CS202884B1 - Preparation method for l-lysine by the fermentation - Google Patents
Preparation method for l-lysine by the fermentation Download PDFInfo
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- CS202884B1 CS202884B1 CS913678A CS913678A CS202884B1 CS 202884 B1 CS202884 B1 CS 202884B1 CS 913678 A CS913678 A CS 913678A CS 913678 A CS913678 A CS 913678A CS 202884 B1 CS202884 B1 CS 202884B1
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- Prior art keywords
- fermentation
- lysine
- ethanol
- acetic acid
- soil
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- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 title claims description 30
- 238000000855 fermentation Methods 0.000 title claims description 25
- 230000004151 fermentation Effects 0.000 title claims description 25
- 238000002360 preparation method Methods 0.000 title claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 24
- 235000019766 L-Lysine Nutrition 0.000 claims description 14
- 239000004472 Lysine Substances 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- 238000004519 manufacturing process Methods 0.000 claims description 10
- 239000002689 soil Substances 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 229930006000 Sucrose Natural products 0.000 claims description 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 7
- 229910052799 carbon Inorganic materials 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- UKAUYVFTDYCKQA-UHFFFAOYSA-N -2-Amino-4-hydroxybutanoic acid Natural products OC(=O)C(N)CCO UKAUYVFTDYCKQA-UHFFFAOYSA-N 0.000 claims description 5
- UKAUYVFTDYCKQA-VKHMYHEASA-N L-homoserine Chemical compound OC(=O)[C@@H](N)CCO UKAUYVFTDYCKQA-VKHMYHEASA-N 0.000 claims description 5
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 5
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims description 5
- 230000001419 dependent effect Effects 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- DXRKKNDPLKMIOR-UHFFFAOYSA-N 2-(aminomethyl)butanethioic S-acid Chemical compound CCC(CN)C(S)=O DXRKKNDPLKMIOR-UHFFFAOYSA-N 0.000 claims description 3
- 239000011707 mineral Substances 0.000 claims description 3
- 235000010755 mineral Nutrition 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 2
- 238000009630 liquid culture Methods 0.000 claims description 2
- 125000000218 acetic acid group Chemical class C(C)(=O)* 0.000 claims 1
- 239000002609 medium Substances 0.000 description 7
- 241000186226 Corynebacterium glutamicum Species 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 241000186216 Corynebacterium Species 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 150000001242 acetic acid derivatives Chemical class 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000012499 inoculation medium Substances 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 235000017060 Arachis glabrata Nutrition 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000018262 Arachis monticola Nutrition 0.000 description 1
- 241000186249 Corynebacterium sp. Species 0.000 description 1
- GHSJKUNUIHUPDF-BYPYZUCNSA-N L-thialysine Chemical compound NCCSC[C@H](N)C(O)=O GHSJKUNUIHUPDF-BYPYZUCNSA-N 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- ZXVOCOLRQJZVBW-UHFFFAOYSA-N azane;ethanol Chemical compound N.CCO ZXVOCOLRQJZVBW-UHFFFAOYSA-N 0.000 description 1
- ALSPKRWQCLSJLV-UHFFFAOYSA-N azanium;acetic acid;acetate Chemical compound [NH4+].CC(O)=O.CC([O-])=O ALSPKRWQCLSJLV-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 125000000741 isoleucyl group Chemical class [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 239000007793 ph indicator Substances 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
Vynález se týká způsobu fermentační přípravy L-lysinu, a to kultivací kmenů Corynebacterium produkujících L-lysin v tekuté živné půdě, která obsahuje na počátku fermentace jako hlavní zdroj uhlíku soli kyseliny octové, komplexní zdroje dusíku >a minerální živné soli.The present invention relates to a process for the fermentative preparation of L-lysine by culturing L-lysine-producing Corynebacterium strains in a liquid nutrient broth which initially contains acetic acid salts, complex nitrogen sources and mineral nutrient salts as the main carbon source.
Je známo více způsobů fermentační přípravy L-lysinu, při kterých se původně používaný zdroj asimilovatelného uhlíku, tj. sacharosa, nahrazuje jinými levnějšími a dostupnějšími zdroji, i za cenu o něco nižší produkce L-lýsinu. Tak například je popsána fermentace kmene Corynebacterium glutamicum v půdě s ethanolem jako jediným zdrojem uhlíku pro růst produkčního mikroorganismu a produkci L-lysinu a močovinou a NZ-aminem /obchodní produkt neurčeného složení/ jako zdroji dusíku. V průběhu fermentace se do půdy dávkuje směs ethanolu a močoviny v určitých poměrech v závislosti na pH-indikátoru, který je přítomen ve fermentačním médiu /australský pat. spis.č. 51577/.Several methods for fermenting L-lysine are known in which the initially used assimilable carbon source, i.e. sucrose, is replaced by other cheaper and more affordable sources, even at the cost of somewhat lower L-lysine production. For example, fermentation of the Corynebacterium glutamicum strain in soil with ethanol as the sole carbon source for the growth of the producing microorganism and the production of L-lysine and urea and NZ-amine (a commercial product of unspecified composition) as a nitrogen source is described. During fermentation, a mixture of ethanol and urea is added to the soil in certain proportions depending on the pH indicator present in the fermentation medium / Australian pat. spis.č. 51577 /.
Analogický postup s použitím fermentačního média jiného složení uvádí americký pat. spis č. 3 595 751. Dále je popsán technologický postup biosyntézy L-lysinu s produkčními kmeny Brevibacterium lactofermentum, užívající jako zdroje uhlíku a dusíku směsi kyselina octová-octan amonný nebo ethanol-čpavek /NSR pat. spis č. 2 321 461/.. Aktuální koncentrace ethanolu ve fermentačním médiu je sledována plynovou čhromatografii.An analogous procedure using a fermentation medium of a different composition is disclosed in U.S. Pat. No. 3,595,751. Further, a technological process for L-lysine biosynthesis with production strains of Brevibacterium lactofermentum using acetic acid-ammonium acetate or ethanol-ammonia / NSR pat. The actual concentration of ethanol in the fermentation broth is monitored by gas chromatography.
líkolem vynálezu bylo najít vhodné zdroje asimilovatelného uhlíku, které by pokud možno nahradily v produkční fázi drahou a deficitní sacharosu bez podstatného zásahu do obvyklé technologie a bez podstatného snížení výtěžků L-lysinu. Podařilo se vypracovat způsob fermentační přípravy L-lysinu kultivací kmenů Corynebacterium produkujících L-lysin v tekuté živné půdě obsahující na počátku fermentace jako hlavní zdroj uhlíku soli kyseliny octové, komplexní zdroje dusíku a minerální živné soli, podle vynálezu, jehož podstata spočívá V tom, že se v průběhu fermentace přidává do půdy po dosažení hodnoty pH 7,8 až 8,0 ve více dávkách obsahující 600 ml ethanolu, 100 ml kyseliny octové, 66 g. sacharosy a vodu do objemu 1 000 ml, v takových časových intervalech, aby koncentrace ethanolu v půdě činila 0,60 až 0,75 Z hmot. a hodnota pH 7,0 až 7,2, načež se po dosažení maxima produkce fermentace ukončí. Jako kmeneThe object of the invention was to find suitable sources of assimilable carbon which would, if possible, replace the pathway and deficient sucrose in the production phase without substantially interfering with conventional technology and without substantially reducing the yields of L-lysine. We have succeeded in developing a process for the fermentation preparation of L-lysine by culturing L-lysine-producing Corynebacterium strains in a liquid culture medium containing acetic acid salts, complex nitrogen sources and mineral nutrient salts as the main carbon source according to the invention. is added to the soil during the fermentation after reaching a pH of 7.8 to 8.0 in multiple portions containing 600 ml of ethanol, 100 ml of acetic acid, 66 g of sucrose and water to a volume of 1000 ml, at intervals such that % of ethanol in the soil was 0.60 to 0.75 wt. and a pH of 7.0 to 7.2, after which the fermentation production is terminated after reaching the maximum. Like a tribe
Corynebacterium, vhodného pro fermentací podle vynálezu, lze použít kmene Corynebacterium glutamicum, dependentního na homoserin a leucin a rezistentního k S-aminoethylcyateinu, popřípadě kmene, který je dependentní na homoserin a leucin a rezistentní k aminomethylthiomáselné kyselině.Corynebacterium suitable for fermentation according to the invention may be a strain of Corynebacterium glutamicum dependent on homoserine and leucine and resistant to S-aminoethylcyatein, or a strain which is dependent on homoserine and leucine and resistant to aminomethylthiobutyric acid.
Podle povahy použitého produkčního kmene lze způsobem podle vynálezu dosáhnout po 72 hodinách fermentace produkce L—lysinu'16 až 26 g/litr média, při podstatné úspoře sacharosy·Depending on the nature of the production strain used, the production of L-lysine 16 to 26 g / liter of medium can be achieved after 72 hours of fermentation with a substantial saving of sucrose.
Bližší podrobnosti jsou patrny z příkladů provedení.Further details can be seen from the examples.
Příklady provedení:Examples:
PřikladlHe did
Kmenem Corynebacterium sp. se zaoěkuje 60 ml inokulační půdy, umístěné v 500 ml varné baňce, tohoto složení:The strain Corynebacterium sp. Inoculate 60 ml of inoculum broth, contained in a 500 ml flask, of the following composition:
octan sodný 1 , 5 g sacharosa 2,0 g kukuřičný extrakt /60Zní/ 3,0 g voda do 100,0 ml pH půdy před sterilizací 7,2, sterilizace 30 minut při 120 °C, Po zaočkování se kultivuje na rotační třepačce /240 ot/min/ 18 hodin při teplotě 29 °C. Kulturou vyrostlou v baňce se zaoěkuje 800 ml půdy ve dvoulitrových fermentačních tancích, jež má toto složení:sodium acetate 1,5 g sucrose 2,0 g corn extract (60Zní / 3,0 g water to 100,0 ml pH of soil before sterilization 7,2, sterilization for 30 minutes at 120 ° C, After inoculation, cultivate on a rotary shaker / 240 rpm / 18 hours at 29 ° C. Inoculate the culture grown in the flask with 800 ml of soil in two-liter fermentation tanks having the following composition:
pH půdy před sterilizací 7,2, sterilizace 30 minut při 120 °C. Po zaočkování se kultivuje při teplotě 29 až 30 °C, vzdušnění stejným objemem vzduchu/min, míchání 800 ot/min. V průběhu fermentace se po dosažení pH 7,8 až 8,0 dávkuje v intervalech po 6 hodinách a v dávkách po ml ethanolová směs tohoto složení: ethanol 600 ml, kyselina octová 100 ml, sacharosa 66 g, voda do 1 000 ml. Během 72 hodin fermentace se do tanků přidá celkem ,00 ml uvedené směsi, která obsahuje zdroje uhlíku v tomto celkovém množství /přepočítáno na 1 litr média/: ethanol g, kyselina octová 11 g a sacha.rosa 7 g. Produkce L-lysinu po 72 hodinách fermentace činí až 18 g/litr fermentačního média.pH of the soil before sterilization 7.2, sterilization for 30 minutes at 120 ° C. After inoculation, it is cultured at 29-30 ° C, aerated with the same volume of air / min, stirring at 800 rpm. During the fermentation, an ethanol mixture of the following composition: ethanol 600 ml, acetic acid 100 ml, sucrose 66 g, water to 1000 ml, are dosed at intervals of 6 hours and in ml portions after fermentation. During 72 hours of fermentation, a total of 00 ml of said mixture containing carbon sources in this total amount (calculated on 1 liter of medium) was added to the tanks: ethanol g, acetic acid 11 g and sucrose 7 g. up to 18 g / liter of fermentation medium.
Příklad 2Example 2
Kmenem Corynebacterium glutamicum, dependentním na homoserin a leucin a rezistentním k analogu lysinu, S-aminoethylcysteinu, se zaoěkuje 60 ml inokulačního média, umístěného v 500 ml varné baňce, stejného složení jako v příkladu 1 a kultivuje se za stejných-podmínek jako v tomto příkladu. Laboratorní dvoulitrové fermentační tanky se naplní po 800 ml fermentačního média stejného složení jako v tomto příkladu. Analogicky se dávkuje ethanolová směs.A lysine analog, S-aminoethylcysteine, resistant to homoserine and leucine-dependent strain Corynebacterium glutamicum is inoculated with 60 ml of inoculation medium in a 500 ml boiling flask of the same composition as in Example 1 and cultured under the same conditions as in this example. . Laboratory 2-liter fermentation tanks are filled with 800 ml of fermentation medium of the same composition as in this example. The ethanol mixture is dosed analogously.
Po 72 hodinách fermentace se proces ukončí. Produkce L—lysinu činí 21 až 26 g/litr fermentační ho média.After 72 hours of fermentation, the process is terminated. The production of L-lysine is 21-26 g / liter of fermentation medium.
Příklad 3Example 3
Kmenem Corynebacterium glutamicum, dependentním na homoserin a leucin a rezistentním k analogu isoleucinu, aminomethylthiomáselné kyselině, se zaoěkuje 60 ml inokulačního média umístěného v 500 ml varné baňce, stejného složení jak,o v příkladu 1 a dále se kultivuje zaA strain of Corynebacterium glutamicum, dependent on homoserine and leucine and resistant to the isoleucine analogue, aminomethylthiobutyric acid, is inoculated with 60 ml of inoculation medium placed in a 500 ml boiling flask of the same composition as in Example 1 and further cultured under
Stejných podmínek jako V tomto příkladu. Obdobně se vede fermentace ve 21 laboratorních dvoulitrových tancích, rovněž i dávkování ethanolové směsi. Produkce L-lysinu po 72 hodinách fermentace činí 19 až 21 g/litr.Same conditions as in this example. Similarly, fermentation was carried out in 21 laboratory 2-liter tanks as well as the ethanol mixture. The production of L-lysine after 72 hours of fermentation is 19 to 21 g / liter.
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Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS913678A CS202884B1 (en) | 1978-12-29 | 1978-12-29 | Preparation method for l-lysine by the fermentation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS913678A CS202884B1 (en) | 1978-12-29 | 1978-12-29 | Preparation method for l-lysine by the fermentation |
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| Publication Number | Publication Date |
|---|---|
| CS202884B1 true CS202884B1 (en) | 1981-02-27 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CS913678A CS202884B1 (en) | 1978-12-29 | 1978-12-29 | Preparation method for l-lysine by the fermentation |
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| Country | Link |
|---|---|
| CS (1) | CS202884B1 (en) |
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1978
- 1978-12-29 CS CS913678A patent/CS202884B1/en unknown
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