary and impossible to enumerate all the embodiments here. And the obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for preparing taurine by multi-enzyme catalysis synthesis, characterized in that: include: S1, respectively cloning the genes encoding cysteine ​​dioxygenase, cysteine ​​sulfenic acid decarboxylase and flavin-containing monooxygenase 1 into the vector pET-28a to obtain the recombinant plasmids corresponding to the respective enzymes; S2, transforming the recombinant plasmid into a host cell to obtain a recombinant strain corresponding to each enzyme; S3, after the recombinant strain is propagated and cultured until its OD600 value is 0.6-0.8, IPTG is added for induction culture, and then the bacteria are collected by centrifugation, and then a buffer solution is added to resuspend the bacteria, and the cells are ultrasonically disrupted, and after centrifugation and purification, recombinant cysteine ​​dioxygenase, recombinant cysteine ​​sulfenic acid decarboxylase and recombinant flavin-containing monooxygenase 1 are obtained respectively; S4, using cysteine ​​as a substrate, utilizing the recombinant cysteine ​​dioxygenase, the recombinant cysteine ​​sulfenic acid decarboxylase and the recombinant flavin-containing monooxygenase 1 to jointly carry out a catalytic reaction to prepare taurine.

2. The method for preparing taurine by multi-enzyme catalysis synthesis according to claim 1, characterized in that: The gene encoding cysteine ​​dioxygenase is derived from humans, Cupriavidus, Bacillus or mice, and its corresponding amino acid sequences are shown in SEQ ID NOs. 1 to 4, respectively.

3. The method for preparing taurine by multi-enzyme catalysis synthesis according to claim 1, characterized in that: The gene encoding cysteine ​​sulfenic acid decarboxylase is derived from Rattus norvegicus, and its corresponding amino acid sequence is shown in SEQ ID NO.

5.

4. The method for preparing taurine by multi-enzyme catalysis synthesis according to claim 1, characterized in that: The gene encoding flavin-containing monooxygenase 1 is derived from Homo sapiens, and its corresponding amino acid sequence is shown in SEQ ID NO.

6.

5. The method for preparing taurine by multi-enzyme catalysis synthesis according to claim 1, characterized in that: In the step S3, the IPTG concentration is 0.2-0.6 mM, the induction culture temperature is 22-27° C., and the culture time is 8-16 hours.

6. The method for preparing taurine by multi-enzyme catalysis synthesis according to claim 1, characterized in that: The recombinant cysteine ​​dioxygenase is 19-22 kDa, the recombinant cysteine ​​sulfenic acid decarboxylase is 55 kDa, and the recombinant flavin-containing monooxygenase 1 is 60 kDa.

7. The method for preparing taurine by multi-enzyme catalysis synthesis according to claim 1, characterized in that: In the step S4, the order of adding the enzymes is: recombinant cysteine ​​dioxygenase→recombinant cysteine ​​sulfenic acid decarboxylase→recombinant flavin-containing monooxygenase 1.

8. The method for preparing taurine by multi-enzyme catalysis synthesis according to claim 1, characterized in that: The catalytic reaction system of step S4 comprises: 0.5-1 mL of a buffer solution with a pH of 7.0-9.0, 0.1-1 mg / mL of recombinant cysteine ​​dioxygenase, 0.1-1 mg / mL of recombinant cysteine ​​sulfenic acid decarboxylase, 0.1-1 mg / mL of recombinant flavin-containing monooxygenase 1, and 1-50 mM of cysteine.

9. The method for preparing taurine by multi-enzyme catalysis synthesis according to claim 8, characterized in that: The catalytic reaction system of step S4 further includes: 1-5 mM Fe 2+ and 0.01-0.1 mM nicotinamide adenine dinucleotide.

10. The method for preparing taurine by multi-enzyme catalysis synthesis according to claim 1, characterized in that: In the catalytic reaction, the reaction temperature is controlled to be 10-50° C., and the reaction time is 0.5-16 hours.

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