CN107586814A - A kind of method of glutathion production by fermentation - Google Patents
A kind of method of glutathion production by fermentation Download PDFInfo
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Abstract
The invention provides a kind of method of Pichia yeast engineering glutathion production by fermentation, specifically include：The culture medium for being available for fermentation is seeded to after Pichia yeast engineering is activated, fermented under conditions of growth is available for, during the fermentation, supplemented medium 1 and supplemented medium 2 are added to culture medium, cultivates into unit volume zymotic fluid after thalline production GSH amounts reach highest and stop fermentation.The fermentation process yield is high, with short production cycle, fermentation process is easily controllable, available for scale fermenting and producing.
Description
Technical field
The present invention relates to biofermentation production glutathione field, and in particular to a kind of structure of Pichia yeast engineering and
The method of its glutathion production by fermentation.
Background technology
Glutathione (Glutathione, GSH) is that glutamic acid, cysteine and glycine contain through what peptide bond condensation formed
There is the active kyrine of γ-amido link and sulfydryl.GSH is widely present in nature, is contained in animal's liver, yeast and wheat embryo
There is abundant GSH.In organism, reduced form GSH has spectrum removing toxic substances, antioxidation, both can be used for medicine, but also as
The base-material of functional food, the extensive use in the functional food such as anti-aging, strengthen immunity, antitumor.
At present, the production method of glutathione mainly has extraction, chemical synthesis, enzymatic conversion method and microbial fermentation
Method, wherein extraction are not suitable for industrialized production due to the limitation of raw material.It is multiple cost of material height, technique to be present in chemical synthesis
The problems such as miscellaneous, product is raceme.Microbe fermentation method have synthetic route is simple, raw material is cheap and easy to get, it is with short production cycle and
The advantages such as yield height receive much concern.Glutathione Production by Microbial Fermentation mainly uses mutation breeding with genetic engineering breeding.Pass through biography
System breeding, although glutathione yield improves, but overall yield is still than relatively low.In terms of genetic engineering breeding, making
In brewer yeast, two key enzymes of GSH biosynthesis pathways be gamma glutamyl cysteine synthetase (γ-
Glutamylcysteine synthetase, GSHI) and glutathione synthetase (glutathione synthetase,
GSHII).CN10225402A is obtained using glutathione synthetases GSHI and the GSHII gene integration being placed under GAP starts
High GSH genetic engineering bacteriums, screening and culturing 72h, zymotic fluid production GSH yield reach 400mg/L.The comprehensive profit such as Zhejiang University's Liu Jian ripples
With protein expression and the Pichia yeast engineering of gene knockout means structure high-yield glutathione, synthesized by overexpression GSH
Sub- sGEX1 is transported in enzyme (sGSH1 and sGSH2) and saccharomyces cerevisiae and knocks out the sub- OPT1 of absorption and transport, recombinant bacterial strain is sent out in 8L
Fermentation tank GSH-PX activity yield reaches 173mg/L (structure [D] the Hangzhoupro of Liu Jian ripple Pichia pastoris high-yield glutathione engineering bacterias
State：Zhejiang University's Life Science College, 2014：1-43).
After the bacterial strain of the plant height of seed selection one production, the optimization to bacterium culture medium and condition of culture then can further improve yield,
Wherein, addition accumulation of the optimization to biomass of carbon source, nitrogen source and inorganic salts, the culture medium of trace element and precursor substance and
GSH generation is very crucial.Lu xun such as prolongs at the total output and cell quantity and glutathion inside cell quality using glutathione
Fractional relationship, by adding the synthesis of substrate glycine, glutamic acid and high stimulation without potassium chloride promotion glutathione, Optimal Experimental
Condition causes GSH yield, and up to 257.3mg/L, (Lu xun prolongs application Central composite designs method optimization glutathione biosynthesis bar
Part [J] amino acid and living resources, 2010,32 (2)：45-48).Domestic fermentation method production GSH is in type approval test rank more
Section, optimization and high density fermentation study on regulation to fermentation medium need deeply, to meet the needs of to GSH yield.
The content of the invention
The invention provides a kind of side for the Pichia yeast engineering glutathion production by fermentation that yield is high, fermentation period is short
Method, methods described include：Pichia yeast engineering is activated, is seeded to the culture medium for being available for fermentation, is being available for the condition of growth
Under fermented, during the fermentation, supplemented medium 1 and supplemented medium 2 are added to culture medium, cultivate to unit volume send out
Thalline production GSH amounts stop fermentation after reaching highest in zymotic fluid；
Wherein, the supplemented medium 1 includes glycerine, (NH4)2SO4And MgSO4·7H2O；Optionally, the feed supplement training
Support base 2 and include cysteine, glycine and glutamic acid, adjust pH2.0~4.0.
In some versions, the supplemented medium 1 includes about 450~550g/L glycerine, 4.0~8.0g/L (NH4)2SO4And 1.0~3.0g/L MgSO4·7H2O；The supplemented medium 2 includes 40.0~60.0g/L cysteines, 10.0
~40.0g/L glycine and 50.0~70.0g/L glutamic acid, regulation pH 2.0-4.0.
In some versions, the glycerol content of the supplemented medium 1 be about 460,480,500,520,540g/L,
(NH4)2SO4Content be about 4.5,5.0,5.5,6.5,7.5g/L, MgSO4·7H2O content is about 1.5,1.8,2.0,2.2,
2.5g/L。
Preferably, the supplemented medium 1 includes 500g/L glycerine, 5.0g/L (NH4)2SO4And 2.0g/L MgSO4·
7H2O。
In some versions, the supplemented medium 2 includes about 50.0g/L cysteines, the sweet ammonia of 10.0~30.0g/L
Acid and 60.0g/L glutamic acid, adjust pH2.0~4.0.
In some versions, the cysteine content of the supplemented medium 2 is about 50.0g/L, the content of glycine
About 10.0,15.0,20.0,25.0,30.0g/L, and the content of glutamic acid is about 60.0g/L, regulation pH to 2.8,3.0 or
3.2。
In an arrangement, the supplemented medium 2 include 50.0g/L cysteines, 15.0g/L glycine and
60.0g/L glutamic acid, adjust pH3.0.
In an arrangement, the supplemented medium 1 includes 500g/L glycerine, 5.0g/L (NH4)2SO4And 2.0g/L
MgSO4·7H2O；The supplemented medium 2 includes 50.0g/L cysteines, 15.0g/L glycine and 60.0g/L glutamic acid,
Adjust pH3.0.
Preferably, the additional amount of supplemented medium 1 is 2%~4% volume ratio, preferably 3%.
Preferably, the additional amount of supplemented medium 2 is 0.5%~1.5% volume ratio, preferably 1%.
More preferably, the additional amount of supplemented medium 1 is 3% (v/v) every time, and the additional amount of supplemented medium 2 is every time 1%
(v/v)。
Preferably, feed supplement three times, is added supplemented medium 1 in 24h, 36h and 48h respectively and mended simultaneously in incubation
Expect culture medium 2.
In some versions, being available for the culture medium of fermentation includes：15~25g/L glycerine, 1.025g/L MgSO4·7H2O,
0.5g/L NaCl, 2.94g/L KH2PO4, 9.14g/L K2HPO4, 0.0004g/L biotins and selected from (NH4)2SO4、NH4Cl
And NH4NO3Nitrogen source；Preferably, the nitrogen source is (NH4)2SO4；More preferably, described (NH4)2SO4Content is 5~10g/L.
In some versions, being available for the culture medium of fermentation includes：20g/L glycerine, 1.025g/L MgSO4·7H2O, 0.5g/
L NaCl, (NH4)2SO48g/L, 2.94g/L KH2PO4, 9.14g/L K2HPO4, 0.0004g/L biotins.
Preferably, the inoculum concentration is 2%~10% (v/v), preferably 5%~10%.
Preferably, the condition for being available for growth is 25 DEG C~37 DEG C of temperature, shaking speed 100rpm~300rpm.
More preferably, the condition for being available for growth is 33 DEG C of temperature, shaking speed 200rpm.
Preferably, the time for stopping fermentation being typically smaller than 60 hours (h), preferably 56 hours.
On the other hand, the invention provides a kind of Pichia yeast engineering ferment tank production glutathione method,
Methods described includes：Pichia yeast engineering is activated, is seeded to fermentation tank culture medium, is sent out under conditions of growth is available for
Ferment, during the fermentation, the supplemented medium of 2%~4% supplemented medium 1,0.5%~1.5% is added into culture medium
2, cultivate into unit volume zymotic fluid after thalline production GSH amounts reach highest and stop fermentation；
Wherein, the supplemented medium 1 includes 500g/L glycerine, 5.0g/L (NH4)2SO4And 2.0g/L MgSO4·
7H2O；The supplemented medium 2 include 40.0~60.0g/L cysteines, 10.0~40.0g/L glycine and 50.0~
70.0g/L glutamic acid, adjust pH3.0.
Preferably, the supplemented medium 2 include 50.0g/L cysteines, 10.0~30.0g/L glycine and
60.0g/L glutamic acid, adjust pH3.0；
More preferably, the supplemented medium 2 includes 50.0g/L cysteines, 15.0g/L glycine and 60.0g/L paddy
Propylhomoserin, adjust pH3.0.
Optionally, inoculum concentration is 2%~10% (v/v).
Preferably, the additional amount of supplemented medium 1 is 3%.
Preferably, the additional amount of supplemented medium 2 is 1%.
More preferably, the additional amount of supplemented medium 1 is 3%, and the additional amount of supplemented medium 2 is 1%.
In some versions, when the beginning feed supplement time of supplemented medium 1 is that dissolved oxygen rebounds, supplemented medium 2 starts feed supplement
Time for cell density OD values up to 30~38 when.
In some versions, the fermentation tank culture medium includes 20g/L glycerine, 1.025g/L MgSO4·7H2O, 0.54g/
L NaCl, 32.4g/L KH2PO4, 9.14g/L K2HPO4, 21.6g/L (NH4)2SO4, 0.0004g/L biotins.
In some versions, described to be available under conditions of growing being 28 DEG C~33 DEG C of temperature, ventilate 8~12L/min, maintains
Dissolved oxygen 35%~40%, rotating speed 200rpm~700rpm, dissolved oxygen level is maintained by adjusting ventilation and tank pressure, tank pressure must not be high
In 0.1MPa.
In one aspect, the present invention provides a kind of Pichia yeast engineering culture medium, it is characterised in that including the first feed supplement
Culture medium and the second supplemented medium, first supplemented medium include about 450~550g/L glycerine, 4.0~8.0g/L
(NH4)2SO4And 1.0~3.0g/L MgSO4·7H2O, preferably comprising about 500g/L glycerine, 5.0g/L (NH4)2SO4And
2.0g/L MgSO4·7H2O；It is sweet that second supplemented medium includes 40.0~60.0g/L cysteines, 10.0~40.0g/L
Propylhomoserin and 50.0~70.0g/L glutamic acid, pH 2.0~4.0, preferably comprising about 50.0g/L cysteines, 10.0~
30.0g/L glycine and 60.0g/L glutamic acid, pH2.0~4.0, more preferably comprising 50.0g/L cysteines, 15.0g/L
Glycine and 60.0g/L glutamic acid, pH3.0.
On the other hand, the invention provides a kind of fermentation tank culture medium of Pichia yeast engineering, the culture medium to include
20g/L glycerine, 1.025g/L MgSO4·7H2O, 0.54g/L NaCl, 32.4g/L KH2PO4, 9.14g/L K2HPO4,
21.6g/L(NH4)2SO4, 0.0004g/L biotins.
The construction method of Pichia yeast engineering in the present invention：Clonal expansion derives from the key enzyme GSH I of saccharomyces cerevisiae
With GSH II, digestion, carrier pGAPZaA is connected, obtains recombinant plasmid pGAPZaA-GSH I, pGAPZaA-GSH II；Clone comes
PPic9k gene cassette His is come from, is connected with carrier pGAPZaA-GSH I, obtains the recombinant plasmid pGAPZaA- of integration
His-GSH I；Carrier pGAPZaA-His-GSH I are connected with the GSH II obtained after carrier pGAPZaA-GSH II double digestions and obtained
Take recombinant plasmid pGAPZaA-His-GSH I-GSH II；Recombinant plasmid the pGAPZaA-His-GSH I, pGAPZaA- of structure
GSH II, pGAPZaA-His-GSH I-GSH II convert Pichia pastoris P.pastoris GS115, obtain recombinant bacterium
P.pastoris GS115/His-GSH I, P.pastoris GS115/GSH II, P.pastoris GS115/His-GSH
I-GSH II。
The Pichia yeast engineering of the present invention is key enzyme GSH I and the GSH II of integrant expression Saccharomyces cerevisiae base
Because of engineering bacteria.Preferably, Pichia yeast engineering include recombinant plasmid pGAPZaA-His-GSH I or pGAPZaA-GSH II or
pGAPZaA-His-GSH I-GSH II.Preferably, the Pichia yeast engineering includes recombinant plasmid pGAPZaA-His-GSH
I-GSH II Pichia pastoris G115 bacterial strains.
The method that the present invention provides glutathion production by fermentation, it has been found, surprisingly, that the technique effect of the present invention
Yield, yield height are including but not limited to obtained, easy to operate, fermentation period is short, the low fermentation process of production cost.Especially,
Using the supplemented medium of the present invention, after the certain time of fermentation carries out sequentially feed supplement, the production of glutathione greatly improved
Amount and yield, the time of fermenting and producing is greatly reduced in method compared with prior art, and reduces production cost.The hair of the present invention
Ferment technique can be used for ferment tank and large-scale industrial production.
Brief description of the drawings
Fig. 1 construction of recombinant plasmid flow charts
The amplification of Fig. 2 fermentation tanks adds the growth of thalline weight in wet base and GSH formation curves after supplemented medium 2 in cultivating
Embodiment
The method for providing Pichia yeast engineering production glutathione, methods described include：Pichia yeast engineering is lived
Change, the culture medium for being available for fermentation be seeded to 2%~15% (v/v) inoculum concentration, fermented under conditions of growth is available for,
During the fermentation, the benefit of the volume ratio of supplemented medium 1 and 0.5%~1.5% of 2%~4% volume ratio is added to culture medium
Expect culture medium 2, cultivate into unit volume zymotic fluid after thalline production GSH amounts reach highest and stop fermentation；
Wherein, the supplemented medium 1 includes glycerine, (NH4)2SO4And MgSO4·7H2O；The supplemented medium 2 wraps
Containing cysteine, glycine and glutamic acid.
Optionally, the supplemented medium 2 includes cysteine, glycine and glutamic acid, adjusts pH2.0~4.0.
In some embodiments, the supplemented medium 1 includes about 450~550g/L glycerine, 4.0~8.0g/L
(NH4)2SO4And 1.0~3.0g/L MgSO4·7H2O；The supplemented medium 2 include 40.0~60.0g/L cysteines,
10.0~40.0g/L glycine and 50.0~70.0g/L glutamic acid, adjust pH 2.0~4.0.
Preferably, the glycerol content of the supplemented medium 1 be about 460,480,500,520,540g/L, (NH4)2SO4's
Content is about 4.5,5.0,5.5,6.5,7.5g/L, MgSO4·7H2O content is about 1.5,1.8,2.0,2.2,2.5g/L.
More preferably, the supplemented medium 1 includes 500g/L glycerine, 5.0g/L (NH4)2SO4And 2.0g/L MgSO4·
7H2O。
In some embodiments, the supplemented medium 2 includes about 50.0g/L cysteines, 10.0~30.0g/L
Glycine and 60.0g/L glutamic acid, adjust pH2.0~4.0.
Preferably, the cysteine content of the supplemented medium 2 is about 50.0g/L, and the content of glycine is about
10.0th, 15.0,20.0,25.0,30.0g/L, and the content of glutamic acid is about 60.0g/L, adjusts pH to 2.8,3.0 or 3.2.
It is highly preferred that the supplemented medium 2 includes 50.0g/L cysteines, 15.0g/L glycine and 60.0g/L
Glutamic acid, adjust pH3.0.
In some embodiments, the supplemented medium 1 includes 500g/L glycerine, 5.0g/L (NH4)2SO4And
2.0g/L MgSO4·7H2O, and the supplemented medium 2 include 50.0g/L cysteines, 15.0g/L glycine and
60.0g/L glutamic acid, adjust pH3.0.
Optionally, the additional amount of supplemented medium 1 is 3% (v/v).
Optionally, the additional amount of supplemented medium 2 is 1% (v/v).
In some embodiments, the additional amount of supplemented medium 1 is every time 3%, and the additional amount of supplemented medium 2 is every
Secondary 1%.Preferably, feed supplement three times, adds supplemented medium 1 and feed supplement simultaneously in 24h, 36h and 48h respectively in incubation
Culture medium 2.
In some embodiments, the culture medium for being available for fermentation includes：15~25g/L glycerine, 1.025g/L
MgSO4·7H2O, 0.5g/L NaCl, 2.94g/L KH2PO4, 9.14g/L K2HPO4, 0.0004g/L biotins and it is selected from
(NH4)2SO4、NH4Cl and NH4NO3Nitrogen source.Preferably, the nitrogen source is (NH4)2SO4, it is highly preferred that (the NH4)2SO4
Content is 5~10g/L.
Preferably, the culture medium for being available for fermentation includes：20g/L glycerine, 1.025g/L MgSO4·7H2O, 0.5g/L
NaCl, 8g/L (NH4)2SO4, 2.94g/L KH2PO4, 9.14g/L K2HPO4, 0.0004g/L biotins.
Optionally, the inoculum concentration is 5%~10% (v/v).
Optionally, the condition for being available for growing is 25 DEG C~37 DEG C, shaking speed 100rpm~300rpm of temperature, preferably
Ground, the condition for being available for growth is 33 DEG C of temperature, shaking speed 200rpm.
In some embodiments, the time for stopping fermentation being typically smaller than 60h, preferably 56h.
On the other hand, there is provided a kind of method of Pichia yeast engineering ferment tank production glutathione, methods described
Including：Pichia yeast engineering is activated, fermentation tank culture medium is seeded to 2%~10% inoculum concentration, is being available for growth
Under the conditions of fermented, during the fermentation, 2%~4% supplemented medium 1,0.5%~1.5% is added into culture medium
Supplemented medium 2, cultivate into unit volume zymotic fluid after thalline production GSH amounts reach highest and stop fermenting；
Wherein, the supplemented medium 1 includes 500g/L glycerine, 5.0g/L (NH4)2SO4And 2.0g/L MgSO4·
7H2O；The supplemented medium 2 include 40.0~60.0g/L cysteines, 10.0~40.0g/L glycine and 50.0~
70.0g/L glutamic acid, adjust pH3.0.
In some embodiments, the supplemented medium 2 includes 50.0g/L cysteines, the sweet ammonia of 10.0~30.0g/L
Acid and 60.0g/L glutamic acid, adjust pH3.0.In some embodiments, the material culture medium 2 includes the Guang ammonia of 50.0g/L half
Acid, 15.0g/L glycine and 60.0g/L glutamic acid, adjust pH3.0.
In some embodiments, the additional amount of supplemented medium 1 is 3% (v/v).
In some embodiments, the additional amount of supplemented medium 2 is 1% (v/v).
In some embodiments, the additional amount of supplemented medium 1 is 3%, and the additional amount of supplemented medium 2 is
1%.
In some embodiments, when the beginning feed supplement time of supplemented medium 1 is that dissolved oxygen rebounds, supplemented medium 2 starts
The feed supplement time for cell density OD values up to 30~38 when.
In some embodiments, the fermentation tank culture medium includes 20g/L glycerine, 1.025g/L MgSO4·
7H2O, 0.54g/L NaCl, 32.4g/L KH2PO4, 9.14g/L K2HPO4, 21.6g/L (NH4)2SO4, 0.0004g/L biologies
Element.
In some embodiments, described to be available under conditions of growing being 28 DEG C~33 DEG C of temperature, ventilate 8~12L/
Min, dissolved oxygen 35%~40% is maintained, rotating speed 200rpm~700rpm, dissolved oxygen level, tank are maintained with tank pressure by adjusting ventilation
Must not press and be higher than 0.1MPa.
Optionally, the Pichia yeast engineering is key enzyme GSH I and the GSH II of integrant expression Saccharomyces cerevisiae
Genetic engineering bacterium.
Optionally, the Pichia yeast engineering includes recombinant plasmid pGAPZaA-His-GSH I or pGAPZaA-GSH
II or pGAPZaA-His-GSH I-GSH II.
Optionally, the Pichia yeast engineering includes recombinant plasmid pGAPZaA-His-GSH I-GSH II complete red ferment
Female G115 bacterial strains.
With reference to specific embodiment, the present invention is further illustrated, the embodiment be explanation of the invention and
It is not limitation.Experimental method used in following examples, it is conventional method unless otherwise specified；Used reagent,
Material, unless otherwise specified, commercially obtain.
The plasmid construction of embodiment 1
Respectively with saccharomyces cerevisiae genome and plasmid pPIC9K amplification gene gshI (Genebank numbering Gene ID:
And gshII (Genebank numbering Gene ID 853344):854108) and his, primer are shown in Table 2.Restriction enzyme is used respectively
Gene gshI, gshII and His are connected with expression vector pGAPZaA, structure plasmid pGAPZaA-His-GSHI, pGAPZaA-
GSHII, pGAPZaA-His-GSHI-GSHII.By recombinant plasmid pGAPZaA-His-GSHI, the pGAPZaA-GSHII of structure,
PGAPZaA-His-GSHI-GSHII converts Pichia pastoris P.pastoris, obtains recombinant bacterium P.pastoris GS115/His-
GSHI (PGshI), P.pastoris GS115/GSHII (PGshII), P.pastoris GS115/His-GSHI-GSHII
(PGshI-PGshII)。
Primer used in 1 research of table
Note：Underscore represents the restriction enzyme used
The Pichia pastoris of embodiment 2 is electroporated
The preparation of Pichia pastoris competent cell
(1) picking Pichia pastoris single bacterium colony is inoculated in 25mL/250mL YPD culture mediums, and 30 DEG C of 250rpm cultures are about
14h。
(2) with 1% inoculum concentration switching 50mL/500mL YPD fluid nutrient mediums, 30 DEG C, 250rpm cultivates about 12h.
(3) at 4 DEG C, 5min is centrifuged under the conditions of 5000rpm, abandons supernatant, with 1/2 volume ice precooling sterilized water by thalline weight
It is outstanding.
(4) at 4 DEG C, 10min is centrifuged under the conditions of 5000rpm, abandons supernatant, with 1/2 volume ice precooling sterilized water by thalline weight
It is outstanding.
(5) at 4 DEG C, 10min is centrifuged under the conditions of 5000rpm, abandons supernatant, then the sorb with the 1/2 sterile 1M of volume ice precooling
Alcohol washs 1 time.
(6) the 1/200 sterile 1M of volume ice precooling sorbierite is dissolved in, is distributed into 100 μ L/ pipes in case conversion.
The electroporated step of Pichia pastoris：
(1) the plasmid 1-5 μ g of KpnI or XmaJI restriction enzyme sites linearisation are added in 80 μ L competent yeast cells.
(2) it is transferred to after placing 5min on ice in 0.2cm precoolings electric shock cup.
(3) 1mL precooling 1M sorbierites are rapidly added after shocking by electricity, gently pressure-vaccum is mixed and is transferred in 1.5mL sterile tubes.
(4) 30 DEG C of stationary incubation 1-2h, 10-200 μ L are taken to apply YPDS plates (containing 100 μ g/mL Zeocin).
(5) flat board is inverted culture 2-3 days in 30 DEG C, until growing monoclonal.
(6) 10-20 monoclonal is selected to be used to verify in ruling on fresh YPD solid plates.
Pichia pastoris competence prepares and electroporated culture medium：
YPD solid mediums (g/L)：Dusty yeast 10.0, peptone 20.0, glucose 20.0, agar powder 15.0-20.0.
YPD culture mediums (g/L)：Dusty yeast 10.0, peptone 20.0, glucose 20.0.
YPDS culture mediums (g/L)：Dusty yeast 10.0, peptone 20.0, glucose 20.0, sorbierite 182.2.
The seed culture of embodiment 3
Seed activation：The single bacterium colony of the engineered strain of picking structure is in plate streaking, 28 DEG C of culture 60-72h；
Seed culture：The thalline of picking flat board activation, 28 DEG C, 200rpm, 16-18h is cultivated, fermentation medium of transferring.
Wherein, slant medium (g/L)：Glycerine 20.0, dusty yeast 10.0, peptone 10.0, agar powder 15.0 are natural
pH；Seed culture medium (g/L)：Glycerine 20.0, peptone 20.0, dusty yeast 10.0.Liquid amount 50mL/250mL, natural pH.
4 three kinds of recombinant bacterium shake flask fermentations of embodiment
The recombinant bacterial strain for expressing the gshI and gshII that there are different genes is cultivated, transferred with 5% inoculum concentration, culture
During, add the and of supplemented medium 1 of 3.2% volume ratio into shaking flask respectively at 24h, 36h and 48h using sterile working
The supplemented medium 2 of 1% volume ratio, into unit volume zymotic fluid, thalline production GSH amounts stop a fermentation (56h left sides after reaching highest
It is right), determine GSH contents.
Fermentation medium (g/L)：Glycerine 20, MgSO4·7H2O 1.025, NaCl 0.5, KH2PO42.94 K2HPO4
9.14, (NH4)2SO45.0, biotin 0.0004, liquid amount 50mL/500mL, natural pH；Supplemented medium 1 (g/L)：Glycerine
500.0 (NH4)2SO45.0, MgSO4·7H2O 2.0, natural pH；Supplemented medium 2 (g/L)：Cysteine 50.0, glycine
30.0, glutamic acid 60.0, pH 3.0.
As shown in Table 1, GSH yield is 0.07g/L in starting strain P.pastoris GS115, illustrates P.pastoris
Complete GSH route of synthesis in GS115 be present.Single expression gshI, gshII and the recombinant bacterium for co-expressing gshI and gshII
Middle GSH yield is respectively 0.35g/L, 0.24g/L and 0.76g/L, and 5.0 times, 3.4 times and 10.9 have been respectively increased than starting strain
Times, illustrate that express GSH route of synthesis key enzyme has facilitation to GSH synthesis.
The GSH yield of the different strains of table 1 fermentation
The recombinant bacterial strain PGshI-PGshII shake flask fermentations of different initial concentration glycerine in the culture medium of embodiment 5
Recombinant bacterial strain PGshI-PGshII is transferred with 5% inoculum concentration, in incubation, using sterile working respectively at 24h,
36h and 48h adds the supplemented medium 2 of the volume ratio of supplemented medium 1 and 1% of 3.2% volume ratio into shaking flask, and fermentation is extremely
56h or so, determine GSH contents.Initial glycerol concentration is respectively 16g/L, 20g/l and 24g/L in fermentation medium, and other are sent out
Ferment culture medium and feed-batch culture based component are the same as embodiment 4.
Recombinant bacterial strain PGshI-PGshII GSH yield under the different initial glycerol concentrations of table 2
The recombinant bacterial strain PGshI-PGshII shake flask fermentations of different nitrogen sources in the culture medium of embodiment 6
Recombinant bacterial strain PGshI-PGshII is transferred with 5% inoculum concentration, in incubation, using sterile working respectively at 24h,
36h and 48h adds the supplemented medium 2 of the volume ratio of supplemented medium 1 and 1% of 3.2% volume ratio into shaking flask, and fermentation is extremely
56h or so, determine GSH contents.Nitrogen source selects equimolar different ammonium salt ((NH in fermentation medium4)2SO45.0g/L
(NH4)2Cl 4.1g/L, NH4NO36.0g/L), other fermentation mediums and feed-batch culture based component are the same as embodiment 4.
Recombinant bacterial strain PGshI-PGshII GSH yield under the different nitrogen sources of table 3
The recombinant bacterial strain PGshI-PGshII shake flask fermentations of the different additions of supplemented medium 1 of embodiment 7
Recombinant bacterial strain PGshI-PGshII is transferred with 5% inoculum concentration, in incubation, using sterile working respectively at 24h,
36h and 48h adds the feed supplement of 2.5%, 3.2% and 3.9% volume ratio supplemented medium 1 and 1% volume ratio into shaking flask respectively
Culture medium 2, ferment to 56h or so, determine GSH contents.Fermentation medium (g/L)：Glycerine 20, MgSO4·7H2O 1.025,
NaCl 0.5, KH2PO42.94 K2HPO49.14, (NH4)2SO48.0, biotin 0.0004, liquid amount 50mL/500mL, from
Right pH；Supplemented medium 1 (g/L)：Glycerine 500.0, (NH4)2SO45.0, MgSO4·7H2O 2.0, natural pH；Supplemented medium
2(g/L)：Cysteine 50.0, glycine 30.0, glutamic acid 60.0, pH 3.0.
Recombinant bacterial strain PGshI-PGshII GSH yield under the different additions of supplemented medium 1 of table 4
The recombinant bacterial strain PGshI-PGshII shake flask fermentations of the different supplemented mediums 2 of embodiment 8
Recombinant bacterial strain PGshI-PGshII is transferred with 5% inoculum concentration, in incubation, using sterile working respectively at 24h,
36h and 48h adds the supplemented medium 2 of the volume ratio of supplemented medium 1 and 1% of 3.2% volume ratio into shaking flask, and fermentation is extremely
56h or so, determine GSH contents.Fermentation medium (g/L)：Glycerine 20, MgSO4·7H2O 1.025, NaCl 0.5, KH2PO4
2.94 K2HPO49.14, (NH4)2SO45.0, biotin 0.0004, liquid amount 50mL/500mL, natural pH；Supplemented medium 1
(g/L)：Glycerine 500.0, (NH4)2SO45.0, MgSO4·7H2O 2.0, natural pH；Supplemented medium 2 is divided into by component difference
Three groups, respectively A groups (mol ratio cysteine：Glycine：Glutamic acid=1:1:1)；B groups (mol ratio cysteine：Sweet ammonia
Acid：Glutamic acid=1:1:0.5)；C groups (mol ratio cysteine：Glycine：Glutamic acid=1:0.5:1).
The recombinant bacterial strain PGshI-PGshII of supplemented medium 2 of the different component of table 5 GSH yield
The recombinant bacterial strain PGshI-PGshII shake flask fermentations of the different additions of supplemented medium 2 of embodiment 9
Recombinant bacterial strain PGshI-PGshII is transferred with 5% inoculum concentration, in incubation, using sterile working respectively at 24h,
36h and 48h adds 3.2% volume ratio supplemented medium 1 and 0.8%, 1.0% and 1.2% volume ratio into shaking flask respectively
Supplemented medium 2, ferment to 56h or so, determine GSH contents.Fermentation medium (g/L)：Glycerine 20, MgSO4·7H2O
1.025, NaCl 0.5, KH2PO42.94 K2HPO49.14, (NH4)2SO48.0, biotin 0.0004, liquid amount 50mL/
500mL, natural pH；Supplemented medium 1 (g/L)：Glycerine 500.0, (NH4)2SO45.0, MgSO4·7H2O 2.0, natural pH；Mend
Expect culture medium 2 (g/L)：Cysteine 50.0, glycine 15.0, glutamic acid 60.0, pH 3.0.
The recombinant bacterial strain PGshI-PGshII of the different additions of supplemented medium 2 of table 6 GSH yield
The recombinant bacterium PGshI-PGshII of embodiment 10 shake flask fermentation
Recombinant bacterial strain PGshI-PGshII is transferred with 10% inoculum concentration, in incubation, using sterile working respectively at
It is 3.0% that 24h, 36h and 48h, which add material culture medium 1 additional amount into shaking flask, and the additional amount of supplemented medium 2 is 1.0%, fermentation
To 56h or so, determine GSH contents.Fermentation medium (g/L)：Glycerine 20, MgSO4·7H2O 1.025、NaCl 0.5、KH2PO4
2.94、K2HPO4 9.14、(NH4)2SO48.0, biotin 0.0004, liquid amount 50mL/500mL, initial pH 6.0.Feed supplement is trained
Support base 1 (g/L)：Glycerine 500.0, (NH4)2SO45.0, MgSO4·7H2O 2.0, natural pH；Supplemented medium 2 (g/L)：Half Guang
Propylhomoserin 50.0, glycine 15.0, glutamic acid 60.0, pH 3.0.GSH maximum outputs reach 2.14 ± 0.05g/L, in experimental point
Higher level, 0.76 ± 0.03g/L of highest before relatively optimizing improve 268.4%.
The recombinant bacterial strain PGshI-PGshII of embodiment 11 15L fermentation tanks amplification culture
Seed liquor is seeded in 15L fermentation tanks with 5% inoculum concentration, liquid amount 9L.Condition of culture：28 DEG C of temperature, lead to
Gas 10L/min, dissolved oxygen 35%-40%, initial speed 200rpm are maintained, rotating speed is improved when dissolved oxygen is down to 35%, highest maintains
700rpm, dissolved oxygen level is maintained with tank pressure by adjusting ventilation, tank pressure must not be higher than 0.1MPa.When dissolved oxygen rebounds (about
24h) start feed supplement, the additional amount of supplemented medium 1 is 3.0%, when the OD values of cell density to be determined are more than 35, starts to add
Supplemented medium 2, additional amount and supplemented medium 1 are that ratio is 1:3, into unit volume zymotic fluid, thalline production GSH amounts are up to most
Stop fermentation (fermentation 50h or so) after height, thalline weight in wet base and GSH contents is measured by sampling in every 2~3h.
Fermentation tank culture medium (g/L)：Glycerine 20, MgSO4·7H2O 1.025, NaCl 0.54, KH2PO432.4 K2HPO4
91.4, (NH4)2SO421.6, biotin 0.004；Supplemented medium 1 (g/L)：Glycerine 500.0, (NH4)2SO45.0
MgSO4·7H2O 2.0, natural pH；Supplemented medium 2 (g/L)：Cysteine 50.0, glycine 15.0, glutamic acid 60.0, pH
3.0。
Strain growth and GSH yield significantly improve after feed supplement, illustrate to utilize constitutive promoter expression key enzyme, purpose production
Thing GSH yield and strain growth direct proportionality, yield is up to 4.90g/L, is understood with reference to Fig. 2, with the feed supplement time
Extension, unit weight in wet base thalline, as the extension of feed supplement time gradually steps up, highest reaches in 23h, now to GSH yield
GSH yield highests.
The glutathione detection method of embodiment 12
Glutathione：1mL samples are centrifuged into 3min under the conditions of 12000rpm, abandon supernatant, thalline is resuspended with 1mL pure water,
Boiling water bath 6min, is cooled to room temperature, shakes up, 12000rpm centrifugation 3min, 0.22 μm of membrane filtration of supernatant, through Peroxide paddy
After the sweet peptide reduction of Guang, glutathione is detected with HPLC.
Oxidized form of glutathione reduces：The μ L 0.25M DTT+50 μ L 0.1M Tris-HCl (pH of 100 μ L samples+100
8.5), 4 DEG C of standing 30min.
HPLC is analyzed.Chromatographic condition：Column type number：TSK gel ODS, 5 μm, 4mm × 250mm, UV-detector wavelength：
210nm, sample size：20 μ L mobile phases：A phase 0.025mol/L NaH2PO4(H3PO4PH is adjusted to 3.15), B phases 60% (v/v) second
The nitrile aqueous solution.Column temperature：30 DEG C, flow velocity：1.0mL/min.Gradient：0-13min A 100%B 0%；13-18min A 0%B
100%；18-23min A 0%B 100%；23-25min A 100%B 0%；25-35min A 100%B 0%.
All publications and patents application mentioned in this specification indicates ordinary skill of the art
It is horizontal.All publications and patents application is hereby incorporated herein by, and cited degree is just as each single
Publication or patent application are by specifically as independently providing and being hereby incorporated herein by.Through each specific excellent
The embodiment and technology of choosing describe the present invention.It is understood that can on the premise of being maintained at the spirit and scope of the present invention
To make change or modification.
Claims (10)
1. a kind of method of Pichia yeast engineering production glutathione (GSH), methods described include：By Pichia yeast engineering
Activation, the culture medium for being available for fermentation is seeded to, is fermented under conditions of growth is available for, during the fermentation, to culture medium
Supplemented medium 1 and supplemented medium 2 are added, cultivates into unit volume zymotic fluid after thalline production GSH amounts reach highest and stops hair
Ferment；
Wherein, the supplemented medium 1 includes glycerine, (NH4)2SO4And MgSO4·7H2O；The supplemented medium 2 includes half
Cystine, glycine and glutamic acid.
2. according to the method for claim 1, the supplemented medium 1 includes about 450~550g/L glycerine, 4.0~8.0g/
L(NH4)2SO4And 1.0~3.0g/L MgSO4·7H2O, preferably comprising about 500g/L glycerine, 5.0g/L (NH4)2SO4And
2.0g/L MgSO4·7H2O；The supplemented medium 2 includes 40.0~60.0g/L cysteines, the sweet ammonia of 10.0~40.0g/L
Acid and 50.0~70.0g/L glutamic acid, adjust pH 2.0~4.0, preferably comprising about 50.0g/L cysteines, 10.0~
30.0g/L glycine and 60.0g/L glutamic acid, adjust pH2.0~4.0, more preferably comprising 50.0g/L cysteines,
15.0g/L glycine and 60.0g/L glutamic acid, adjust pH3.0.
3. according to the method for claim 1, the supplemented medium 1 includes 500g/L glycerine, 5.0g/L (NH4)2SO4With
And 2.0g/L MgSO4·7H2O；The supplemented medium 2 include 50.0g/L cysteines, 15.0g/L glycine and
60.0g/L glutamic acid, adjust pH3.0.
4. according to method according to any one of claims 1 to 3, the additional amount of supplemented medium 1 is 3%, feed-batch culture
The additional amount of base 2 is 1%, it is preferable that feed supplement three times, adds feed-batch culture simultaneously in 24h, 36h and 48h respectively in incubation
Base 1 and supplemented medium 2.
5. according to method according to any one of claims 1 to 4, the inoculum concentration is 5%~10% (v/v), described to be available for
The condition of growth is 25 DEG C~37 DEG C of temperature, preferably 33 DEG C, shaking speed 100rpm~300rpm, preferably 200rpm.
6. according to method according to any one of claims 1 to 5, the time for stopping fermentation being typically smaller than 60h, is preferably
56h。
7. according to the method described in any one of claim 1~6, the Pichia yeast engineering comes for integrant expression saccharomyces cerevisiae
Key enzyme GSH I and the GSH II in source genetic engineering bacterium or the Pichia yeast engineering include recombinant plasmid pGAPZaA-
His-GSH I-GSH II genetic engineering bacterium, preferably described Pichia pastoris are G115 bacterial strains.
8. a kind of method of Pichia yeast engineering ferment tank production glutathione, methods described include：By Pichia pastoris
Engineering bacteria is activated, and fermentation tank culture medium is seeded to 2%~10% inoculum concentration, is fermented under conditions of growth is available for,
During the fermentation, the supplemented medium 2 of 2%~4% supplemented medium 1,0.5%~1.5% is added into culture medium, is trained
Support into unit volume zymotic fluid after thalline production GSH amounts reach highest and stop fermentation；
Wherein, the supplemented medium 1 includes 500g/L glycerine, 5.0g/L (NH4)2SO4And 2.0g/L MgSO4·7H2O；Institute
State supplemented medium 2 and include 40.0~60.0g/L cysteines, 10.0~40.0g/L glycine and 50.0~70.0g/L paddy
Propylhomoserin, pH3.0 is adjusted, preferably comprises 50.0g/L cysteines, 10.0~30.0g/L glycine and 60.0g/L glutamic acid,
PH3.0 is adjusted, more preferably comprising 50.0g/L cysteines, 15.0g/L glycine and 60.0g/L glutamic acid, adjusts pH3.0.
9. according to the method for claim 8, the additional amount of supplemented medium 1 is 3%, the additional amount of supplemented medium 2
For 1%, supplemented medium 1 starts the feed supplement time when being that dissolved oxygen rebounds, and supplemented medium 2 starts the feed supplement time as cell density
When OD values are up to 30~38.
10. a kind of culture medium of Pichia yeast engineering, it is characterised in that including the first supplemented medium and the second feed-batch culture
Base, first supplemented medium include about 450~550g/L glycerine, 4.0~8.0g/L (NH4)2SO4And 1.0~3.0g/L
MgSO4·7H2O, preferably comprising about 500g/L glycerine, 5.0g/L (NH4)2SO4And 2.0g/L MgSO4·7H2O；Described
Two supplemented mediums include 40.0~60.0g/L cysteines, 10.0~40.0g/L glycine and 50.0~70.0g/L paddy
Propylhomoserin, adjust pH 2.0~4.0, preferably comprising about 50.0g/L cysteines, 10.0~30.0g/L glycine and
60.0g/L glutamic acid, adjust pH2.0~4.0, more preferably comprising 50.0g/L cysteines, 15.0g/L glycine and
60.0g/L glutamic acid, adjust pH3.0.
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| CN112662700A (en) * | 2021-02-04 | 2021-04-16 | 南华大学 | Construction method of recombinant plasmid for glutathione biosynthesis |
| CN114032266A (en) * | 2021-12-03 | 2022-02-11 | 江西诚志生物工程有限公司 | Process for producing glutathione by fermentation method |
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