L-glutathione bifunctional synthetase and preparation method thereof

Abstract

The invention discloses an L-glutathione bifunctional synthetase and a preparation method thereof. The invention provides an application of the protein in at least one of the following steps: A1) synthesizing glutathione; a2) is used as a glutathione bifunctional synthetase; a3) synthesizing glutathione by taking glutamic acid, cysteine and glycine as substrates; according to the invention, a bifunctional enzyme for synthesizing GSH by catalyzing a substrate through a one-step method is obtained through an artificial intelligence screening method based on big data. After protein is purified, the amount of zymoprotein is adjusted to reach the same concentration, and the GshF-FIH can efficiently catalyze glutamic acid, cysteine and glycine to synthesize glutathione.

Description

Technical Field

[0001] The invention belongs to the fields of genetic engineering and enzyme catalysis, and relates to L-glutathione bifunctional synthetase and a preparation method thereof. Background Art

[0002] L-glutathione (GSH) is a tripeptide with strong antioxidant activity. It is synthesized from three amino acid substrates: L-glutamic acid (L-Glu), L-cysteine ​​(L-Cys), and glycine (Gly). GSH is widely present in organisms and possesses antioxidant and detoxifying properties, as well as functions in maintaining a normal immune system. GSH is not only used in pharmaceuticals but also as a base for functional foods, with applications in anti-aging, immunity-boosting, and anti-tumor properties.

[0003] Currently, methods for producing GSH domestically and internationally include chemical synthesis, fermentation, and enzymatic methods. Chemical synthesis, however, is plagued by issues such as low purity of optical isomers and environmental pollution from organic reagents, and has been gradually replaced by fermentation and enzymatic methods. The core of fermentation and enzymatic methods, based on modern bioengineering technology, is the development of highly active GSH synthases. Although fermentation and enzymatic methods for GSH synthesis have been industrialized in recent years, the predominantly two-step process is employed. This involves synthesizing the target product GSH from the substrates L-Glu, L-Cys, and Gly via a two-step enzymatic reaction involving γ-glutamylcysteine ​​synthetase (γ-GCS) and glutathione synthetase (GS). This method is limited by the synergistic cascade required for the two enzymes to exert their activity and the substrate inhibition effect, resulting in high production costs for GSH and hindering the widespread use of industrialized GSH products.

[0004] Therefore, there is a need to find enzymes and methods that can efficiently synthesize GSH. Summary of the Invention

[0005] The technical problem solved by the present invention is how to efficiently synthesize L-glutathione and the bifunctional synthase used.

[0006] In order to solve the above technical problems, the first aspect of the present invention provides the use of a protein in at least one of the following:

[0007] A1) synthesis of glutathione;

[0008] A2) acts as a bifunctional glutathione synthetase;

[0009] A3) Synthesizes glutathione using glutamate, cysteine, and glycine as substrates;

[0010] The protein is any of the following:

[0011] a1) The protein includes the amino acid residues shown in SEQ ID NO: 3;

[0012] a2) a protein that has an amino acid residue identity of greater than 80% with the protein in a1) and has the same function;

[0013] The protein shown in a3) includes a protein shown in the sequence obtained by connecting a tag to the N-terminus or / and C-terminus of any protein shown in a1)-a2).

[0014] In the above, the glutathione bifunctional synthetase can realize the function of γ-glutamylcysteine ​​synthetase (γ-GCS) catalyzing L-Glu and L-Cys to synthesize γ-Glu-Cys, and can also realize the function of glutathione synthetase (GS) catalyzing γ-Glu-Cys and Gly to synthesize Gsh.

[0015] The above synthesis of glutathione using glutamate, cysteine ​​and glycine as substrates is a one-step synthesis.

[0016] In certain embodiments, the protein represented by a1) is the amino acid residue represented by SEQ ID NO:3.

[0017] In the above description, the tag refers to a polypeptide or protein that is fused with the target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag can be a Flag tag, His tag, MBP tag, HA tag, Myc tag, GST tag, and / or SUMO tag, etc.

[0018] In the above description, identity refers to amino acid sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, the identity of a pair of amino acid sequences can be calculated by searching in Advanced BLAST 2.1 using blastp as the program, setting the Expect value to 10, all filters to OFF, BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively. The identity value (%) can then be obtained.

[0019] In the above proteins, the above 80% or greater identity may be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0020] In a second aspect, the present invention provides a use of a biomaterial related to the mutant protein described in the first aspect in at least one of the following:

[0021] B1) synthesis of glutathione;

[0022] B2) preparing glutathione bifunctional synthetase;

[0023] B3) synthesizes glutathione using glutamate, cysteine ​​and glycine as substrates;

[0024] The biological material is any one of the following C1) to C4):

[0025] C1) a nucleic acid molecule encoding the protein of the first aspect;

[0026] C2) an expression cassette containing the nucleic acid molecule described in C1);

[0027] C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

[0028] C4) A recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3).

[0029] In the applications described above,

[0030] C1) The nucleic acid molecule is any one of the following:

[0031] c1) The nucleotide sequence of the nucleic acid molecule includes SEQ ID NO: 4;

[0032] c2) a cDNA molecule or a DNA molecule that has 75% or more identity with the nucleotide sequence defined in c1) and encodes the protein described in the first aspect;

[0033] c3) a cDNA molecule or a DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in any one of c1) to c2) and encodes the protein described in the first aspect.

[0034] In the above application, the glutathione is L-glutathione.

[0035] The term "identity" refers to sequence similarity to a natural nucleic acid sequence. Identity can be assessed visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess the identity between related sequences. The identity of more than 75% can be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%.

[0036] In certain embodiments, the nucleotide sequence of the nucleic acid molecule represented by c1) is SEQ ID NO:4.

[0037] In certain embodiments, the expression cassette described in C2) containing the nucleic acid molecule described in C1) refers to DNA capable of expressing the protein described in the above application in a host cell. This DNA may include not only a promoter for initiating transcription of the protein-encoding gene, but also a terminator for terminating transcription of the protein-encoding gene. Furthermore, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters.

[0038] In certain embodiments, the recombinant vector shown in C3) is a recombinant vector obtained by introducing the nucleic acid molecule described in C1) into a plasmid expression vector, and contains the nucleic acid molecule described in C1) or the expression cassette described in C2).

[0039] In certain embodiments, the plasmid expression vector may be pET-28a(+).

[0040] In one embodiment, the recombinant vector is pET-28a-GshF-FIH.

[0041] In certain embodiments, the recombinant microorganism shown in C4) is obtained by introducing the recombinant vector described in C3) into a host microorganism.

[0042] In certain embodiments, the host microorganism can be yeast, bacteria, algae or fungi.

[0043] In certain embodiments, the bacteria may specifically be Escherichia coli.

[0044] In one embodiment, the recombinant microorganism shown in C4) is BL21 / pET-28a-GshF-FIH.

[0045] In a third aspect, the present invention provides a method for synthesizing glutathione, comprising the following steps: using the protein described in the first aspect as an enzyme to catalyze a substrate with the assistance of ATP to synthesize glutathione;

[0046] The substrates are glutamate, cysteine ​​and glycine.

[0047] In the above method, the glutathione is L-glutathione.

[0048] In the above method, the catalytic substrate is a one-step catalytic substrate.

[0049] In certain embodiments, the catalytic reaction system comprises the protein described in the first aspect, ATP, glutamate, cysteine, glycine and a buffer.

[0050] In certain embodiments, the buffer may be Tris-HCl buffer.

[0051] In certain embodiments, the catalytic reaction system further comprises metal ions.

[0052] In certain embodiments, the metal ion is a Mg ion.

[0053] In a certain embodiment, the metal ion is present in the form of MgCl2.

[0054] In certain embodiments, in the catalytic reaction system, the concentration of the protein is 1-5 mg / L.

[0055] In certain embodiments, in the catalytic reaction system, the concentration of the protein is 1, 2, 3, 4 or 5 mg / L.

[0056] In certain embodiments, in the catalytic reaction system, the concentrations of glutamate, cysteine, and glycine are all 20 mM.

[0057] In certain embodiments, the catalytic pH is 8±1.

[0058] In certain embodiments, the catalysis time is 20-30 min.

[0059] In a certain embodiment, the catalysis time is 20 minutes.

[0060] In certain embodiments, the catalytic temperature is 35-40°C.

[0061] In a certain embodiment, the catalytic temperature is 37°C.

[0062] In a fourth aspect, the present invention provides the biomaterial according to the second aspect.

[0063] Beneficial effects of the present invention: The present invention obtains a bifunctional enzyme with a one-step catalytic substrate synthesis of GSH through an artificial intelligence screening method based on big data. After purifying the protein, the amount of the enzyme protein is adjusted to the same concentration, and the catalytic performance of the enzymes at the same concentration is compared. After 20 minutes of catalysis, the concentration of glutathione synthesized by GshF-CP (protein expressed by Escherichia coli BL21 transformed with SEQ ID NO: 2) is 0.12 g / L, the concentration of glutathione synthesized by GshF-FIH (protein expressed by Escherichia coli BL21 transformed with SEQ ID NO: 4) is 0.21 g / L, the concentration of glutathione synthesized by BL21 / pET-28a-GshF-RJN (protein expressed by Escherichia coli BL21 transformed with SEQ ID NO: 6) is 0.06 g / L, and the concentration of glutathione synthesized by BL21 / pET-28a-GshF-RJP (protein expressed by Escherichia coli BL21 transformed with SEQ ID NO: 8) is 0.17 g / L. Therefore, GshF-FIH has the best catalytic activity and can efficiently catalyze the synthesis of glutathione from glutamate, cysteine ​​and glycine. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 This is the protein gel result of recombinant bacterial protein expression.

[0065] Figure 2 The protein gel results before and after purification of the catalytic reaction protein. DETAILED DESCRIPTION

[0066] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0067] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0068] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0069] In the present invention, the terms "glutathione bifunctional synthetase", "glutathione synthetase", and "bifunctional glutathione synthetase" have the same meaning and refer to L-glutathione bifunctional synthetase (GshF).

[0070] The culture medium involved in the following examples: LB medium (1 L): 10 g NaCl, 10 g tryptone, 5 g yeast extract, and the balance water.

[0071] Example 1. Preparation of high substrate affinity glutathione bifunctional synthetase GshF

[0072] 1. Screening of a high-substrate-affinity glutathione bifunctional synthetase GshF

[0073] The Kcat of glutamate, cysteine ​​and glycine, three substrates of GshF, were predicted for 253 putative GshFs on Uniprot, and the four GshFs with the highest affinity for the three substrates were obtained. The results are shown in Table 1.

[0074] Table 1 shows the Kcat of four GshFs for three substrates

[0075]

[0076] 2. Expression and Purification of GshF

[0077] 1. Construction of recombinant Escherichia coli expressing GshF

[0078] Four GshF gene sequences were synthesized by Beijing Qingke Biotechnology Co., Ltd.: GshF-CP gene (SEQ ID NO: 2), GshF-FIH gene (SEQ ID NO: 4), GshF-RJN gene (SEQ ID NO: 6), and GshF-RJP gene (SEQ ID NO: 8). The proteins encoded by them were named GshF-CP protein, GshF-FIH protein, GshF-RJN protein, and GshF-RJP protein, respectively, and their amino acid sequences were SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, respectively.

[0079] At the same time, the four synthesized GshF gene sequences were separately constructed and inserted into the kanamycin-resistant pET-28a(+) expression vector (which also contains a 6×His protein purification tag) by Sangon Biotech (Shanghai) Co., Ltd., resulting in recombinant plasmids containing each GshF gene. The 3' end of each GshF gene sequence was fused to the 6×His protein purification tag of the vector. The recombinant plasmids were named: pET-28a-GshF-CP, pET-28a-GshF-FIH, pET-28a-GshF-RJN, and pET-28a-GshF-RJP.

[0080] The above recombinant plasmids were transformed into Escherichia coli BL21 (DE3) to obtain recombinant Escherichia coli BL21 / pET-28a-GshF-CP, BL21 / pET-28a-GshF-FIH, BL21 / pET-28a-GshF-RJN, and BL21 / pET-28a-GshF-RJP, respectively.

[0081] 2. Expression and purification of GshF

[0082] The recombinant Escherichia coli BL21 / pET-28a-GshF-CP, BL21 / pET-28a-GshF-FIH, BL21 / pET-28a-GshF-RJN, and BL21 / pET-28a-GshF-RJP were inoculated into 50 mL of LB liquid medium, cultured at 37 °C and 200 rpm overnight, and 2% inoculum was inoculated into 50 mL of LB liquid medium and cultured until OD 600 =0.8, add the inducer IPTG sterilized by filtration through a 0.22μm filter to a final concentration of 0.5mM, then culture at 18°C ​​and 200rpm for 18h, and collect the cells by centrifugation at 12000rpm for 5min. Wash the cells three times with 100mmol / L Tris-HCl (pH8.0) solution, resuspend the collected cells in 100mmol / LTris-HCl (pH8.0), and disrupt the cells with an ultrasonic disruptor, with E. coli cells disrupted for 1s on and 1s off for a total of 15min. Centrifuge at 12000rpm for 10min, and collect the supernatant (crude enzyme solution) for protein gel electrophoresis to detect protein expression. The above crude enzyme solutions are GshF-CP crude enzyme solution, GshF-FIH crude enzyme solution, GshF-RJN crude enzyme solution, and GshF-RJP crude enzyme solution.

[0083] The crude enzyme solution was purified by a protein purification nickel column to obtain a purified enzyme solution (the solvent was 0.01 M PBS buffer with a pH of about 7.4), which was recorded as GshF-CP purified enzyme solution, GshF-FIH purified enzyme solution, GshF-RJN purified enzyme solution, and GshF-RJP purified enzyme solution.

[0084] The purified enzyme solutions were verified by protein gel electrophoresis. The above-mentioned purified enzyme solutions were purified GshF-CP, GshF-FIH, GshF-RJN, and GshF-RJP.

[0085] The results are as follows Figure 1As shown, M: protein molecular weight standard, CP: crude enzyme solution before purification of GshF-CP protein (1) and enzyme solution after purification (2), FIH: crude enzyme solution before purification of GshF-FIH protein (3) and enzyme solution after purification (4), RJN: crude enzyme solution before purification of GshF-RJN protein (5) and enzyme solution after purification (6), RJP: crude enzyme solution before purification of GshF-RJP protein (7) and enzyme solution after purification (8); it can be seen that all four enzyme proteins have soluble specific protein bands before and after purification, and the size is 85.5KD, which is the same as expected.

[0086] Example 2: Detection and comparison of GshF catalytic reaction and enzyme activity

[0087] 1. Purification and expression of GshF

[0088] The recombinant Escherichia coli BL21 / pET-28a-GshF-CP, BL21 / pET-28a-GshF-FIH, BL21 / pET-28a-GshF-RJN, and BL21 / pET-28a-GshF-RJP were inoculated into 50 mL of LB liquid medium, cultured at 37 °C and 200 rpm overnight, and 2% inoculum was inoculated into 50 mL of LB liquid medium and cultured until OD 600 =0.6-0.8, add the inducer IPTG sterilized by filtration with a 0.22 μm filter membrane to a final concentration of 0.5 mM, then culture at 18°C ​​and 200 rpm for 18 h, collect the cells by centrifugation at 12000 rpm for 5 min, and use a hammer super lysis solution (purchased from ACE Biotechnology, catalog number BR0005-03) to lyse the cells at 4°C for 10 min to obtain a lysate (crude enzyme solution). The lysates were then purified by a protein nickel column to obtain purified enzyme solutions (solvent: 0.01 M PBS buffer with a pH of about 7.4), recorded as GshF-CP purified enzyme solution (570 μg / mL), GshF-FIH purified enzyme solution (680 μg / mL), GshF-RJN purified enzyme solution (330 μg / mL), and GshF-RJP purified enzyme solution (680 μg / mL).

[0089] Each lysate and each purified enzyme solution were verified by protein gel electrophoresis.

[0090] The results are as follows Figure 2As shown, M: protein molecular weight standard, CP: GshF-CP protein lysate before purification (left) and enzyme solution after purification (right), FIH: GshF-FIH protein lysate before purification (left) and enzyme solution after purification (right), RJN: GshF-RJN protein lysate before purification (left) and enzyme solution after purification (right), RJP: GshF-RJP protein lysate before purification (left) and enzyme solution after purification (right). It can be seen that all four enzyme proteins have soluble specific protein bands before and after purification, and the size is 85.5KD.

[0091] 2. Enzyme activity detection method

[0092] 1mL pure protease activity detection reaction system: 100mmol / L Tris-HCl (pH8.0), 20mmol / L sodium glutamate, 20mmol / L L-cysteine, 20mmol / L L-glycine, 20mmol / L MgCl2, 10mmol / L ATP, 5mg / L purified GshF protein, react at 37℃ for 20min, then add 100uL 2mol / L hydrochloric acid solution to terminate the reaction, centrifuge at 12000rpm for 5min, and take the supernatant as the test sample solution for L-glutathione determination.

[0093] The above-mentioned GshF purified protein is the purified enzyme solution of GshF-CP, GshF-FIH, GshF-RJN, and GshF-RJP prepared in 1 above.

[0094] L-glutathione content detection method: Glutathione content was determined using the DTNB method. Take glutathione (purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S20186, CAS number 70-18-8) standard solution (glutathione powder dissolved in 100mmol / L Tris-HCl (pH8.0)) or 0.5mL of the sample solution to be tested, add it to 1.5mL of 0.15mol / L NaOH aqueous solution, then add 0.5mL of 3% formaldehyde solution, and react at pH8.0 and 25℃ for 2min. After the reaction is completed, take 0.5mL of the reaction solution and add it to 2.5mL of DTNB analysis solution (composed of 1 volume of 0.01mol / L DTNB solution (weigh DTNB powder (purchased from Sigma-Aldrich, product number D8130, CAS number 69-78-3) and dissolve it in 0.05mol / L A 100-volume phosphate buffer (pH 7) was added to 100 ml of 0.25 mol / L Tris-HCl buffer (pH 8). The mixture was reacted at 25°C for 5 minutes. The absorbance was measured at 412 nm and used as the absorbance of the experimental group. A blank control was used without the addition of purified protein. The difference between the absorbance of the experimental group and the absorbance of the blank control was calculated and applied to the standard curve to calculate the glutathione content, as shown in Table 2.

[0095] Definition of enzyme activity unit: The amount of enzyme required to produce 1 μmol of glutathione per minute under the conditions of pH = 8 and temperature 37°C is defined as 1 unit (U).

[0096] The enzymatic activity results of the four glutathione synthetases are shown in Table 2 below. All results were obtained after subtracting the blank control (i.e., without adding purified protein).

[0097] Table 2 shows the glutathione content and corresponding enzyme activity of the four GshFs catalyzed in 20 min.

[0098] Protein name Glutathione (g / L) Specific enzyme activity (U / mg) GshF-CP 0.12 7.82 GshF-FIH 0.21 10.8 GshF-RJN 0.06 6.07 GshF-RJP 0.17 8.78

[0099] These results demonstrate that the present invention constructed E. coli expression strains using four GshF genes derived from different species. The catalytic product content was measured using the DTNB method, and the catalytic activity of the different GshFs was compared. The results showed that GshF-FIH exhibited the highest activity in L-glutathione synthesis. This provides a new GshF enzyme resource for enzymatic L-glutathione synthesis.

[0100] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Use of the protein in at least one of the following: A1) synthesis of glutathione; A2) acts as a bifunctional glutathione synthetase; A3) Synthesizes glutathione using glutamate, cysteine, and glycine as substrates; The protein is any of the following: a1) The protein includes the amino acid residues shown in SEQ ID NO: 3; a2) a protein that has an amino acid residue identity of greater than 80% with the protein in a1) and has the same function; The protein shown in a3) includes a protein shown in the sequence obtained by connecting a tag to the N-terminus or / and C-terminus of any protein shown in a1)-a2).

2. Use of a biomaterial related to the mutant protein according to claim 1 in at least one of the following: B1) synthesis of glutathione; B2) preparing glutathione bifunctional synthetase; B3) synthesizes glutathione using glutamate, cysteine ​​and glycine as substrates; The biological material is any one of the following C1) to C4): C1) a nucleic acid molecule encoding the protein according to claim 1; C2) an expression cassette containing the nucleic acid molecule described in C1); C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) A recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3).

3. The application according to claim 2, characterized in that: C1) The nucleic acid molecule is any one of the following: c1) The nucleotide sequence of the nucleic acid molecule includes SEQ ID NO: 4; c2) a cDNA molecule or a DNA molecule that has 75% or more identity with the nucleotide sequence defined in c1) and encodes the protein of claim 1; c3) A cDNA molecule or DNA molecule that hybridizes with the nucleotide sequence defined in any one of c1) to c2) under stringent conditions and encodes the protein according to claim 1.

4. The use according to any one of claims 1 to 3, characterized in that: The glutathione is L-glutathione.

5. A method for synthesizing glutathione, comprising the steps of: using the protein of claim 1 as an enzyme to catalyze a substrate with the assistance of ATP to synthesize glutathione; The substrates are glutamate, cysteine ​​and glycine.

6. The method according to claim 5, characterized in that: The glutathione is L-glutathione.

7. The biomaterial according to any one of claims 2 to 4.

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