CN116376751A - Fermentation production method of L-selenomethionine - Google Patents
Fermentation production method of L-selenomethionine Download PDFInfo
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- CN116376751A CN116376751A CN202310138777.3A CN202310138777A CN116376751A CN 116376751 A CN116376751 A CN 116376751A CN 202310138777 A CN202310138777 A CN 202310138777A CN 116376751 A CN116376751 A CN 116376751A
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- 238000000855 fermentation Methods 0.000 title claims abstract description 45
- 230000004151 fermentation Effects 0.000 title claims abstract description 45
- RJFAYQIBOAGBLC-BYPYZUCNSA-N Selenium-L-methionine Chemical compound C[Se]CC[C@H](N)C(O)=O RJFAYQIBOAGBLC-BYPYZUCNSA-N 0.000 title claims abstract description 28
- 229960002718 selenomethionine Drugs 0.000 title claims abstract description 28
- 238000004519 manufacturing process Methods 0.000 title description 4
- 241000186226 Corynebacterium glutamicum Species 0.000 claims abstract description 32
- UKAUYVFTDYCKQA-VKHMYHEASA-N L-homoserine Chemical compound OC(=O)[C@@H](N)CCO UKAUYVFTDYCKQA-VKHMYHEASA-N 0.000 claims abstract description 24
- UKAUYVFTDYCKQA-UHFFFAOYSA-N -2-Amino-4-hydroxybutanoic acid Natural products OC(=O)C(N)CCO UKAUYVFTDYCKQA-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 12
- 239000000758 substrate Substances 0.000 claims abstract description 12
- PPXAQNPNOPCBRG-UHFFFAOYSA-M [Se](=O)(=O)(OC)[O-].[Na+] Chemical compound [Se](=O)(=O)(OC)[O-].[Na+] PPXAQNPNOPCBRG-UHFFFAOYSA-M 0.000 claims abstract description 10
- VRDKYJSLDJDLML-UHFFFAOYSA-N methylselenol Chemical compound [Se]C VRDKYJSLDJDLML-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000011669 selenium Substances 0.000 claims description 23
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 claims description 22
- 229910052711 selenium Inorganic materials 0.000 claims description 22
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 13
- 239000001888 Peptone Substances 0.000 claims description 10
- 108010080698 Peptones Proteins 0.000 claims description 10
- 235000019319 peptone Nutrition 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 8
- 229920001817 Agar Polymers 0.000 claims description 7
- 239000008272 agar Substances 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 235000002639 sodium chloride Nutrition 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 230000009466 transformation Effects 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 5
- 229940099596 manganese sulfate Drugs 0.000 claims description 4
- 235000007079 manganese sulphate Nutrition 0.000 claims description 4
- 239000011702 manganese sulphate Substances 0.000 claims description 4
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 4
- 238000009423 ventilation Methods 0.000 claims description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 239000011573 trace mineral Substances 0.000 claims description 3
- 235000013619 trace mineral Nutrition 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 2
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 2
- 239000003125 aqueous solvent Substances 0.000 claims description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims 1
- YDAKJMZYJQLRNA-UHFFFAOYSA-N methyl hydrogen selenate Chemical compound CO[Se](O)(=O)=O YDAKJMZYJQLRNA-UHFFFAOYSA-N 0.000 claims 1
- 125000002327 selenol group Chemical class [H][Se]* 0.000 claims 1
- 239000011734 sodium Substances 0.000 claims 1
- 229910052708 sodium Inorganic materials 0.000 claims 1
- 238000011218 seed culture Methods 0.000 abstract description 5
- 230000004913 activation Effects 0.000 abstract description 3
- 239000002609 medium Substances 0.000 description 24
- 229940091258 selenium supplement Drugs 0.000 description 22
- 239000001963 growth medium Substances 0.000 description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 6
- 235000015278 beef Nutrition 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 235000013305 food Nutrition 0.000 description 5
- 238000005273 aeration Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 239000002054 inoculum Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241000233866 Fungi Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108010074686 Selenoproteins Proteins 0.000 description 2
- 102000008114 Selenoproteins Human genes 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- HUGQWUJVICPBEX-UHFFFAOYSA-N [Na].C[SeH] Chemical compound [Na].C[SeH] HUGQWUJVICPBEX-UHFFFAOYSA-N 0.000 description 2
- 239000000679 carrageenan Substances 0.000 description 2
- 229920001525 carrageenan Polymers 0.000 description 2
- 229940113118 carrageenan Drugs 0.000 description 2
- 235000010418 carrageenan Nutrition 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 231100000053 low toxicity Toxicity 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 235000011121 sodium hydroxide Nutrition 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 2
- 125000000659 L-selenomethionine group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C([H])([H])C([H])([H])[Se]C([H])([H])[H] 0.000 description 1
- RJFAYQIBOAGBLC-UHFFFAOYSA-N Selenomethionine Natural products C[Se]CCC(N)C(O)=O RJFAYQIBOAGBLC-UHFFFAOYSA-N 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
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- 238000001514 detection method Methods 0.000 description 1
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- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- -1 preferably 0.0001g/L Chemical compound 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229940065287 selenium compound Drugs 0.000 description 1
- 150000003343 selenium compounds Chemical class 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000000153 supplemental effect Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 231100000816 toxic dose Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
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Abstract
The invention relates to the technical field of biological fermentation, in particular to a corynebacterium glutamicum and a method for preparing L-selenomethionine by using the strain fermentation, wherein the screened corynebacterium glutamicum is subjected to (1) plate activation (2) seed culture (3) fermentation culture and bioconversion steps, and is subjected to primary or multistage culture, L-homoserine, sodium methylselenate or methylselenol is used as a substrate, and L-selenomethionine is generated by fermentation.
Description
Technical Field
The invention belongs to the technical field of L-selenomethionine production, and particularly relates to corynebacterium glutamicum and a method for preparing L-selenomethionine by using the strain for fermentation production.
Background
Selenomethionine molecular formula, C 5 H 11 NO 2 Se, which is white or white-like crystal powder in appearance, has optical rotation of +17.0 DEG to +19.5 deg. An organic selenium compound in which the organic form of selenium is present in nature mainly in plants. Compared with inorganic selenium, L-selenomethionine has the advantages of low toxicity, high bioavailability and the like (Environment research,2005, 98 (1): 46-54). At present, many countries in Europe and America have promoted the use of organic selenium, for example, sweden requires that organic selenium must be used in the feed of suckling pigs; the use of inorganic selenium in feeds has been regulated in japan. The L-selenomethionine has stable property and obvious efficacy (Current protein and peptide science,2014, 15 (6): 598-607), and has higher practical value and wide popularization and application prospect.
Selenium has been identified by the world health organization as one of the essential trace elements for animals and humans. The current selenium source food mainly comprises selenium-enriched yeast, seleno carrageenan, selenoprotein, selenium-enriched edible fungus powder, L-selenomethionine and L-selenomethylselenocysteine. The L-selenomethionine structure contains about 40% of selenium element, and is the main existing form of natural selenium.
Disclosure of Invention
Summary of The Invention
In order to solve the above problems, the first aspect of the present invention provides Corynebacterium glutamicum Corynebacterium glutamicum OMK-88, accession No.: CCTCC M20222075, date of preservation: 2022, 12, 23. Preservation unit: the China center for type culture Collection (CCTCC, university of Wuhan) has been subjected to biological preservation.
In a second aspect, the present invention proposes the use of the corynebacterium glutamicum Corynebacterium glutamicum OMK-88 of the first aspect for the preparation of L-selenomethionine, comprising fermenting the corynebacterium glutamicum in a fermentation medium comprising a reaction substrate to obtain L-selenomethionine.
In some embodiments, the use of second aspects Corynebacterium glutamicum OMK-88 in the preparation of L-selenomethionine, the fermentation substrate is any one or more of L-homoserine, sodium methylselenate, or methylselenol, and further, the L-homoserine is 50-70g/L, and the sodium methylselenate or methylselenol is 50-70g/L.
In some embodiments, the second aspect Corynebacterium glutamicum OMK-88 uses, the fermentation medium comprises one or more of a carbon source, a nitrogen source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, sodium chloride, ammonium sulfate, trace elements.
Wherein the carbon source comprises glucose,
the nitrogen source comprises one or more of yeast powder and peptone.
In some embodiments, the second aspect Corynebacterium glutamicum OMK-88 application includes the steps of:
s1: corynebacterium glutamicum is cultured in a plate medium,
s2: s1, performing primary or multistage culture and expansion culture on the flat mycelium,
s3: s2, performing biological transformation on the expansion culture seed solution.
In some embodiments, the second aspect Corynebacterium glutamicum OMK-88 uses a seed liquid cultivated in one or more stages to a bioconversion broth volume ratio of 0.9:10-0.9:20, preferably 0.9:15, particularly preferably 0.9: 13. 0.9: 12. 0.9: 11. 0.9:10.
in some embodiments, the second aspect Corynebacterium glutamicum OMK-88 uses, the plate medium pH is 7.4-7.6, preferably 7.4, 7.5, 7.6, the rotational speed rpm of the expansion step is 300-400, preferably 300, 350, 400, the cultivation temperature is 36.5-37.5 ℃, preferably 36.5 ℃,37 ℃, 37.5 ℃, the cultivation time is 13-16h, preferably 14-16h, particularly preferably 15h. The fermentation temperature in the bioconversion step is 37 ℃, the stirring rotation speed is 400-500rpm, and the ventilation ratio is 1:0.1-1:0.4, preferably 1:0.1, 1:0.2, 1:0.3, 1:0.4, the bioconversion time is 12-20h, preferably 12-16h, particularly preferably 12h, 16h, the reaction substrate L-homoserine, sodium methylselenate or methylselenol is added in one portion or in portions, and the addition in portions is each time.
In some embodiments, the second aspect Corynebacterium glutamicum OMK-88 application, the fermentation medium comprises 2-5g/L, preferably 3-5g/L, particularly preferably 3-4g/L, peptone 1-10g/L, preferably 2g/L-9g/L, particularly preferably 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, agar 10-30g/L, preferably 15-25g/L, particularly preferably 15g/L, 20g/L, 25g/L, potassium dihydrogen phosphate 1.0-2.5g/L, preferably 1.2g/L-2.0g/L, particularly preferably 1.2g/L, 1.5g/L, 2.0g/L, 0.5g/L to 10g/L, preferably 0.8g/L to 5g/L, particularly preferably 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 20g/L, preferably 10g/L to 15g/L, particularly preferably 10g/L, 11g/L, 12g/L, 13g/L, 14g/L, 15g/L, 0.1 g to 0.3g/L, preferably 0.2g to 0.3g/L, particularly preferably 0.2g/L, 3g to 6g/L, preferably 4g to 5g/L, particularly preferably 5g/L, 2.0g to 2.6g/L, preferably 2.2 g to 2.4g/L, particularly preferably 2.4g/L, 0.0001g to 0.0003g/L of manganese sulfate, preferably 0.0001g/L, glucose 10-40g/L, preferably 20-30g/L, particularly preferably 20g/L, 25g/L, 30g/L, pH between 6.8-7.6, preferably 7, which may be adjusted with either buffered saline solution, caustic soda, and aqueous solvent.
The third aspect of the invention also provides the organic selenium prepared by the preparation method in the second aspect.
The reagents used in the invention are purchased in public legal markets and are not further purified.
Compared with the prior art, the invention has the following advantages:
(1) L-selenomethionine has the advantage of low toxicity of organic selenium compared with inorganic selenium.
(2) L-selenomethionine has the advantages of clear structure, stable content, clear metabolism mechanism in human body and the like. The selenium content in the products such as selenium-enriched yeast, selenylation carrageenan, selenoprotein, selenium-enriched edible fungus powder and the like is different to a certain extent from batch to batch, and is not fixed. Because the effective supplemental dose of selenium is relatively close to the toxic dose, other selenium source foods present a certain risk for food safety.
(3) L-selenomethionine and L-selenium-methylselenocysteine are the only two selenium-containing amino acids in nature, and are also known as third generation varieties of selenium supplements. Compared with other forms of selenium-source foods, if the L-selenomethionine is used as a main source of selenium in animal foods, the L-selenomethionine has higher edible safety.
(4) The L-selenomethionine is mainly obtained by a chemical synthesis method, and the invention develops a biological fermentation method, so that the market customer acceptance is higher.
(5) The concentration of L-selenomethionine in the fermentation liquor is more than 70g/L, and the fermentation liquor has higher efficiency.
Definition of terms:
in the context of this document, all numbers disclosed herein are approximations, whether or not the word "about" or "about" is used. Based on the numbers disclosed, there is a possibility that the values of each number may differ by less than + -10% or a reasonable difference as recognized by those skilled in the art, such as + -1%, + -2%, + -3%, + -4%, or + -5%.
The term "and/or" is understood to mean any one of the selectable items or a combination of any two or more of the selectable items.
The term "wt%" refers to mass percent.
The term "% vol" means volume percent.
The term "aeration ratio" refers to the volume of gas introduced per unit volume of fermentation broth per unit time (typically per minute). For example, "1: a aeration ratio of 0.4 "i.e. 0.4L of gas was aerated per minute for 1L of fermentation broth. The term "inoculum size" in the expansion culture refers to the volume percentage of the volume of the medium containing C.glutamicum relative to the total volume of seed medium and medium containing C.glutamicum after inoculation. The term "inoculum size" in fermentation culture refers to the volume percentage of the seed solution to the total volume of the seed solution and the fermentation medium after the seed solution is inoculated into the fermentation medium.
The term "OD 600 "means the optical density at 600nm wavelength of detection
In order for those skilled in the art to better understand the teachings herein, certain non-limiting examples are further disclosed below to provide further details herein. The reagents used herein are all commercially available or can be prepared by the methods described herein. The term "rpm" means the rotational speed "revolutions per minute"; "°c" means the unit of temperature "degrees celsius"; "L" means the volume unit "liter"; "mL" means the volume unit "milliliter"; "pH" means pH value; "g" means the mass unit "gram"; "Mpa" means the unit of pressure "Mpa".
The formulation of the media used in the examples below, unless otherwise indicated, is as follows:
drawings
FIG. 1 shows the results of bioconversion of Corynebacterium glutamicum Corynebacterium glutamicum
FIG. 2 shows the results of bioconversion of Corynebacterium glutamicum Corynebacterium glutamicum OMK-88
Detailed Description
Table 1.Corynebacterium glutamicum OMK-88 solid seed Medium (g/L)
Table 2.Corynebacterium glutamicum OMK-88 seed Medium (g/L)
Table 3.Corynebacterium glutamicum OMK-88 fermentation Medium (g/L)
The inventor successfully screens out a strain Corynebacterium glutamicum which can biologically transform L-homoserine and methyl selenol (sodium) to generate L-selenomethionine. The laboratory adopts a single chemical mutagenesis reagent and a mixed chemical mutagenesis reagent to carry out multiple rounds of traditional chemical mutagenesis on the strain, and simultaneously adopts a reasonable and efficient strain screening mode to finally screen a mutant strain Corynebacterium glutamicum OMK-88 which can efficiently convert L-homoserine and methyl selenol or sodium methyl selenol into L-selenomethionine. And a corresponding L-selenomethionine fermentation synthesis method is developed according to the method.
Fermentation culture process
(1) Plate activation
And (3) refrigerating glycerol strains in a refrigerator at the temperature of minus 80 ℃, marking the strains on a beef extract peptone agar plate, and culturing the plate in a 37 ℃ incubator until the clones are full.
(2) Seed culture
Picking up monoclonal seeds from the activation plate, inoculating the monoclonal seeds into seed shake flasks, and culturing Corynebacterium glutamicum OMK-88 in shaking tables at 37 ℃ and 200rpm for 12 hours respectively; after microscopic examination of the sterile seeds, they were ready for transfer to seed shake flasks.
(3) Fermentation culture and bioconversion
Inoculating the shake flask seeds into a fermenter (shake flask) at 10% inoculum size, culturing at 37deg.C, automatically controlling pH at 6.8-7.0,Corynebacterium glutamicum OMK-88 with 30% sodium hydroxide solution for 12-16 hr, and measuring OD with spectrophotometer 600 When OD 600 No longer increases and enters the bioconversion stage. The substrate L-homoserine 70g/L and sodium methylselenate 70g/L or the substrate sodium methylselenate is added in batch. Every 4h, the sample is measured, and when the substrate reaction is complete or the reaction is not continued, the conversion is ended.
Example 1
In this example, the seed medium and fermentation medium were the same as the medium compositions and concentrations shown in tables 1, 2, and Corynebacterium glutamicum OMK-88, unless otherwise specified.
Preparation of plate medium:
washing, drying, sterilizing, placing the culture medium which is not solidified just after sterilization in a sterile box or an ultra-clean workbench, holding a triangular flask with the left hand, burning the triangular flask mouth by flame, pouring the sterilized or melted culture medium cooled to 55 ℃ of about 15mL, rapidly covering a dish cover, placing the dish on a table, lightly rotating the dish to ensure that the culture medium is uniformly distributed at the bottom of the whole dish, and condensing to obtain the flat-plate culture medium.
The culture medium comprises the following components: beef extract 3g/L, peptone 10g/L, agar 15-25g/L, sodium chloride 15g/L in this example, pH 7.4-7.6 in this example.
Seed preparation:
glycerol tubes Corynebacterium glutamicum OMK-88 were transferred to fresh, sterile beef extract peptone agar plates at-80℃under sterile conditions and incubated at 37℃for 24h (mycelium).
And (3) performing expansion culture:
the mycelium is picked up and inoculated in a 3L bioreactor filled with 1.8L seed culture medium, and the mycelium is cultured for 14 to 16 hours at 37 ℃ under the condition of ventilation and stirring at 300rpm, so as to obtain seed liquid.
The seed culture solution comprises 20g/L glucose, 15g/L yeast powder, 10g/L peptone, 2.0 potassium dihydrogen phosphate, 2.5g/L, pH =7.0 sodium chloride.
Fermentation and bioconversion:
preparing a fermentation medium, which comprises the following components: 30g/L glucose, 10.0g/L yeast powder, 1.0g/L peptone, 0.2g/L magnesium sulfate, 1.2g/L potassium dihydrogen phosphate, 0.8g/L sodium chloride, 5.0g/L sulfuric acid, 2.4g/L dipotassium hydrogen phosphate and 0.0001g/L manganese sulfate.
10.2L of the prepared fermentation medium is put into a 20L bioreactor, sterilization is carried out for 30min at 121 ℃, then 1.8L of the cultured seed liquid is inoculated into the 20L bioreactor for fermentation, the fermentation temperature is 37 ℃, the stirring rotation speed is 400rpm, and the aeration ratio is 1:0.4. after 20h of fermentation culture, the microorganism OD 600 No more increase, starting to add substrate L-homoserine 50g/L and sodium methylselenate 50g/L, and starting to enter into bioconversionIn the stage, the transformation is carried out for 16 hours. The concentration of L-selenomethionine in the fermentation broth was determined to be 70g/L by HPLC at the end of bioconversion.
Example 2
In this example, the seed medium and fermentation medium were the same as the medium compositions and concentrations shown in tables 1, 2, and Corynebacterium glutamicum OMK-88, unless otherwise specified.
Preparation of plate medium:
washing, drying, sterilizing, placing the culture medium which is not solidified just after sterilization in a sterile box or an ultra-clean workbench, holding a triangular flask with the left hand, burning the triangular flask mouth by flame, pouring about 15mL of the culture medium which is sterilized or melted and cooled to 60 ℃, rapidly covering a dish cover, placing the dish on a table, lightly rotating the dish to ensure that the culture medium is uniformly distributed at the bottom of the whole dish, and condensing to obtain the flat-plate culture medium.
The culture medium comprises the following components: beef extract 3g/L, peptone 10g/L, agar 15-25g/L, 25g/L in this example, sodium chloride 5g/L, and pH 7.4-7.6 in this example.
Seed preparation:
transferring glycerol Corynebacterium glutamicum OMK-88 at-80deg.C onto fresh, sterile beef extract peptone agar plate under aseptic condition, and culturing at 37deg.C for 24 hr.
And (3) performing expansion culture:
the mycelium is picked up and inoculated in a 3L bioreactor filled with 1.8L seed culture medium, and the seed liquid is obtained by culturing for 13 hours at 37 ℃ with the rotation speed of 400rpm of ventilation and stirring.
Fermentation and bioconversion:
10.2L of the prepared fermentation medium is put into a 20L bioreactor, sterilization is carried out for 30min at 121 ℃, then 1.8L of the cultured seed liquid is inoculated into the 20L bioreactor for fermentation, the fermentation temperature is 30 ℃, the stirring rotation speed is 500rpm, and the aeration ratio is 1:0.4. after 12h of fermentation, the microorganism OD 600 The addition of the substrate L-homoserine 70g/L and sodium methylselenate 70g/L is started, the transformation stage is started to enter, the transformation is carried out for 16 hours, and the transformation is ended until the concentration of the product L-selenomethionine is not increased. Determination of the end of bioconversion by HPLCThe concentration of L-selenomethionine in the fermentation broth is 91g/L.
While the methods of this application have been described in terms of preferred embodiments, it will be apparent to those of skill in the relevant art that variations and combinations of the methods and applications described herein can be made to practice and use the techniques of this application within the spirit and scope of the application. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included herein.
Claims (10)
1.Corynebacterium glutamicum, accession No.: cctccc M20222075.
2. Use of a corynebacterium glutamicum according to claim 1 for the preparation of L-selenomethionine, comprising fermenting a corynebacterium glutamicum according to claim 1 in a fermentation medium comprising a reaction substrate to obtain L-selenomethionine.
3. The use according to claim 2, wherein the fermentation substrate is any one or more of L-homoserine, sodium methylselenate or methylselenate.
4. Use according to claim 3, characterized in that the L-homoserine is 50-70g/L, sodium or selenol is 50-70g/L.
5. The use according to claim 2, wherein the fermentation medium comprises one or more of a carbon source comprising glucose, a nitrogen source comprising one or more of yeast powder, peptone, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, sodium chloride, ammonium sulfate, trace elements.
6. Use according to any of claims 2-5, characterized by the steps of:
s1: corynebacterium glutamicum is cultured in a plate medium,
s2: s1, performing primary or multistage culture and expansion culture on the flat mycelium,
s3: s2, performing biological transformation on the expansion culture seed solution.
7. The use according to claim 6, wherein the ratio of the seed liquid after one or more stages of culture to the volume of the bioconversion fermentation liquid is 0.9:10-0.9:20, preferably 0.9:15, particularly preferably 0.9: 13. 0.9: 12. 0.9: 11. 0.9:10.
8. the use according to any of claims 6 to 7, characterized in that the plate medium has a pH of 7.4 to 7.6, preferably 7.4 to 7.5, particularly preferably 7.5, and the rotational speed rpm of the expansion step is 300 to 500, preferably 300 to 400, particularly preferably 350, 400, the cultivation temperature is 36.5 to 37.5 ℃, preferably 36.5 to 37 ℃, particularly preferably 37 ℃, the cultivation time is 13 to 16h, preferably 14 to 16h, particularly preferably 15h. The fermentation temperature in the bioconversion step is 37 ℃, the stirring rotation speed is 400-500rpm, and the ventilation ratio is 1:0.1-1:0.4, preferably 1:0.1, 1:0.2, 1:0.3, 1:0.4, the bioconversion time is 12-20h, preferably 12-16h, particularly preferably 12h, 16h, the reaction substrate L-homoserine, sodium methylselenate or methylselenol being added in one portion or in portions.
9. The use according to any of claims 2 to 8, comprising, based on the total volume of the fermentation medium, 2 to 5g/L, preferably 3 to 5g/L, particularly preferably 3 to 4g/L, 1 to 10g/L, preferably 2g/L to 9g/L, particularly preferably 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10 to 30g/L, preferably 15 to 25g/L, particularly preferably 15g/L, 20g/L, 25g/L, 1.0 to 2.5g/L, preferably 1.2g/L to 2.0g/L, particularly preferably 1.2g/L, 1.5g/L, 2.0g/L of agar, 0.5g/L to 10g/L, preferably 0.8g/L to 5g/L, particularly preferably 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 20g/L, preferably 10g/L to 15g/L, particularly preferably 10g/L, 11g/L, 12g/L, 13g/L, 14g/L, 15g/L, 0.1 g to 0.3g/L, preferably 0.2g to 0.3g/L, particularly preferably 0.2g/L, 3g to 6g/L, preferably 4g to 5g/L, particularly preferably 5g/L, 2.0g to 2.6g/L, preferably 2.2 g to 2.4g/L, particularly preferably 2.4g/L, 0.0001g to 0.0003g/L, preferably 0.0001g/L, glucose 10-40g/L, preferably 20-30g/L, particularly preferably 20g/L, 25g/L, 30g/L, pH between 6.8-7.6, preferably 7, and an aqueous solvent.
10. An organic selenium prepared by the method of any of claims 2-9.
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