L-glutathione bifunctional synthetase and method for preparing the same

Abstract

The application discloses L-glutathione bifunctional synthetic enzyme and a preparation method thereof. The application provides application of a protein in at least one of the following: A1) synthesis of glutathione; A2) as glutathione bifunctional synthetic enzyme; A3) synthesis of glutathione with glutamic acid, cysteine and glycine as substrates. The application obtains the bifunctional enzyme with one-step catalysis of substrate synthesis of GSH through an artificial intelligence screening method based on big data. After the protein is purified, the amount of enzyme protein is adjusted to reach the same concentration, and GshF-FIH can efficiently catalyze glutamic acid, cysteine and glycine to synthesize glutathione.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme catalysis, and relates to L-glutathione bifunctional synthase and its preparation method. Background Technology

[0002] L-Glutathione (GSH) is a tripeptide with strong antioxidant activity, 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, detoxification, and immune-maintaining functions. GSH is not only used in pharmaceuticals but also as a base ingredient in functional foods, widely applied in products for anti-aging, immune-boosting, and anti-tumor purposes.

[0003] Currently, GSH production methods both domestically and internationally include chemical synthesis, fermentation, and enzymatic methods. Chemical synthesis suffers from problems 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. In recent years, although fermentation and enzymatic synthesis of GSH has been industrialized, it mainly employs a two-step method. The synthesis of GSH from substrates L-Glu, L-Cys, and Gly requires two enzymatic catalytic reactions involving γ-glutamylcysteine ​​synthase (γ-GCS) and glutathione synthase (GS). This method is limited by the need for synergistic cascade action of the two enzymes to exert their activity and substrate inhibition effects, resulting in high GSH production costs and difficulties in 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 this invention is how to efficiently synthesize L-glutathione and the bifunctional synthase used.

[0006] To address the aforementioned technical problems, the first aspect of the present invention provides the use of a protein in at least one of the following:

[0007] A1) Synthesize glutathione;

[0008] A2) is a bifunctional glutathione synthase;

[0009] A3) Synthesize glutathione using glutamic acid, cysteine, and glycine as substrates;

[0010] The protein is any one of the following:

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

[0012] a2) Proteins that share more than 80% amino acid residue identity with the proteins shown in a1) and have the same function;

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

[0014] In the above text, the glutathione bifunctional synthase can both perform the function of γ-glutamylcysteine ​​synthase (γ-GCS) catalyzing the synthesis of γ-Glu-Cys from L-Glu and L-Cys, and can also perform the function of glutathione synthase (GS) catalyzing the synthesis of Gsh from γ-Glu-Cys and Gly.

[0015] The synthesis of glutathione using glutamic acid, cysteine, and glycine as substrates described above is a one-step synthesis method.

[0016] In some embodiments, the protein shown in a1) is the amino acid residue shown in SEQ ID NO:3.

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

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

[0019] In the aforementioned proteins, the 80% or more identity can 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 the use of biomaterials related to the mutant protein described in the first aspect in at least one of the following:

[0021] B1) Synthesize glutathione;

[0022] B2) Preparation of a bifunctional glutathione synthase;

[0023] B3) Synthesize glutathione using glutamic acid, cysteine, and glycine as substrates;

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

[0025] C1) The nucleic acid molecule encoding the protein described in 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) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms 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] The nucleotide sequence of the nucleic acid molecule shown in c1) includes SEQ ID NO:4;

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

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

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

[0035] The term "identity" refers to sequence similarity to a natural nucleic acid sequence. Identity can be evaluated 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 evaluate the identity between related sequences. Having more than 75% identity can mean at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity.

[0036] In some embodiments, the nucleotide sequence of the nucleic acid molecule shown in c1) is SEQ ID NO:4.

[0037] In some embodiments, the expression cassette containing the nucleic acid molecule described in C1) as described in C2) refers to DNA capable of expressing the protein described in the above applications in host cells. This DNA may include not only promoters that initiate transcription of protein-coding genes but also terminators that terminate transcription of protein-coding genes. Furthermore, the expression cassette may also include enhancer sequences. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters.

[0038] In some 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, containing the nucleic acid molecule described in C1) or the expression cassette described in C2).

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

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

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

[0042] In some embodiments, the host microorganism may specifically be yeast, bacteria, algae, or fungi.

[0043] In some 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] Thirdly, the present invention provides a method for synthesizing glutathione, comprising the following steps: using the protein described in the first aspect as an enzyme, catalyzing a substrate with the assistance of ATP to synthesize glutathione;

[0046] The substrates are glutamic acid, cysteine, and glycine.

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

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

[0049] In some embodiments, the system for the catalytic reaction includes the protein described in the first aspect, ATP, glutamate, cysteine, glycine, and a buffer solution.

[0050] In some embodiments, the buffer solution may be a Tris-HCl buffer solution.

[0051] In some embodiments, the system of the catalytic reaction also includes metal ions.

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

[0053] In one embodiment, the metal ions exist in the form of MgCl2.

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

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

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

[0057] In some embodiments, the pH value of the catalyst is 8±1.

[0058] In some embodiments, the catalytic time is 20-30 minutes.

[0059] In one embodiment, the catalytic time is 20 minutes.

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

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

[0062] Fourthly, the present invention provides the biomaterials described in the second aspect.

[0063] The beneficial effects of this invention are as follows: This invention obtains a bifunctional enzyme capable of one-step catalytic synthesis of GSH from substrates through an artificial intelligence screening method based on big data. After purifying the protein, the amount of enzyme protein was adjusted to achieve the same concentration, and the catalytic performance of the enzymes at the same concentration was compared. After catalysis for 20 minutes, the concentration of glutathione synthesized by GshF-CP (transformed from E. coli BL21 expressed in SEQ ID NO:2) was 0.12 g / L, by GshF-FIH (transformed from E. coli BL21 expressed in SEQ ID NO:4) was 0.21 g / L, by BL21 / pET-28a-GshF-RJN (transformed from E. coli BL21 expressed in SEQ ID NO:6) was 0.06 g / L, and by BL21 / pET-28a-GshF-RJP (transformed from E. coli BL21 expressed in SEQ ID NO:8) was 0.17 g / L. Therefore, GshF-FIH exhibits the best catalytic activity and can efficiently catalyze the synthesis of glutathione from glutamic acid, cysteine, and glycine. Attached Figure Description

[0064] Figure 1 The results of protein gel expression of recombinant bacterial proteins.

[0065] Figure 2 The results show the protein gel before and after purification of the protein in the catalytic reaction. Detailed Implementation

[0066] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

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

[0068] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

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

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

[0071] Example 1: Preparation of a high substrate affinity glutathione bifunctional synthase GshF

[0072] I. Screening of high substrate affinity glutathione bifunctional synthase GshF

[0073] Kcat was predicted for 253 GshFs on Uniprot that were thought to be GshFs, namely glutamic acid, cysteine ​​and glycine. The four GshFs with the highest affinity for the three substrates were obtained, and the results are shown in Table 1.

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

[0075]

[0076] II. 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 they encode are named GshF-CP protein, GshF-FIH protein, GshF-RJN protein, and GshF-RJP protein, respectively, with their respective amino acid sequences being SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7.

[0079] Simultaneously, Sangon Biotech (Shanghai) Co., Ltd. constructed the four synthesized GshF gene sequences into kanamycin-resistant pET-28a(+) expression vectors (containing a 6×His protein purification tag), obtaining recombinant plasmids containing each GshF gene. The 3' end of each GshF gene sequence was fused with the 6×His protein purification tag of the vector for expression. The recombinant plasmids were named: pET-28a-GshF-CP, pET-28a-GshF-FIH, pET-28a-GshF-RJN, and pET-28a-GshF-RJP, respectively.

[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] 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 and cultured overnight at 37°C and 200 rpm. Then, 2% inoculum was added to 50 mL of LB liquid medium and cultured until OD... 600 =0.8, add IPTG (an inducer for sterilization) to a final concentration of 0.5 mM after filtration through a 0.22 μm filter membrane, and then incubate at 18℃ and 200 rpm for 18 h. Collect the cells by centrifugation at 12000 rpm for 5 min. Wash the cells three times with 100 mmol / L Tris-HCl (pH 8.0) solution. Resuspend the collected cells in 100 mmol / L Tris-HCl (pH 8.0) solution and sonicate them for 15 min, breaking down the cells for 1 second at a time. Centrifuge at 12000 rpm for 10 min, and use 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, respectively.

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

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

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

[0086] Example 2: GshF-catalyzed reaction and detection and comparison of enzyme activity

[0087] 1. Purification and expression of GshF

[0088] 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 and cultured overnight at 37°C and 200 rpm. Then, 2% inoculum was added to 50 mL of LB liquid medium and cultured until OD... 600 =0.6-0.8, add IPTG, the inducing agent for sterilization, to a final concentration of 0.5mM after filtration through a 0.22μm filter membrane, and then incubate at 18℃ and 200rpm for 18h. Collect the bacterial cells by centrifugation at 12000rpm for 5min. The collected bacterial cells are then lysed at 4℃ for 10min using Hammer Super Lysis Buffer (purchased from ACE Biotechnology, catalog number BR0005-03) to obtain lysate (crude enzyme solution). The lysate is then purified by nickel protein column chromatography to obtain purified enzyme solutions (solvent: 0.01M PBS buffer with pH approximately 7.4), labeled as GshF-CP purified enzyme solution (570μg / mL), GshF-FIH purified enzyme solution (680μg / mL), GshF-RJN purified enzyme solution (330μg / mL), and GshF-RJP purified enzyme solution (680μg / mL).

[0089] Each lysis buffer and each purified enzyme solution were subjected to protein gel electrophoresis for verification.

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

[0091] 2. Enzyme activity detection methods

[0092] The reaction system for detecting 1 mL of pure protease activity consisted of 100 mmol / L Tris-HCl (pH 8.0), 20 mmol / L sodium glutamate, 20 mmol / L L-cysteine, 20 mmol / L L-glycine, 20 mmol / L MgCl2, 10 mmol / L ATP, and 5 mg / L GshF purified protein. The reaction was carried out at 37 °C for 20 min, and then 100 μL of 2 mol / L hydrochloric acid solution was added to terminate the reaction. The mixture was centrifuged at 12000 rpm for 5 min, and the supernatant was collected as the test sample solution for L-glutathione determination.

[0093] The purified GshF proteins mentioned above are the purified enzyme solutions of GshF-CP, GshF-FIH, GshF-RJN, and GshF-RJP prepared in step 1 above.

[0094] L-Glutathione Content Determination Method: Glutathione content was determined using the DTNB method. Take 0.5 mL of glutathione (purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S20186, CAS number 70-18-8) standard solution (glutathione powder dissolved in 100 mmol / L Tris-HCl (pH 8.0)) or the sample solution to be tested, and add it to 1.5 mL of 0.15 mol / L NaOH aqueous solution. Then add 0.5 mL of 3% formaldehyde solution. React at pH 8.0 and 25℃ for 2 min. After the reaction, take 0.5 mL of the reaction solution and add it to 2.5 mL of DTNB analytical solution (made by dissolving 1 volume of 0.01 mol / L DTNB solution in 0.05 mol / L Tris-HCl). (Prepared with phosphate buffer (pH=7) and 100 volumes of 0.25 mol / L Tris-HCl buffer (pH=8), the mixture was reacted at 25°C for 5 min, and the absorbance was measured at 412 nm. This absorbance was used as the experimental group absorbance. A blank control was used without the added purified protein. The difference between the experimental group absorbance and the blank control absorbance was calculated and substituted into the standard curve to calculate the glutathione content, as shown in Table 2.

[0095] Enzyme activity unit definition: Under conditions of pH=8 and temperature of 37℃, the amount of enzyme required to produce 1 μmol of glutathione per minute is defined as 1 unit (U).

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

[0097] Table 2 shows the glutathione content and corresponding enzyme activity produced by four GshF catalysis methods at 20 min.

[0098] 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] The above results show that this invention constructed E. coli expression strains using four GshF genes from different species, and used the DTNB method to detect the content of catalytic products and compare the catalytic activities of different GshFs. The results showed that GshF-FIH exhibited the highest catalytic activity for the synthesis of L-glutathione. This provides a new GshF enzyme resource for the enzymatic synthesis of L-glutathione.

[0100] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for synthesizing glutathione, comprising the following steps: using a protein as an enzyme to catalyze a substrate in the presence of ATP to synthesize glutathione; The amino acid sequence of the protein is SEQ ID NO:3; The substrates are glutamic acid, cysteine, and glycine; The catalytic reaction system includes the protein, ATP, glutamate, cysteine, glycine, Tris-HCl buffer, and MgCl2. The concentration of the protein is 5 mg / L; The concentration of ATP is 10 mmol / L; The concentrations of glutamic acid, cysteine, and glycine were all 20 mM. The concentration of MgCl2 was 20 mmol / L; The catalytic pH value is 8; The catalytic time is 20 min; The catalytic temperature is 37°C; The protein was prepared as follows: Recombinant Escherichia coli BL21 / pET-28a-GshF-FIH was inoculated into 50 mL of LB liquid medium and cultured overnight at 37 ℃ and 200 rpm. Then, 2% inoculum was added to 50 mL of LB liquid medium and cultured until OD... 600 =0.6-0.8, add IPTG, the inducing agent for sterilization, to a final concentration of 0.5 mM after filtration through a 0.22 μm filter membrane, and then incubate at 18℃ and 200 rpm for 18 h. Collect the bacterial cells by centrifugation at 12000 rpm for 5 min. The collected bacterial cells are then lysed using Hammer Super Ligation broth at 4℃ for 10 min to obtain the lysate. The lysate is then purified by passing it through a protein nickel column to obtain an enzyme solution purified with 680 µg / mL GshF-FIH, which is the protein. The recombinant Escherichia coli BL21 / pET-28a-GshF-FIH was obtained by introducing the recombinant vector pET-28a-GshF-FIH into Escherichia coli BL21(DE3); The recombinant vector pET-28a-GshF-FIH is a vector obtained by constructing the gene shown in SEQ ID NO:4 into the pET-28a(+) expression vector.

2. The method according to claim 1, characterized in that: The glutathione is L-glutathione.

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