CN112662720A

CN112662720A - Preparation method of recombinant bacillus subtilis and glutathione

Info

Publication number: CN112662720A
Application number: CN202011631928.1A
Authority: CN
Inventors: 韦建国; 王德正; 李志敏; 刘世领
Original assignee: Shanghai Qingping Pharmaceutical Co ltd
Current assignee: Shanghai Qingping Pharmaceutical Co ltd
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2021-04-16

Abstract

The invention relates to a preparation method of recombinant bacillus subtilis and glutathione, which adopts bacillus subtilis genetic engineering strain of over-expression bifunctional glutathione synthetase gene to ferment and produce the glutathione, and has the advantages of high yield and high yield of the glutathione, simple and convenient operation, and simultaneously, the method takes food-grade bacillus subtilis as a production strain, is safer and more reliable, and provides effective reference and reference for industrialized green production of the glutathione.

Description

Preparation method of recombinant bacillus subtilis and glutathione

Technical Field

The invention relates to the field of bioengineering, and particularly relates to a preparation method of recombinant bacillus subtilis and glutathione.

Background

Glutathione (GSH) is an active tripeptide compound formed by condensing L-glutamic acid, L-cysteine and glycine, and is widely present in various organisms. Glutathione of reduced type has a plurality of important functions in living tissues, glutathione can improve human immunity and has anti-aging effect, the effect of glutathione on delayed cells of the old is greater than that of young people, and glutathione has very obvious effect on treating symptoms such as leucopenia caused by radioactive rays and radiopharmaceuticals.

At present, glutathione is prepared by a plurality of methods, which commonly comprise a solvent extraction method, a chemical synthesis method, a biological fermentation method and an enzyme method. At present, glutathione is produced at home and abroad mainly by adopting a fermentation method or an enzyme method, wherein the fermentation method is mainly characterized in that genes for coding and synthesizing a glutathione enzyme system or bifunctional enzymes are cloned into bacteria or yeast, glutathione is obtained by fermentation culture, and the yeast fermentation method in the fermentation method is mature in process. Patents CN201810844388 and CN201680013630 exogenously express glutathione synthesis bifunctional enzyme genes in yeast and escherichia coli respectively to achieve the purpose of producing glutathione by fermentation, but the yield is very low, and the method is not suitable for batch production. The reduced glutathione is widely applied in the fields of medicine and health care, food and cosmetics and the like, and the safety is very important, so that the produced glutathione is safe and reliable enough.

Disclosure of Invention

The invention aims to provide a method for preparing glutathione with high yield.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides a preparation method of glutathione, which adopts a recombinant bacillus subtilis genetic engineering strain for over-expressing a bifunctional glutathione synthetase gene to ferment and produce the glutathione.

Preferably, the bifunctional glutathione synthetase gene is a gshFst gene derived from Streptococcus thermophilus, and the gene sequence is shown as SEQ ID NO. 1.

In the invention, the bifunctional glutathione synthetase gene can be obtained by a conventional genetic engineering means. For example, it can be obtained by PCR amplification and isolation using Streptococcus thermophilus DNA as a template, or by artificial synthesis based on its sequence.

Preferably, the glutathione is produced by a fed-batch fermentation process.

Preferably, glutamic acid, glycine and cysteine are added when the glutathione is produced.

Preferably, the initial concentrations of the glutamic acid, the glycine and the cysteine to be added in the reaction system are respectively 80 to 120mM, more preferably 90 to 110mM, and still more preferably 95 to 105 mM.

The invention synthesizes the externally added glutamic acid, glycine and cysteine into the glutathione by high-density culture and over-expression of the bifunctional glutathione synthetase gene, thereby achieving high yield and high yield.

Preferably, the bacillus subtilis genetic engineering bacteria are inoculated into a fermentation culture medium for fermentation culture, a feed liquid is added in batches during the fermentation culture, the temperature of the fermentation culture is controlled to be 35-40 ℃, the pH value is controlled to be 6.5-7.5, and the fermentation culture is carried out until the OD600 is 45-55; and then adding an inducer, continuously culturing until the OD600 is 60-70, adding glutamic acid, glycine and cysteine, and continuously culturing until the yield of the glutathione is not increased any more.

In the invention, in the fermentation culture process, the fermentation culture time is 25-35 h, and the OD600 reaches 45-55; and (4) continuing to culture for 8-12 hours after adding the inducer, wherein the OD600 reaches 60-70.

In the invention, the method for the expanded culture of the corynebacterium glutamicum genetically engineered bacteria is as follows:

A. plate culture: inoculating the corynebacterium glutamicum genetic engineering bacteria stored in the glycerin tube into an LB culture medium, and culturing at 30 ℃ for 8-12h to obtain first-class seeds.

B. Liquid seed culture: inoculating the genetically engineered bacteria (primary seeds) of the corynebacterium glutamicum cultured by a plate into a secondary seed culture medium, and culturing at 30 ℃ for 6-13h to obtain a secondary seed culture solution. In the present invention, the seed medium is LB liquid medium.

Further preferably, the amount of inoculation in the fermentation culture is controlled to be 5% to 20%, more preferably 7% to 10%.

In the invention, the secondary seed culture solution is inoculated into the fermentation medium in a fermentation tank according to the inoculation amount of 5-20%.

In the present invention, IPTG is used as the inducer, and the concentration of IPTG is 1 mmol/L.

According to one embodiment, the fermentation medium of the fermentation culture comprises the following components: 5-20 g/L of carbon source, 2-8 g/L of nitrogen source, 1.5-35 g/L of inorganic salt and trace elements and 5-50 mu g/L of selectively added growth factors, wherein the carbon source is one or more of corn flour, corn steep liquor, molasses, cane sugar, glucose, glycerol and dextrin; the nitrogen source is one or more of beef extract, industrial peptone, tryptone, yeast powder, peptone and urea; the inorganic salt and the trace elements are one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, ammonium chloride, potassium chloride, sodium chloride, magnesium chloride, calcium carbonate, ammonium sulfate, magnesium sulfate and manganese sulfate; the growth factor is one or more of biotin, vitamin B1 and vitamin B6.

According to an embodiment, the components of the feed liquid include one or more of glucose, yeast extract, peptone, beef extract, yeast powder, tryptone, corn steep liquor, magnesium sulfate and glycerol, and the concentration of the feed liquid is 400-520 g/L.

The second aspect of the present invention also provides a preparation method of the recombinant bacillus subtilis genetic engineering strain, which comprises the following steps:

(1) cloning the gshFst gene derived from Streptococcus thermophilus to pHT-43 plasmid to obtain recombinant plasmid;

(2) transforming the recombinant plasmid into corynebacterium glutamicum to obtain the corynebacterium glutamicum genetic engineering strain.

In the invention, the bifunctional glutathione synthetase gene can be obtained by a conventional genetic engineering means.

Preferably, the specific method of step (1) is: and (2) amplifying DNA by taking the plasmid pET28a-gshFst as a template, carrying out double enzyme digestion on the amplification product or the plasmid by using BamHI and PstI, and then connecting the amplification product or the plasmid with the pHT-43 plasmid subjected to enzyme digestion treatment by using BamHI and PstI to obtain the recombinant plasmid.

In the invention, the recombinant plasmid is transferred into the bacillus subtilis through an electrotransformation method.

The third aspect of the invention also provides a recombinant Bacillus subtilis genetic engineering strain, wherein the Bacillus subtilis overexpresses a gene of the bifunctional glutathione synthetase, and the Bacillus subtilis is Bacillus subtilis str.168.

Preferably, the gene of the bifunctional glutathione synthetase is the gshFst gene derived from Streptococcus thermophilus, the gene sequence is shown as SEQ ID NO.1, and the bifunctional glutathione synthetase gene can be obtained by a conventional genetic engineering means.

The recombinant bacillus subtilis is used for producing glutathione by a fermentation method, so that the operation is simple and convenient, the yield is high, the production concept of resource saving and environment friendliness is met, and reference is provided for industrial production of glutathione by the recombinant bacillus subtilis.

The bacillus subtilis is a food-grade microorganism, is safe and nontoxic, so that the bacillus subtilis is more favorable for producing glutathione by taking the bacillus subtilis as an original strain.

Compared with the prior art, the invention has the following advantages:

the method for preparing the glutathione has the advantages of high yield, simple and convenient operation, and safer and more reliable use of the food-grade bacillus subtilis as a production strain, and provides effective reference and reference for industrialized green production of the glutathione.

Drawings

FIG. 1: the growth curve of the recombinant Bacillus subtilis BS-ST in the fermentation broth of the embodiment 2 of the invention;

FIG. 2 is a drawing: production profile of GSH in the fermentation broth of example 2 of the present invention;

FIG. 3: the growth curve of recombinant Bacillus subtilis BS-ST in the fermentation broth of example 3 of the present invention;

FIG. 4 is a drawing: production profile of GSH in the fermentation broth of example 3 of the present invention;

FIG. 5: the growth curve of recombinant Bacillus subtilis BS-ST in the fermentation broth of example 4 of the present invention;

FIG. 6: production profile of GSH in the fermentation broth of example 4 of the present invention;

FIG. 7: the growth curve of recombinant Bacillus subtilis BS-ST in the fermentation broth of example 5 of the present invention;

FIG. 8: production profile of GSH in the fermentation broth of example 5 of the present invention;

FIG. 9: the growth curve of recombinant Bacillus subtilis BS-ST in the fermentation broth of example 6 of the present invention;

FIG. 10: production profile of GSH in the fermentation broth of example 6 of the invention;

FIG. 11: the growth curve of recombinant Bacillus subtilis BS-ST in the fermentation broth of example 7 of the present invention;

FIG. 12: production profile of GSH in the fermentation broth of example 7 of the present invention;

FIG. 13: the growth curve of recombinant Bacillus subtilis BS-ST in the fermentation broth of example 8 of the present invention;

FIG. 14: production profile of GSH in the fermentation broth of example 8 of the present invention;

FIG. 15: the growth curve of recombinant Bacillus subtilis BS-ST in the fermentation broth of example 9 of the present invention;

FIG. 16: production profile of GSH in the fermentation broth of example 9 of the present invention;

FIG. 17: the growth curve of recombinant Bacillus subtilis BS-ST in the fermentation broth of example 10 of the present invention;

FIG. 18: production profile of GSH in the fermentation broth of example 10 of the present invention;

FIG. 19: the growth curve of recombinant Bacillus subtilis BS-ST in the fermentation broth of example 11 of the present invention;

FIG. 20: production profile of GSH in the fermentation broth of example 11 of the present invention;

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to specific related embodiments. The described embodiments are only some, but not all embodiments of the invention. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without any inventive work belong to the scope of the present invention. The experimental procedure used in the examples below. Unless otherwise specified, all the methods are conventional, and materials, reagents and the like used therein may be commercially available. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1

Construction of recombinant bacillus subtilis engineering bacteria for GSH production

The method comprises the steps of selecting a bifunctional glutathione synthetase gene (SEQ ID NO: 1) derived from Streptococcus thermophilus (gshFst), amplifying a gene fragment of the bifunctional glutathione synthetase by using a recombinant plasmid pET28a-gshFst as a template, carrying out double enzyme digestion by BamHI and SmaI to obtain a linearized plasmid, and carrying out ligation under the action of T4 ligase to obtain a recombinant plasmid pHT-ST.

Preparation of DNA fragments: DNA amplification was carried out using a recombinant plasmid pET28a-gshFst available in the laboratory as a template. The reaction conditions are as follows: pre-denaturation at 98 ℃ for 5 min; denaturation at 98 ℃ for 10 s; annealing at 60 ℃ for 30 s; extension at 72 ℃ for 5min for 30 cycles; after completion, final extension was carried out at 72 ℃ for 10 min. The amplified product was verified by running gel by nucleic acid electrophoresis, purified by PCR Purification Kit, and then digested with DpnI. Finally carrying out double digestion by using BamHI and SmaI.

The enzyme cutting system is as follows: the PCR product was either pHT-43 plasmid 50ul, BamHI and SmaI each 1ul, buffer 10ul, ddH2O38ul in a total volume of 100 ul.

The PCR product after enzyme digestion is connected with pHT-43 plasmid after the same enzyme treatment, and the connecting system is as follows: 4ul of PCR product after enzyme digestion, 4ul of pHT-43 plasmid after enzyme digestion, 1ul of T4 ligase and 1ul of buffer solution, and the recombinant plasmid pHT-ST is obtained through connection. And transferring the recombinant plasmid pHT-ST into a Bacillus subtilis str.168 to obtain a recombinant strain BS-ST.

Example 2:

production of GSH using recombinant bacteria BS-ST

(1) Inoculating the recombinant strain BS-ST engineering bacteria preserved in a glycerol tube into an LB culture medium, and culturing at 30 ℃ for 11h to serve as a first-level seed.

(2) Transferring the primary seeds into an LB liquid culture medium, and culturing at 30 ℃ for 13h to obtain a secondary seed culture solution.

(3) Inoculating the secondary seed culture solution into a 2L fermentation tank according to the inoculation amount of 10% for fed-batch fermentation, wherein the fermentation culture medium adopts 10g/L of corn flour, 2g/L of glucose, 5g/L of industrial peptone, 3g/L of corn steep liquor, 1g/L of urea, 0.5g/L of calcium carbonate, 0.8g/L of magnesium sulfate, 0.5g/L of potassium dihydrogen phosphate and 0.5g/L of dipotassium hydrogen phosphate, the feed solution contains 500g/L of glucose and 2g/L of peptone, the culture temperature is 37 ℃, the culture pH value is 6.8, the fermentation time is 30 hours, the OD600 is 46 at this moment, 1mM of inducer IPTG is added into the culture medium, the culture is continued for 5 hours, and finally 100mM of glutamic acid, glycine and cysteine are added for GSH synthesis, and the fermentation is stopped when the GSH yield is not increased any more.

The absorbance of the fermentation broth at a wavelength of 600nm was measured, and a production curve was plotted with the fermentation time as abscissa and the absorbance as ordinate, and the growth curve of the recombinant Bacillus subtilis BS-ST in the fermentation broth of this example is shown in FIG. 1. The content of GSH in the fermentation broth is measured, and a GSH production curve is drawn, wherein the GSH production curve of the embodiment is shown in figure 2. FIGS. 1 and 2 show that when cultured for 30 hours, the OD600 of the cells was 46, and at this time, 1mM IPTG was added for induction, and after 5 hours of induction, the OD600 reached 63, and GSH synthesis was started by adding three amino acid precursors each at 100mM, and the final GSH yield was 21.8g/L, and it was calculated that the GSH yield was 70.9% and the OD600 was decreased to 52, based on the addition amount of amino acid. The decrease in the cell concentration after the addition of the amino acid precursor may be caused by the decrease in OD600 due to the lysis of a part of the cells caused by the toxic action of cysteine on the cells.

Example 3:

production of GSH using recombinant bacteria BS-ST

(1) Inoculating the recombinant strain BS-ST engineering bacteria preserved in a glycerol tube into an LB culture medium, and culturing at 30 ℃ for 12h to serve as a first-level seed.

(2) Transferring the primary seeds into an LB liquid culture medium, and culturing at 30 ℃ for 10h to obtain a secondary seed culture solution.

(3) Inoculating the secondary seed culture solution into a 2L fermentation tank according to the inoculation amount of 9.5% for fed-batch fermentation, wherein the fermentation culture medium adopts 5g/L of corn flour, 3g/L of glucose, 4g/L of industrial peptone, 3g/L of corn steep liquor, 0.5g/L of urea, 1.5g/L of calcium carbonate, 1.5g/L of magnesium sulfate, 0.4g/L of potassium dihydrogen phosphate and 0.4g/L of dipotassium hydrogen phosphate, the feed solution contains 500g/L of glucose and 1g/L of peptone, the culture temperature is 37 ℃, the culture pH is 6.8, the fermentation time is 32h, the OD600 is 48 at this moment, 1mM of inducer IPTG is added into the culture medium, the culture is continued for 5h, and finally 100mM of glutamic acid, glycine and cysteine are added to synthesize GSH, and the fermentation is stopped when the yield of the GSH is not increased any more.

The growth curve of the recombinant Bacillus subtilis BS-ST in the fermentation liquid of the embodiment is shown in figure 3, and the GSH production curve is shown in figure 4. FIGS. 3 and 4 show that, when cultured for 32 hours, the OD600 of the cells was 48, and at this time, 1mM IPTG was added for induction, and after 5 hours of induction, the OD600 reached 64.3, and GSH synthesis was started by adding three amino acid precursors each at 100mM, and the final GSH yield was 20.25g/L, and it was calculated that the GSH yield was 65.9% and the OD600 was decreased to 55.2, based on the addition amount of amino acid. The decrease in the cell concentration after the addition of the amino acid precursor may be caused by the decrease in OD600 due to the lysis of a part of the cells caused by the toxic action of cysteine on the cells.

Example 4:

production of GSH using recombinant bacteria BS-ST

(1) Inoculating the recombinant strain BS-ST engineering bacteria preserved in a glycerol tube into an LB culture medium, and culturing at 30 ℃ for 10h to serve as a first-level seed.

(2) Transferring the primary seeds into an LB liquid culture medium, and culturing at 30 ℃ for 11h to obtain a secondary seed culture solution.

(3) Inoculating the second-level seed culture solution into a 2L fermentation tank according to the inoculation amount of 8% for fed-batch fermentation, wherein the fermentation culture medium adopts 8g/L glucose, 2g/L beef extract, 4g/L industrial peptone, 3g/L corn steep liquor, 0.5g/L ammonium chloride, 2g/L calcium carbonate and 1g/L magnesium sulfate, 0.2g/L potassium dihydrogen phosphate and 0.2g/L dipotassium hydrogen phosphate, wherein the feed supplement contains 400g/L glucose, 50g/L corn steep liquor and 0.5g/L peptone, the culture temperature is 37 ℃, the culture pH is 7.4, the fermentation is carried out for 29h, the OD600 is 49 at this moment, 1mM inducer IPTG is added into the culture medium, the culture is continued for 5h, finally 100mM of each of glutamic acid, glycine and cysteine are added to carry out GSH synthesis, and the fermentation is stopped when the yield of the GSH is not increased any more.

The growth curve of the recombinant Bacillus subtilis BS-ST in the fermentation liquid of the embodiment is shown in figure 5, and the GSH production curve is shown in figure 6. FIGS. 5 and 6 show that, when cultured for 29 hours, the OD600 of the cells was 49, and at this time, 1mM IPTG was added for induction, and after 5 hours of induction, the OD600 reached 64.1, and GSH synthesis was started by adding three amino acid precursors each at 100mM, and the final GSH yield was 20.95g/L, and based on the addition amount of amino acid, the GSH yield was 68.2%, and the OD600 was decreased to 57.2. The decrease in the cell concentration after the addition of the amino acid precursor may be caused by the decrease in OD600 due to the lysis of a part of the cells caused by the toxic action of cysteine on the cells.

Example 5:

production of GSH using recombinant bacteria BS-ST

(1) Inoculating the recombinant strain BS-ST engineering bacteria preserved in a glycerol tube into an LB culture medium, and culturing at 30 ℃ for 8h to serve as a first-level seed.

(2) Transferring the primary seeds into an LB liquid culture medium, and culturing at 30 ℃ for 12h to obtain a secondary seed culture solution.

(3) Inoculating the secondary seed culture solution into a 2L fermentation tank according to the inoculation amount of 7.5% for fed-batch fermentation, wherein the fermentation culture medium adopts 10g/L of glucose, 1g/L of beef extract, 0.5g/L of corn steep liquor, 2.0g/L of yeast powder, 1g/L of peptone, 1g/L of ammonium chloride, 1.5g/L of calcium carbonate, 2g/L of magnesium sulfate, 0.3g/L of potassium dihydrogen phosphate, 1.5g/L of disodium hydrogen phosphate and 1g/L of manganese sulfate, the feed supplement contains 400g/L of glucose, 1g/L of beef extract and 1g/L of yeast powder, the culture temperature is 37 ℃, the culture pH is 7.3, the fermentation time is 34h, the OD600 is 48 at this moment, 1mM of inducer G is added into the culture medium, the culture is continued for 5h, and finally 100mM of glutamic acid, glycine and cysteine are added to synthesize GSH, the fermentation was stopped when the production of GSH no longer increased.

The growth curve of the recombinant Bacillus subtilis BS-ST in the fermentation liquid of the embodiment is shown in figure 7, and the GSH production curve is shown in figure 8. FIGS. 7 and 8 show that, when cultured for 34 hours, the OD600 of the cells was 48, and at this time, 1mM IPTG was added for induction, and after 5 hours of induction, the OD600 reached 62.1, and GSH synthesis was started by adding three amino acid precursors each at 100mM, and the final GSH yield was 19.55g/L, and it was calculated that the GSH yield was 63.6% and the OD600 was decreased to 52.0, based on the addition amount of amino acid. The decrease in the cell concentration after the addition of the amino acid precursor may be caused by the decrease in OD600 due to the lysis of a part of the cells caused by the toxic action of cysteine on the cells.

Example 6:

production of GSH using recombinant bacteria BS-ST

(3) Inoculating the second-stage seed culture solution into a 2L fermentation tank according to the inoculation amount of 9.5% for fed-batch fermentation, wherein the fermentation culture medium adopts 5g/L glucose, 2g/L glycerol, 3g/L beef extract, 1g/L corn steep liquor, 1g/L yeast powder, 2g/L ammonium chloride and 0.5g/L calcium carbonate, 3g/L of magnesium sulfate, 0.6g/L of potassium dihydrogen phosphate, 3g/L of disodium hydrogen phosphate and 2g/L of manganese sulfate, wherein the feed supplement contains 450g/L of glucose and 1g/L of tryptone, the culture temperature is 37 ℃, the culture pH is 7.5, the fermentation is carried out for 33h, the OD600 is 46.5, 1mM of inducer IPTG is added into the culture medium, the culture is continued for 5h, 100mM of glutamic acid, glycine and cysteine are finally added to carry out GSH synthesis, and the fermentation is stopped when the GSH yield is not increased any more.

The growth curve of the recombinant Bacillus subtilis BS-ST in the fermentation liquid of the embodiment is shown in figure 9, and the GSH production curve is shown in figure 10. FIGS. 9 and 10 show that, when cultured for 33 hours, the OD600 of the cells was 46.5, and at this time, 1mM IPTG was added for induction, and after 5 hours of induction, the OD600 reached 64.8, and GSH synthesis was started by adding three amino acid precursors each at 100mM, and the final GSH yield was 20.99g/L, and based on the addition amount of amino acid, the GSH yield was 68.3%, and the OD600 was decreased to 56.4. The decrease in the cell concentration after the addition of the amino acid precursor may be caused by the decrease in OD600 due to the lysis of a part of the cells caused by the toxic action of cysteine on the cells.

Example 7:

production of GSH using recombinant bacteria BS-ST

(1) Inoculating the recombinant strain BS-ST engineering strain preserved in a glycerol tube into an LB culture medium, and culturing at 30 ℃ for 8.5h to serve as a first-level seed.

(2) Transferring the primary seeds into an LB liquid culture medium, and culturing at 30 ℃ for 11.5h to obtain a secondary seed culture solution.

(3) Inoculating the secondary seed culture solution into a 2L fermentation tank according to the inoculation amount of 8.5% for fed-batch fermentation, wherein the fermentation culture medium adopts 4g/L of corn steep liquor, 1g/L of glycerol, 3g/L of sucrose, 2g/L of tryptone, 2g/L of yeast powder, 3g/L of ammonium chloride, 15g/L of calcium carbonate, 3g/L of magnesium sulfate, 1g/L of sodium chloride, 1g/L of potassium dihydrogen phosphate, 1g/L of dipotassium hydrogen phosphate and 120ug/L of vitamin B, the feed solution contains 400g/L of glucose, 2g/L of yeast extract and 3g/L of magnesium sulfate, the culture temperature is 37 ℃, the culture pH is 7.1, the fermentation time is 28.2h, the OD600 is 47.5 at this time, 1mM of inducer G is added into the culture medium, the culture is continued for 5h, and finally glutamic acid, glycine, acetic acid and acetic acid are added, Synthesis of GSH was carried out with 100mM each of cysteine and the fermentation was stopped until the production of GSH was no longer increased.

The growth curve of the recombinant Bacillus subtilis BS-ST in the fermentation liquid of the embodiment is shown in figure 11, and the GSH production curve is shown in figure 12. FIGS. 11 and 12 show that the cell OD600 was 47.5 at 28.2h after the culture, 1mM IPTG was added for induction, and after 5h of induction, OD600 reached 67.9, 100mM each of the three amino acid precursors was added to start GSH synthesis, and the final GSH yield was 21.69g/L, and based on the amino acid addition, the GSH yield was 70.6% and the OD600 was decreased to 62.9. The decrease in the cell concentration after the addition of the amino acid precursor may be caused by the decrease in OD600 due to the lysis of a part of the cells caused by the toxic action of cysteine on the cells.

Example 8:

production of GSH using recombinant bacteria BS-ST

(1) Inoculating the recombinant strain BS-ST engineering strain preserved in a glycerol tube into an LB culture medium, and culturing at 30 ℃ for 11.5h to serve as a first-level seed.

(3) Inoculating the secondary seed culture solution into a 2L fermentation tank according to the inoculation amount of 7.5% for fed-batch fermentation, wherein the fermentation culture medium adopts 6g/L glucose, 2g/L molasses, 1g/L tryptone, 3g/L yeast powder, 5g/L corn flour, 3g/L ammonium sulfate, 10g/L calcium carbonate, 4g/L magnesium chloride, 2g/L potassium dihydrogen phosphate, 10g/L disodium hydrogen phosphate and 10ug/L biotin, the feed solution contains 400g/L glucose, 100g/L glycerol, 1.5g/L yeast extract and 5g/L magnesium sulfate, the culture temperature is 37 ℃, the culture pH is 7.0, the fermentation time is 30h, the OD600 is 48.5, adding 1mM inducer IPTG into the culture medium, continuously culturing for 5h, finally, 100mM of each of glutamic acid, glycine and cysteine are added to synthesize GSH, and the fermentation is stopped when the yield of GSH is not increased any more.

The growth curve of the recombinant Bacillus subtilis BS-ST in the fermentation liquid of the embodiment is shown in figure 13, and the GSH production curve is shown in figure 14. FIGS. 13 and 14 show that, when cultured for 30 hours, the OD600 of the cells was 48.5, and at this time, 1mM IPTG was added for induction, and after 5 hours of induction, the OD600 reached 66.4, and GSH synthesis was started by adding three amino acid precursors each at 100mM, and the final GSH yield was 21.29g/L, and based on the addition amount of amino acid, the GSH yield was 69.3%, and the OD600 was decreased to 54.2. The decrease in the cell concentration after the addition of the amino acid precursor may be caused by the decrease in OD600 due to the lysis of a part of the cells caused by the toxic action of cysteine on the cells.

Example 9:

production of GSH using recombinant bacteria BS-ST

(2) Transferring the primary seeds into an LB liquid culture medium, and culturing at 30 ℃ for 9h to obtain a secondary seed culture solution.

(3) Inoculating the secondary seed culture solution into a 2L fermentation tank according to the inoculation amount of 9% for fed-batch fermentation, wherein the fermentation culture medium adopts 1g/L beef extract, 1.5g/L corn steep liquor, 1g/L dextrin, 7g/L glucose, 2g/L tryptone, 2g/L yeast powder, 2g/L ammonium sulfate, 5g/L calcium carbonate, 2g/L magnesium sulfate, 1g/L sodium chloride, 1g/L potassium dihydrogen phosphate, 2g/L dipotassium hydrogen phosphate, 20ug/L biotin and 620 ug/L vitamin B, the feed solution contains 450g/L glucose, 1g/L yeast extract, 1g/L tryptone and 5g/L magnesium sulfate, the culture temperature is 37 ℃, the culture pH is 7.2, the fermentation time is 32h, and the OD600 is 51.2 at this time, adding 1mM inducer IPTG into the culture medium, continuing culturing for 5h, finally adding 100mM of each of glutamic acid, glycine and cysteine to synthesize GSH, and stopping fermentation when the yield of the GSH is not increased any more.

The growth curve of the recombinant Bacillus subtilis BS-ST in the fermentation liquid of the embodiment is shown in figure 15, and the GSH production curve is shown in figure 16. FIGS. 15 and 16 show that, when cultured for 32 hours, the OD600 of the cells was 51.2, and at this time, 1mM IPTG was added for induction, and after 5 hours of induction, the OD600 reached 69.4, and GSH synthesis was started by adding three amino acid precursors each at 100mM, and the final GSH yield was 21.76g/L, and it was calculated that the GSH yield was 71.1% and the OD600 was decreased to 60.2, based on the addition amount of amino acid. The decrease in the cell concentration after the addition of the amino acid precursor may be caused by the decrease in OD600 due to the lysis of a part of the cells caused by the toxic action of cysteine on the cells.

Example 10:

production of GSH using recombinant bacteria BS-ST

(2) Transferring the primary seeds into an LB liquid culture medium, and culturing for 6h at 30 ℃ to obtain a secondary seed culture solution.

(3) Inoculating the secondary seed culture solution into a 7L fermentation tank according to the inoculation amount of 7% for fed-batch fermentation, wherein the initial liquid loading amount of the fermentation tank is 3.5L, the formulas of a fermentation culture medium and the feed solution are the same as those in example 2, the culture temperature is 37 ℃, the culture pH is 6.8, the fermentation is carried out for 28h, the OD600 is 50.1, 1mM of inducer IPTG is added into the culture medium, the culture is continued for 5h, 100mM of glutamic acid, glycine and cysteine are finally added for GSH synthesis, and the fermentation is stopped when the GSH yield is not increased any more.

The growth curve of the recombinant Bacillus subtilis BS-ST in the fermentation liquid of the embodiment is shown in figure 17, and the GSH production curve is shown in figure 18. FIGS. 17 and 18 show that the OD600 of the cells was 50.1 at 28 hours of culture, 1mM IPTG was added for induction, OD600 reached 65.3 after 5 hours of induction, 100mM of amino acid precursor was added to start GSH synthesis, and the final GSH yield was 20.08g/L, which was calculated to be 65.3% for GSH yield and 57.8 for OD600 reduction based on the amino acid addition. The decrease in the cell concentration after the addition of the amino acid precursor may be caused by the decrease in OD600 due to the lysis of a part of the cells caused by the toxic action of cysteine on the cells.

Example 11:

production of GSH using recombinant bacteria BS-ST

(3) Inoculating the secondary seed culture solution into a 30L fermentation tank according to the inoculation amount of 9% for fed-batch fermentation, wherein the initial liquid loading amount of the fermentation tank is 15L, the formulas of a fermentation culture medium and the feed solution are the same as those in example 2, the culture temperature is 37 ℃, the culture pH is 6.8, the fermentation is carried out for 31h, the OD600 is 47.1, 1mM of inducer IPTG is added into the culture medium, the culture is continued for 5h, finally 100mM of each of glutamic acid, glycine and cysteine are added for GSH synthesis, and the fermentation is stopped when the GSH yield is not increased any more.

The growth curve of the recombinant Bacillus subtilis BS-ST in the fermentation liquid of the embodiment is shown in figure 19, and the GSH production curve is shown in figure 20. FIGS. 9 and 20 show that the OD600 of the cells was 47.1 after 31 hours of culture, 1mM IPTG was added for induction, OD600 reached 62.3 after 5 hours of induction, and GSH synthesis was started by adding 100mM amino acid precursor, and the final GSH yield was 21.35g/L, and based on the amino acid addition, the GSH yield was 69.5% and the OD600 was reduced to 46.3. The decrease in the cell concentration after the addition of the amino acid precursor may be caused by the decrease in OD600 due to the lysis of a part of the cells caused by the toxic action of cysteine on the cells.

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Sequence listing

<110> Shanghai Qingping pharmaceutical Co., Ltd

<120> preparation method of recombinant bacillus subtilis and glutathione

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2265

<212> DNA

<213> Streptococcus thermophilus bifunctional glutathione synthetase gene (Streptococcus thermophiles gshFst)

<400> 1

atgacattaa accaacttct tcaaaaactg gaagctacca gccctattct ccaagctaat 60

tttggaatcg agcgcgagag tctacgtgtc gataggcaag gacaactggt gcatacacct 120

cacccatcct gtctaggagc tcgtagtttc cacccctata ttcagactga tttttgcgag 180

tttcagatgg aactcatcac accagttgcc aaatctacta ctgaggctcg ccgatttctg 240

ggagctatta ctgatgtagc tggccgctct attgctacag acgaggttct ctggccttta 300

tccatgccac ctcgtctaaa ggcagaggag attcaagttg ctcaactgga aaatgacttc 360

gaacgccatt atcgtaacta tttggctgaa aaatacggaa ctaaactaca agctatctca 420

ggtatccact ataatatgga actgggtaaa gatttagttg aggccttgtt ccaagaaagt 480

ggtcagaccg atatgattgc cttcaaaaac gccctctatc ttaagctggc tcagaactac 540

ttgcgctacc gttgggtgat tacctatctc tttggggcct cacccatcgc cgaacaaggt 600

ttctttgacc aggaagttcc agaacctgtg cgttccttcc gtaacagtga ccacggctat 660

gtcaataagg aagagattca agtatccttt gtaagtctag aagattatgt ctcagccatt 720

gaaacctata tcgaacaagg agatttgaat gcagagaaag aattttactc agctgttcgt 780

ttccgtggac aaaaggttaa tcgttccttc cttgacaaag gaatcaccta cctagagttc 840

cgtaatttcg accttaaccc ttttgagcgt atcggtatta gtcagactac tatggacact 900

gtgcacttac tcattttagc cttcctttgg cttgatagcc ctgaaaatgt cgaccaagct 960

cttgcacaag gccacgcgtt aaatgagaaa attgccctct ctcatcctct agaacctcta 1020

ccttcggagg ctaaaactca ggacattgta actgccctag accaactggt gcaacacttt 1080

ggacttggtg actatcatca agatctggtt aagcaagtta aggcagcctt tgcggatcca 1140

aatcaaacgc tctctgccca gctcttaccc tatatcaaag acaaatctct agccgaattt 1200

gctttaaaca aggctcttgc ctatcatgat tacgactgga ctgcccacta tgctctcaag 1260

ggctatgaag agatggaact ctccacccag atgttgctct ttgatgccat ccaaaagggg 1320

attcactttg aaatattgga tgagcaagat caattcctaa aactttggca ccaagaccat 1380

gttgaatacg tcaaaaacgg taacatgacc tcaaaagaca actacgtggt tccccttgct 1440

atggctaata agaccgtaac caagaagatt ctagcagatg ctggctttcc agttccttca 1500

ggagacgaat ttaccagtct tgaggaagga cttgcctact accctcttat caaggataag 1560

caaattgttg tcaaacccaa gtcaactaac tttggtctgg gaatttccat tttccaagaa 1620

cctgccagtc ttgacaacta tcaaaaagcc cttgaaattg ctttcgcaga agatacctct 1680

gtccttgttg aagaatttat tccaggaacc gaataccgtt tcttcatctt ggatgggcgt 1740

tgtgaggctg ttcttctgcg tgtcgctgcc aatgttattg gtgatggcaa acacaccatt 1800

cgtgaactag tcgctcagaa aaatgctaat ccattgcgtg gccgtgatca ccggtcacct 1860

ctggaaatca ttgagctagg agacatcgaa caactaatgt tagctcaaca gggttacaca 1920

cctgatgata ttctcccaga aggaaaaaag gtcaatctgc gtcgtaattc caacatctct 1980

acaggtggtg actctattga tatcactgag accatggatt cctcttacca agaattagcc 2040

gcagccatgg caactagcat gggcgcctgg gcttgcgggg ttgatctgat aattccagat 2100

gaaactcaaa ttgccaccaa ggaaaatcct cattgcacct gcattgagct caactttaac 2160

ccttcgatgt atatgcacac ctactgtgct gagggtcctg gccaagctat cactactaaa 2220

atcctagata aactttttcc agaaatagtg gctggtcaaa cttaa 2265

Claims

1. A preparation method of glutathione is characterized by comprising the following steps: and (3) fermenting and producing the glutathione by adopting a recombinant bacillus subtilis genetic engineering strain for over-expressing the bifunctional glutathione synthetase gene.

2. The method of claim 1, wherein: the bifunctional glutathione synthetase gene is a gshFst gene derived from Streptococcus thermophilus, and the gene sequence is shown as SEQ ID NO. 1.

3. The method of claim 1, wherein: the glutathione is produced by adopting a fed-batch fermentation method.

4. The method of claim 1, wherein: glutamic acid, glycine and cysteine are added during production of the glutathione, and the initial concentrations of the added glutamic acid, the added glycine and the added cysteine in a reaction system are respectively 80-120 mM.

5. The production method according to claim 1 or 4, characterized in that: inoculating the bacillus subtilis genetic engineering bacteria into a fermentation culture medium for fermentation culture, adding a feed liquid in batches during the fermentation culture, controlling the temperature of the fermentation culture to be 35-40 ℃, controlling the pH to be 6.5-7.5, and performing the fermentation culture until the OD600 is 45-55; and then adding an inducer, continuously culturing until the OD600 is 60-70, adding glutamic acid, glycine and cysteine, and continuously culturing until the yield of the glutathione is not increased any more.

6. The method of claim 5, wherein: and controlling the inoculation amount in fermentation culture to be 5-20%.

7. A preparation method of a recombinant bacillus subtilis genetic engineering strain is characterized by comprising the following steps: the method comprises the following steps:

8. The method of claim 7, wherein: the specific method of the step (1) is as follows: and (2) amplifying DNA by taking the plasmid pET28a-gshFst as a template, carrying out double enzyme digestion on the amplification product or the plasmid by using BamHI and PstI, and then connecting the amplification product or the plasmid with the pHT-43 plasmid subjected to enzyme digestion treatment by using BamHI and PstI to obtain the recombinant plasmid.

9. A recombinant Bacillus subtilis genetic engineering strain is characterized in that a bifunctional glutathione synthetase gene is overexpressed in the Bacillus subtilis, and the Bacillus subtilis is Bacillus subtilis str.168.

10. The genetically engineered strain of claim 9, wherein: the bifunctional glutathione synthetase gene is a gshFst gene derived from Streptococcus thermophilus, and the gene sequence is shown as SEQ ID NO. 1.