A method for producing glutathione, engineering bacteria, reagents and applications

Abstract

The present invention provides a method for generating glutathione, an engineering bacterium, a reagent and an application thereof. The method for producing glutathione comprises the following steps: S1, obtaining an engineering bacterium; the engineering bacterium is a Pichia pastoris cloned with a bifunctional glutathione synthetase gene, and the nucleotide sequence of the bifunctional glutathione synthetase gene is shown in SEQ NO.1. S2, fermenting and culturing the engineering bacterium in a culture medium to obtain a fermentation liquid; S3, performing solid-liquid separation on the fermentation liquid to obtain a supernatant; S4, mixing the supernatant with a glutathione synthesis raw material to react to obtain glutathione. The present invention can make the bifunctional glutathione synthetase secreted outside the cell, and the purification process is simpler and the cost is lower.

Description

Technical Field

[0001] The invention relates to enzymatic synthesis of glutathione, and in particular to a method for generating glutathione, an engineering bacterium, a reagent and an application thereof. Background Art

[0002] Biosynthesis of glutathione is the most widely used production method in recent years; among them, fermentation and enzyme methods are both biosynthesis, but compared with enzyme methods, fermentation methods require a large amount of fermentation, and the waste bacteria and waste residues produced are mixed with glutathione, which increases the difficulty of purification. And after the enzyme is immobilized, it can be used repeatedly in the reaction system, saving production costs.

[0003] A Chinese invention patent application with publication number CN104611396A discloses a method for producing glutathione. Although glutathione is efficiently obtained by an enzymatic method, the method does not achieve the extracellular secretion of glutathione synthetase. The glutathione synthetase can only be obtained after the bacteria are broken, resulting in the release of cell breakage products, increasing the difficulty of obtaining glutathione synthetase, and thus increasing the production cost of glutathione.

[0004] In view of this, it is necessary to provide a method, engineered bacteria, reagent and application for producing glutathione to solve or at least alleviate the technical defect of the above-mentioned prior art that the extracellular secretion of glutathione synthetase cannot be achieved. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for generating glutathione, an engineered bacterium, a reagent and an application thereof, aiming to solve the technical problem that the above-mentioned prior art fails to realize the extracellular secretion of glutathione synthetase, resulting in difficulty in purifying glutathione synthetase.

[0006] To achieve the above object, the present invention provides an engineered bacterium that secretes glutathione synthetase extracellularly. The engineered bacterium is Pichia pastoris cloned with a bifunctional glutathione synthetase gene, and the nucleotide sequence of the bifunctional glutathione synthetase gene is shown in SEQ NO.1.

[0007] The present invention also provides a method for preparing any of the above-mentioned engineered bacteria, comprising: obtaining a recombinant vector containing the bifunctional glutathione synthetase gene; and then transforming competent Pichia pastoris with the recombinant vector to obtain the engineered bacteria.

[0008] The present invention also provides a use of any of the above-mentioned engineering bacteria in obtaining glutathione synthetase outside a cell.

[0009] The present invention also provides a use of any of the above-mentioned engineering bacteria in producing glutathione.

[0010] The present invention also provides a reagent for producing glutathione, comprising any of the above-mentioned engineering bacteria.

[0011] The present invention also provides a method for producing glutathione, comprising the steps of:

[0012] S1, obtaining any of the engineered bacteria described above;

[0013] S2, fermenting and culturing the engineered bacteria to obtain a fermentation liquid;

[0014] S3, performing solid-liquid separation on the fermentation liquid to obtain a supernatant;

[0015] S4, mixing the supernatant and glutathione synthesis raw materials to react to obtain glutathione.

[0016] Furthermore, the fermentation culture includes a glycerol culture stage and a methanol induction stage; the duration of the glycerol culture stage is 27-37 hours, and the temperature of the glycerol culture stage is 30±0.5°C; the duration of the methanol induction stage is 88-110 hours, and the temperature of the methanol induction stage is 28±0.5°C.

[0017] Furthermore, the culture medium used in the fermentation culture includes phosphoric acid, calcium sulfate dihydrate, potassium sulfate, magnesium sulfate heptahydrate, potassium hydroxide, glycerol, and trace elements.

[0018] Furthermore, the raw materials for glutathione synthesis include glutamic acid, glycine, L-cysteine, ATP, MgCl 2 .

[0019] Furthermore, the duration of the mixed reaction is 8-30 minutes, and the temperature of the mixed reaction is 36-38°C.

[0020] Compared with the prior art, the present invention has at least the following advantages:

[0021] The present invention constructs a recombinant Pichia pastoris and clones the nucleotide sequence shown in SEQ ID NO.1 into Pichia pastoris, so that the bifunctional glutathione synthetase can be secreted outside the cell, the purification process is simpler, the cost is lower, and the production of glutathione is simpler. Specifically, the host bacteria used in the present invention is X-33 Pichia pastoris, and after cloning, the produced enzyme is secreted and expressed outside the cell, and the enzyme that catalyzes the production of glutathione can be directly present in the fermentation supernatant; in the case of synthesizing glutathione by enzymatic method, the effective synthesis of glutathione can be achieved, and the introduction of cell fragments can be avoided. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0023] Figure 1 The plasmid map constructed in Example 1 of the present invention;

[0024] Figure 2 is an agarose gel electrophoresis diagram of the transformant in Example 1 of the present invention; wherein M is Maker, and 1-8 are 8 cloned PCR products;

[0025] Figure 3 The figure is a Western Blot identification diagram of the target protein expressed by 7 positive strains in Example 1 of the present invention; wherein, M is Maker, 1 is 1# positive strain, 2 is 2# positive strain, 3 is 3# positive strain, 4 is 4# positive strain, 5 is 5# positive strain, 6 is 7# positive strain, 7 is 8# positive strain, and 8 is a positive control (EGFP-28KD);

[0026] Figure 4 for Figure 3 Western Blot identification diagram of the target protein before purification of the supernatant corresponding to the 2# positive strain; where M is Maker, 1 is the purified protein, and 2 is the positive control (EGFP-28KD);

[0027] Figure 5 for Figure 3 Western Blot identification of the target protein after purification of the supernatant corresponding to the 2# positive strain; where M is Maker, 1 is the purified protein, 2 is the effluent, 3 is the washing solution, and 4 is the positive control (EGFP-28KD);

[0028] Figure 6 for Figure 3 SDS-PAGE identification diagram of the target protein after purification of the supernatant corresponding to the 2# positive strain; where M is Maker, 1 is the purified protein, 2 is the effluent, 3 is the washing solution, and 4 is the positive control (EGFP-28KD);

[0029] Figure 7 This is a high performance liquid chromatogram of the glutathione standard (10 ug / ml) in Example 1 of the present invention;

[0030] Figure 8 This is a high performance liquid chromatogram of glutathione synthesized by the reaction in Example 1 of the present invention;

[0031] Fig. 9 It is an agarose gel electrophoresis diagram of the transformant (corresponding to Streptococcus agalactiae gshF) in Comparative Example 1 of the present invention; wherein M is Maker, and 1-8 are 8 cloned PCR products;

[0032] Fig.10 It is a Western Blot identification diagram of the expression of the target protein by 7 positive strains (corresponding to Streptococcus agalactiae gshF) in Comparative Example 1 of the present invention; wherein, M is Maker, 1 is 2# positive strain, 2 is 3# positive strain, 3 is 4# positive strain, 4 is 5# positive strain, 5 is 6# positive strain, 6 is 7# positive strain, 7 is 8# positive strain, and 8 is a positive control (EGFP-28KD);

[0033] Fig.11 It is an agarose gel electrophoresis diagram of the transformant (corresponding to Bacillus cereus gshF) in Comparative Example 1 of the present invention; wherein M is Maker, and 1-8 are 8 cloned PCR products;

[0034] Fig.12 It is a Western Blot identification diagram of the expression of the target protein by 7 positive strains (corresponding to Bacillus cereus gshF) in Comparative Example 1 of the present invention; wherein, M is Maker, 1 is 1# positive strain, 2 is 2# positive strain, 3 is 3# positive strain, 4 is 4# positive strain, 5 is 5# positive strain, 6 is 6# positive strain, 7 is 7# positive strain, and 8 is a positive control (EGFP-28KD).

[0035] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with the implementation methods and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Moreover, the technical solutions between the various embodiments of the present invention can be combined with each other, but they must be based on the ability of ordinary technicians in the field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention. In the present invention, the clone numbers of PCR identification and protein identification do not necessarily correspond one to one; the clone numbers for PCR identification are 8, and the clone numbers for protein identification (positive strains) are 7; limited by the number of electrophoresis wells (9), since positive controls were introduced during protein identification, only 7 clones with more obvious brightness during PCR identification were selected for subsequent protein expression identification.

[0038] In order to achieve the extracellular secretion of glutathione synthetase, the present invention provides an engineered bacterium for extracellular secretion of glutathione synthetase, wherein the engineered bacterium is Pichia pastoris cloned with a bifunctional glutathione synthetase gene, and the nucleotide sequence of the bifunctional glutathione synthetase gene is shown in SEQ ID NO.1.

[0039] The sequence of SEQ ID NO.1 is:

[0040]

[0041] It should be noted that, according to the applicant's research, not all glutathione synthetase genes can be expressed extracellularly in Pichia pastoris. The present application uses Pichia pastoris to secrete and express bifunctional glutathione synthetase to the extracellular space. The purification process is simpler and less expensive than that of Escherichia coli, with fewer fermentation batches and less waste gas from bacterial residues. In theory, glutathione synthetase can be continuously obtained, which has unexpected technical effects. In addition, generally speaking, in high-density fermentation, the concentration and expression of Pichia pastoris are higher than those of Escherichia coli. Therefore, in order to avoid a significant impact on the yield of glutathione synthetase, it is also necessary to explore genes that can secrete glutathione synthetase extracellularly in Pichia pastoris and clone them into Pichia pastoris.

[0042] To obtain the above-mentioned engineered bacteria, the present invention also provides a method for preparing any of the above-mentioned engineered bacteria, comprising: obtaining a recombinant vector containing the bifunctional glutathione synthetase gene; then, using the recombinant vector to transform competent Pichia pastoris to obtain the engineered bacteria.

[0043] Specifically, the present invention inserts the bifunctional glutathione synthetase gene gshF gene (SEQ ID NO.1) into the plasmid pPicZαA, and the target gene is adjacent to the vector α-factor to construct a recombinant vector pPicZαA-gshF; then, the recombinant vector is introduced into Pichia pastoris X-33 to obtain a recombinant genetically engineered bacterium.

[0044] The present invention also provides a use of any of the above-mentioned engineering bacteria in obtaining glutathione synthetase outside a cell.

[0045] The present invention also provides a use of any of the above-mentioned engineering bacteria in producing glutathione.

[0046] The present invention also provides a reagent for producing glutathione, comprising any of the above-mentioned engineering bacteria.

[0047] The present invention also provides a method for producing glutathione, comprising the steps of:

[0048] S1, obtaining any of the engineered bacteria described above; the engineered bacteria may be derived from any of the reagents described above, or may be prepared using any of the preparation methods described above.

[0049] S2, fermenting and culturing the engineered bacteria to obtain a fermentation liquid; the fermentation and culturing includes a glycerol culturing stage and a methanol induction stage, wherein the glycerol culturing stage is in the first stage and the methanol induction stage is in the second stage.

[0050] The duration of the glycerol culture stage is 27-37 hours, and the temperature of the glycerol culture stage is 30±0.5°C. Specifically, the glycerol culture stage is divided into the first stage and the second stage; the first stage: the first 20-26 hours of glycerol batch fermentation; the second stage: the glycerol feeding stage of 7-11 hours; the glycerol feeding stage includes: controlling the dissolved oxygen in the fermentation process to be maintained at 20-30% by adding glycerol.

[0051] The temperature of the methanol induction stage is 28±0.5°C, and the duration of the methanol induction stage is 88-110 hours; the methanol induction stage controls the dissolved oxygen in the fermentation process to be maintained at 20-30%. There is usually a transition stage between the glycerol culture stage and the methanol induction stage, and the transition stage is a transition period in which the carbon source is replaced by methanol. The temperature of the transition stage can be 30±0.5°C, and the duration of the transition stage can be 0.8-3 hours.

[0052] As an optional implementation, the fermentation culture duration is 120-140 hours, depending on whether the enrichment culture of the engineered bacteria can be achieved.

[0053] The inoculation amount of the fermentation culture can be 8-12%, that is, the bacterial liquid of the engineered bacteria is added to the culture medium at a volume ratio of 8-12%. After the fermentation culture is completed, the wet weight in the fermentation liquid reaches 220-260g / L; as another option, in order to fully utilize the engineered bacteria in the fermentation liquid, the engineered bacteria in the fermentation liquid can also be in a logarithmic growth phase, which is subject to actual industrial needs.

[0054] In order to ensure that the engineered bacteria can grow normally and secrete glutathione synthetase extracellularly, the culture medium used in the fermentation culture may include BSM culture medium, and the BSM culture medium may include 85% phosphoric acid, calcium sulfate dihydrate, potassium sulfate, magnesium sulfate heptahydrate, potassium hydroxide, glycerol, PMT1 trace elements, and ammonia water to adjust the pH to 5.0; specifically, the culture medium may be composed of 26.7 ml / L 85% phosphoric acid, 0.93 g / L calcium sulfate dihydrate, 18.2 g / L potassium sulfate, 14.9 g / L magnesium sulfate heptahydrate, 4.13 g / L potassium hydroxide, 40 g / L glycerol, 4.0 ml / L PMT1 trace elements, and ammonia water to adjust the pH to 5.0.

[0055] S3, performing solid-liquid separation on the fermentation liquid to obtain a supernatant.

[0056] It should be noted that, since the engineered bacteria can secrete glutathione synthetase extracellularly, there is no need to crush the engineered bacteria. It is only necessary to perform the solid-liquid separation treatment on the fermentation broth to obtain the separated bacteria and the supernatant containing glutathione synthetase, thereby avoiding the mixing of cell crushed materials and the glutathione synthetase and ensuring the activity of the engineered bacteria.

[0057] The solid-liquid separation method may include centrifugal separation, the rotation speed of the centrifugal separation may be 3000-5000 rpm, and the duration of the centrifugal separation may be 10-20 min.

[0058] S4, mixing the supernatant and glutathione synthesis raw materials to react to obtain glutathione.

[0059] The raw materials for glutathione synthesis include glutamic acid, glycine, L-cysteine, ATP, MgCl 2 6H 2 O; in order to facilitate the reaction, the glutathione synthesis raw material may also include a buffer solution, the buffer solution is a Tris-HCl solution; the pH of the glutathione synthesis system may be 7.0; glutamic acid, glycine, L-cysteine, ATP, MgCl 2 6H 2 O are present in the buffer.

[0060] In the present invention, the glutamic acid may be sodium glutamate, the L-cysteine ​​may be L-cysteine ​​hydrochloride; the L-cysteine ​​hydrochloride is L-cysteine ​​hydrochloride anhydrate, and the ATP is adenosine triphosphate.

[0061] In the present invention, sodium glutamate, glycine, L-cysteine ​​hydrochloride, ATP, MgCl 2 6H 2The concentrations of O in the buffer can be 50 mM / L sodium glutamate, 50 mM / L glycine, 50 mM / L cysteine ​​hydrochloride, 100 mM / L ATP, 100 mM / L MgCl 2 6H 2 O 20mM / L. In the present invention, the volume ratio of the supernatant to the buffer solution may be 1:4-6, specifically 1:5.

[0062] In order to ensure that the mixing reaction is fully carried out, the duration of the mixing reaction can be 8-30 minutes, and the temperature of the mixing reaction can be 36-38°C.

[0063] The following are specific examples of the present invention:

[0064] Example 1

[0065] 1. Gene Synthesis

[0066] 1) Search the gene sequence of Streptococcus salivarius gshF gene sequence in NCBI (GenBank: CP009913.1);

[0067] 2) Codon optimization was performed according to the Pichia pastoris expression system to avoid the SacI restriction site to obtain the target gene, the nucleotide sequence of which is shown in SEQ ID NO.1;

[0068] 3) The obtained target gene was synthesized into pPICZαA, and the target gene was next to the vector α-factor. The plasmid map constructed in this example is as follows Figure 1 shown.

[0069] The amino acid sequence of the target protein corresponding to the target gene is shown in SEQ ID NO.2, and the sequence of SEQ ID NO.2 is:

[0070]

[0071] 2. Plasmid electroporation into Pichia pastoris

[0072] 2.1 Linearization of target vector

[0073] Prepare the vector enzyme digestion system according to the table below and perform enzyme digestion at 37°C overnight.

[0074] Components Dosage Plasmid (5-10ug) 6ug 10×buffer 5ul SacI 1ul <![CDATA[ddH 2 The]]> Make up to 50ul

[0075] 2.2 Linearized vector purification and recovery

[0076] 1) Configure the vector purification system according to the following table:

[0077]

[0078]

[0079] 2) Stand at -20℃ for more than 35 minutes;

[0080] 3) Centrifuge at 12000 rpm for 15 min at 4°C and discard the supernatant;

[0081] 4) Add 400ul pre-cooled 80% ethanol to resuspend the precipitate;

[0082] 5) Centrifuge at 4°C, 12000 rpm for 10 min, discard the supernatant, open the lid and dry to obtain the linearized vector (linearized pPicZαA-GshF);

[0083] 6) Add 10ul ddH 2 O dissolve the precipitate (linearized vector).

[0084] 2.3 Preparation of competent yeast

[0085] 1) Add 5 ml YPD liquid medium to a 50 ml centrifuge tube, inoculate the X-33 strain, and culture at 30°C overnight;

[0086] 2) Transfer 50ul of bacterial solution to a 250ml conical flask containing 50ml YPD liquid medium and culture at 30℃ overnight until OD 600 =1.5;

[0087] 3) Centrifuge at 4000 rpm for 5 min at 4°C to collect the cells, resuspend in 10 ml of buffer A, incubate in a 30°C water bath for 15 min, and add pre-cooled sterile water to make up to 50 ml;

[0088] Buffer A: 20 ml YPD liquid medium + 2 ml 2M HEPES (pH = 8.0, filter sterilized) + 0.5 ml 1M DTT (filter sterilized);

[0089] 4) Centrifuge at 4000 rpm for 5 min at 4°C and resuspend in 50 ml of pre-cooled sterile water (containing 0.3 ml of 2M HEPES, pH = 8.0);

[0090] 5) Centrifuge at 4000 rpm for 5 min at 4°C and resuspend in 4 ml of pre-cooled 1 M sorbitol;

[0091] 6) Centrifuge at 4000 rpm for 5 min at 4°C, resuspend with 100 ul of pre-cooled 1 M sorbitol, dispense into 80 ul / tube, and place on ice.

[0092] 2.4 Electroporation of linearized vector into yeast

[0093] 1) Take 80ul competent cells, add 6ug linearized pPicZαA-GshF, mix well and transfer to a pre-cooled 0.2cm electroporation cuvette;

[0094] 2) Place on ice for 5 minutes;

[0095] 3) Yeast electric shock parameter setting (1.5kV, 25uF, 200Ω), electric shock;

[0096] 4) Immediately add 2 ml of pre-cooled 1M sorbitol + HEPES (10 ml 1M sorbitol + 100 ul 2M HEPES, pH = 8.0) and transfer to a 2 ml sterile centrifuge tube;

[0097] 5) Incubate at 30°C for 1.5 h;

[0098] 6) After diluting 5-fold, 10-fold, and 100-fold, 300 ul / plate was spread on a 15 cm YPD plate containing 100 mg / l Zeocin, and cultured at 30°C until clones grew.

[0099] 3. Screening and identification of transformants

[0100] 1) Randomly select 8 clones from the plate and perform colony PCR;

[0101] 2) After PCR, 2 μl of each sample was taken for agarose electrophoresis. The results are shown in Figure 2 , the molecular weights of clones 1, 2, 3, 4, 5, 7, and 8 were all correct;

[0102] 3) Inoculate clones 1, 2, 3, 4, 5, 7, and 8 (positive strains) into 25 ml of BMGY liquid culture medium, and culture at 30°C and 220 rpm until the wet weight reaches 40 g / L (approximately 24 h);

[0103] 4) Transfer the culture medium to a 50 ml centrifuge tube, centrifuge at 4000 rpm for 5 min, and discard the supernatant;

[0104] 3) Resuspend the cells in 25 ml BMMY medium, transfer to a new sterile 250 ml conical flask, add methanol to a final concentration of 0.75%, and culture at 28°C and 220 rpm for 6 days;

[0105] 4) Methanol was added every 24 h (final concentration was 0.75%);

[0106] 5) On the 6th day of culture, the bacterial culture was collected, centrifuged at 4000 rpm for 5 min, and the supernatant was collected;

[0107] 6) Take 20 μl of supernatant for Western Blot analysis. Figure 3, all 7 clones were expressed successfully.

[0108] 4. Protein Purification

[0109] 1) Select the No. 2 monoclonal clone identified by Western Blot, inoculate it into 50 mL of BMGY liquid culture medium, and culture it at 30°C and 220 rpm until the wet weight reaches 40 g / L (about 24 h);

[0110] 2) Centrifuge at 4000 rpm for 5 min and discard the supernatant;

[0111] 3) Resuspend the cells in 50 mL of BMMY medium and transfer to a new conical flask; add methanol to a final concentration of 0.75% and culture at 28°C and 220 rpm for 6 days;

[0112] 4) Methanol was added every 24 h (final concentration was 0.75%);

[0113] 5) On the morning of the 6th day, collect the bacterial culture, centrifuge at 4000 rpm for 5 min, collect the supernatant, and perform Western Blot identification. Figure 4 , the results showed that the target protein was expressed.

[0114] 6) Purify the target protein with Ni-NTA beads and detect it with SDS-PAGE and Western Blot. Figure 5-6 ,The results showed that the target protein was purified successfully.

[0115] 5. 3L Fermentation Tank Culture

[0116] 1) Select clone No. 2, inoculate it into YPD seed solution, and culture it at 30°C and 220 rpm for 24 hours; the bacterial concentration after 24 hours of culture is 40 g / L (wet weight);

[0117] 2) inoculating into a 3L fermentation tank at an inoculum volume of 10%; the fermentation medium used was BSM medium, which included 26.7 ml / L 85% phosphoric acid, 0.93 g / L calcium sulfate dihydrate, 18.2 g / L potassium sulfate, 14.9 g / L magnesium sulfate heptahydrate, 4.13 g / L potassium hydroxide, 40 g / L glycerol, 4.0 ml / L PMT1 trace elements, and ammonia water to adjust pH to 5.0;

[0118] 3) Initial rotation speed 400 rpm, ventilation 1 L / min (air), temperature 30°C, pH 5.0, initial culture volume 1.5 L;

[0119] 4) When the dissolved oxygen drops to 30%, increase the speed by 50 rpm each time. When the maximum speed reaches 900 rpm, increase the air flow by 0.5 L / min each time, up to 3 L / min, and then increase the tank pressure. The maximum tank pressure shall not exceed 0.08 Mpa.

[0120] 5) When dissolved oxygen rebounds after 22 hours of cultivation, glycerol is added. During the glycerol addition stage, the dissolved oxygen is maintained at 20%-30%;

[0121] 6) After 8 hours of glycerol addition, stop adding glycerol;

[0122] 7) After the addition of glycerol was stopped, the dissolved oxygen began to rise to 100%. After the dissolved oxygen was maintained at 100% for 1 hour, methanol (containing 12% PTM1) was slowly added;

[0123] 8) When the dissolved oxygen drops to 20%, adjust the methanol flow rate to maintain the dissolved oxygen at 20%-30%. At this time, the temperature is controlled at 28°C.

[0124] 9) After 90 hours of methanol addition, the fermentation was stopped;

[0125] 10) The fermentation broth was collected and centrifuged at 4000 rpm for 20 minutes to obtain the supernatant.

[0126] 6. Catalytic synthesis of glutathione

[0127] 5 mL of the supernatant was added to 25 mL of glutathione synthesis raw material to form a reaction system; then the reaction was carried out at 37°C for 10 min at a speed of 110 rpm.

[0128] The raw materials for glutathione synthesis are glycine, sodium glutamate, L-cysteine ​​hydrochloride anhydrate, adenosine triphosphate, MgCl 2 6H 2 O composition; the buffer used for glutathione synthesis raw material is Tris-HCl solution, and the pH of glutathione synthesis raw material is adjusted to 7.0.

[0129] The concentrations of various substances in the raw materials for glutathione synthesis are: sodium glutamate 50mM / L, glycine 50mM / L, L-cysteine ​​hydrochloride anhydrate 50mM / L, adenosine triphosphate 100mM / L, MgCl 2 6H 2 O 20mM / L.

[0130] 7. HPCL detection of glutathione

[0131] After the synthesis reaction is completed, glutathione is detected by high performance liquid chromatography under the following conditions:

[0132] Chromatographic column: C18 column (4.6mm×250mm, 5μm)

[0133] Mobile phase: phosphate buffer: methanol = 95:5

[0134] Detection: UV 210nm

[0135] Flow rate: 1.0ml / min

[0136] Temperature: 30℃

[0137] See also Figure 7-8 It is understood that the peak time of glutathione standard is 8.532, and the reaction system of this embodiment has an obvious single peak at 8.502, indicating that there is glutathione in the reaction system. Therefore, the supernatant of the engineered bacteria constructed by the present invention has glutathione synthetase activity. Figure 7-8 The vertical axis is mAU.

[0138] Comparative Example 1

[0139] 1) The gshF gene sequences of Streptococcus agalactiae and Bacillus cereus were searched in NCBI (GenBank: AE009948.1 and ACLS01000213.1, respectively); after codon optimization, the target genes were synthesized into pPICZαA; the two target genes were recorded as Streptococcus agalactiae gshF and Bacillus cereus gshF, respectively.

[0140] The nucleotide sequence of Streptococcus agalactiae gshF is shown in SEQ ID NO.3. The nucleotide sequence of SEQ ID NO.3 is:

[0141]

[0142] The nucleotide sequence of Bacillus cereus gshF is shown in SEQ ID NO.4. The nucleotide sequence of SEQ ID NO.4 is:

[0143]

[0144] 2) As in the second and third parts of Example 1, the plasmid was linearized and then transferred into competent cells.

[0145] See also Figure 9-12 As shown, although positive clones were obtained after transformant screening, the target protein was not detected in the supernatant by WB after expression induction, indicating that the secretory expression of gshF from Bacillus cereus and Streptococcus agalactiae failed.

[0146] Comparative Example 2

[0147] 1) Select clone No. 2 in Example 1, inoculate it into YPD seed solution, and culture it at 30° C. and 220 rpm for 24 h;

[0148] 2) Inoculate into a 3L fermentation tank at a 10% inoculation rate, and inoculate 3 parallel tanks (to explore the effect of different induction temperatures); the fermentation medium used is BSM medium, which includes 26.7 ml / L 85% phosphoric acid, 0.93 g / L calcium sulfate dihydrate, 18.2 g / L potassium sulfate, 14.9 g / L magnesium sulfate heptahydrate, 4.13 g / L potassium hydroxide, 40 g / L glycerol, 4.0 ml / L PMT1 trace elements, and ammonia water to adjust pH to 5.0;

[0149] 3) Initial speed 400 rpm, ventilation 1 L / min, temperature 30°C, pH 5.0, initial culture volume 1.5 L;

[0150] 4) When the dissolved oxygen drops to 30%, increase the speed by 50 rpm each time. When the maximum speed reaches 900 rpm, increase the air flow by 0.5 L / min each time, up to 3 L / min, and then increase the tank pressure. The maximum tank pressure shall not exceed 0.08 Mpa.

[0151] 5) When the culture reaches about 22 hours, glycerol is added when the dissolved oxygen rebounds. During the glycerol addition stage, the dissolved oxygen is maintained at 20%-30%;

[0152] 6) After 8 hours of glycerol addition, stop adding glycerol;

[0153] 7) After the addition of glycerol was stopped, the dissolved oxygen began to rise to 100%. After the dissolved oxygen was maintained at 100% for 1 hour, methanol (containing 12% PTM1) was slowly added;

[0154] 8) When the dissolved oxygen drops to 20%, the methanol flow rate is adjusted to maintain the dissolved oxygen at 20%-30%. At this time, the temperature is controlled at the set induction temperature. The induction temperatures set in this comparative example are 30° C., 28° C. and 25° C. respectively;

[0155] 9) After 90 hours of methanol addition, the fermentation was stopped;

[0156] 10) Collect the fermentation broth, centrifuge at 4000 rpm for 20 minutes, and harvest the supernatant;

[0157] 11) Synthesize glutathione according to the sixth part of Example 1, and detect glutathione according to the seventh part of Example 1.

[0158] The content of glutathione under various conditions is shown in the following table:

[0159] Induction temperature Glutathione content (mg / L) 30℃ 127.92 28℃ 206.66 25℃ 73.90

[0160] The above technical solutions of the present invention are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. All equivalent structural changes made using the contents of the present invention specification under the technical concept of the present invention, or directly / indirectly applied in other related technical fields, are included in the patent protection scope of the present invention.

Claims

1. An engineered bacterium that secretes glutathione synthetase extracellularly, characterized in that: The engineered bacteria is Pichia pastoris cloned with a bifunctional glutathione synthetase gene, and the nucleotide sequence of the bifunctional glutathione synthetase gene is shown in SEQ NO.1; In the engineered bacteria, the construction process of the recombinant vector containing the bifunctional glutathione synthetase gene is as follows: inserting the bifunctional glutathione synthetase gene into the plasmid pPicZαA, with the target gene next to the vector α-factor, to construct the recombinant vector pPicZαA-gshF.

2. A method for preparing the engineered bacteria according to claim 1, characterized in that: include: Obtaining a recombinant vector containing the bifunctional glutathione synthetase gene; then, using the recombinant vector to transform competent Pichia pastoris to obtain the engineered bacteria; The construction process of the recombinant vector containing the bifunctional glutathione synthetase gene is as follows: inserting the bifunctional glutathione synthetase gene into the plasmid pPicZαA, with the target gene next to the vector α-factor, to construct the recombinant vector pPicZαA-gshF.

3. Use of the engineered bacteria as claimed in claim 1 in obtaining glutathione synthetase outside cells.

4. Use of the engineered bacteria as claimed in claim 1 in producing glutathione.

5. A reagent for producing glutathione, characterized in that Including the engineered bacteria as described in claim 1.

6. A method for producing glutathione, characterized in that Includes steps: S1, obtaining the engineered bacteria as claimed in claim 1; S2, fermenting and culturing the engineered bacteria to obtain a fermentation liquid; S3, performing solid-liquid separation on the fermentation liquid to obtain a supernatant; S4, mixing the supernatant and glutathione synthesis raw materials to react to obtain glutathione.

7. The method for producing glutathione according to claim 6, characterized in that The fermentation culture includes a glycerol culture stage and a methanol induction stage; the duration of the glycerol culture stage is 27-37 hours, and the temperature of the glycerol culture stage is 30±0.5°C; the duration of the methanol induction stage is 88-110 hours, and the temperature of the methanol induction stage is 28±0.5°C.

8. The method for producing glutathione according to claim 6, characterized in that The culture medium used in the fermentation culture comprises phosphoric acid, calcium sulfate dihydrate, potassium sulfate, magnesium sulfate heptahydrate, potassium hydroxide, glycerol and trace elements.

9. The method for producing glutathione according to claim 6, characterized in that The raw materials for glutathione synthesis include glutamic acid, glycine, L-cysteine, ATP and MgCl2.

10. The method for producing glutathione according to claim 6, characterized in that The duration of the mixing reaction is 8-30 minutes, and the temperature of the mixing reaction is 36-38°C.

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