JPS63209593A - Production of l-threonine by fermentation method - Google Patents
Production of l-threonine by fermentation methodInfo
- Publication number
- JPS63209593A JPS63209593A JP4233687A JP4233687A JPS63209593A JP S63209593 A JPS63209593 A JP S63209593A JP 4233687 A JP4233687 A JP 4233687A JP 4233687 A JP4233687 A JP 4233687A JP S63209593 A JPS63209593 A JP S63209593A
- Authority
- JP
- Japan
- Prior art keywords
- threonine
- acetic acid
- producing
- culture
- salt
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 title claims abstract description 78
- 238000000034 method Methods 0.000 title claims description 14
- 238000004519 manufacturing process Methods 0.000 title claims description 9
- 238000000855 fermentation Methods 0.000 title claims description 4
- 230000004151 fermentation Effects 0.000 title claims description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 54
- 239000004473 Threonine Substances 0.000 claims abstract description 43
- 229960002898 threonine Drugs 0.000 claims abstract description 43
- 150000003839 salts Chemical class 0.000 claims abstract description 11
- 244000005700 microbiome Species 0.000 claims description 13
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 5
- 235000011054 acetic acid Nutrition 0.000 abstract description 18
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 10
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 7
- 229910052799 carbon Inorganic materials 0.000 abstract description 7
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 abstract description 6
- 239000005695 Ammonium acetate Substances 0.000 abstract description 6
- 235000019257 ammonium acetate Nutrition 0.000 abstract description 6
- 229940043376 ammonium acetate Drugs 0.000 abstract description 6
- 241000894006 Bacteria Species 0.000 abstract description 5
- 241000588722 Escherichia Species 0.000 abstract description 5
- 229940024606 amino acid Drugs 0.000 abstract description 5
- 150000001413 amino acids Chemical class 0.000 abstract description 5
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 5
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 abstract description 4
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 abstract description 3
- 239000001632 sodium acetate Substances 0.000 abstract description 3
- 235000017281 sodium acetate Nutrition 0.000 abstract description 3
- 241000588724 Escherichia coli Species 0.000 abstract description 2
- 239000012535 impurity Substances 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 abstract description 2
- 235000011056 potassium acetate Nutrition 0.000 abstract description 2
- 239000006228 supernatant Substances 0.000 abstract description 2
- 235000015097 nutrients Nutrition 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 12
- 235000002639 sodium chloride Nutrition 0.000 description 8
- 238000012258 culturing Methods 0.000 description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 5
- 239000013587 production medium Substances 0.000 description 5
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 150000007524 organic acids Chemical class 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- GMKMEZVLHJARHF-UHFFFAOYSA-N (2R,6R)-form-2.6-Diaminoheptanedioic acid Natural products OC(=O)C(N)CCCC(N)C(O)=O GMKMEZVLHJARHF-UHFFFAOYSA-N 0.000 description 3
- LGVJIYCMHMKTPB-UHFFFAOYSA-N 3-hydroxynorvaline Chemical compound CCC(O)C(N)C(O)=O LGVJIYCMHMKTPB-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 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 description 3
- 239000004201 L-cysteine Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- GMKMEZVLHJARHF-SYDPRGILSA-N meso-2,6-diaminopimelic acid Chemical compound [O-]C(=O)[C@@H]([NH3+])CCC[C@@H]([NH3+])C([O-])=O GMKMEZVLHJARHF-SYDPRGILSA-N 0.000 description 3
- 235000005985 organic acids Nutrition 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 241000186146 Brevibacterium Species 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 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
- 239000001888 Peptone Chemical class 0.000 description 2
- 108010080698 Peptones Chemical class 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 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
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- OJCKRNPLOZHAOU-RSXXJMTFSA-N (1r,2r)-2-[(2s,4e,6e,8r,9s,11r,13s,15s,16s)-7-cyano-8,16-dihydroxy-9,11,13,15-tetramethyl-18-oxo-1-oxacyclooctadeca-4,6-dien-2-yl]cyclopentane-1-carboxylic acid Chemical compound O1C(=O)C[C@H](O)[C@@H](C)C[C@@H](C)C[C@@H](C)C[C@H](C)[C@@H](O)\C(C#N)=C\C=C\C[C@H]1[C@H]1[C@H](C(O)=O)CCC1 OJCKRNPLOZHAOU-RSXXJMTFSA-N 0.000 description 1
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 125000000022 2-aminoethyl group Chemical group [H]C([*])([H])C([H])([H])N([H])[H] 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- OJCKRNPLOZHAOU-BNXNOGCYSA-N Borrelidin Natural products CC1CC(C)CC(C)C(O)C(=C/C=C/CC(OC(=O)CC(O)C(C)C1)C2CCCC2C(=O)O)C#N OJCKRNPLOZHAOU-BNXNOGCYSA-N 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- 108010043075 L-threonine 3-dehydrogenase Proteins 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 150000001243 acetic acids Chemical class 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 150000003863 ammonium salts Chemical class 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
- 239000003674 animal food additive Substances 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 229940111685 dibasic potassium phosphate Drugs 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- GVALZJMUIHGIMD-UHFFFAOYSA-H magnesium phosphate Chemical compound [Mg+2].[Mg+2].[Mg+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O GVALZJMUIHGIMD-UHFFFAOYSA-H 0.000 description 1
- 239000004137 magnesium phosphate Substances 0.000 description 1
- 229960002261 magnesium phosphate Drugs 0.000 description 1
- 229910000157 magnesium phosphate Inorganic materials 0.000 description 1
- 235000010994 magnesium phosphates Nutrition 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical class [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 239000006174 pH buffer Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明は、L−スレオニン生産能を有する微生物を酢酸
を添加した培地中で培養するL−スレオニンの製造法に
関する。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for producing L-threonine by culturing a microorganism capable of producing L-threonine in a medium supplemented with acetic acid.
L−スレオニンは、アミノ酸製剤などの医薬品として有
用であるだけでな(、飼料添加用としても利用できるア
ミノ酸である。L-threonine is an amino acid that is not only useful as a medicine such as amino acid preparations (but also can be used as a feed additive).
従来の技術
従来、発酵法によるL−スレオニンの製造法としては、
エッシエリヒア属に属し、ボレリジン感受性を示す微生
物を用いる方法(特公昭51−6752号公報)、エツ
シェリヒア属に属し、ジアミノピメリン酸とメチオニン
の要求性を示し、かつスレオニンの生合成系カスレオニ
ンのフィードバック阻害に対して抵抗性を示す微生物を
用いる方法(特公昭56−10037号公報)、セラチ
ア属に属し、スレオニン脱水素酵素が欠損し、かつスレ
オニン代謝拮抗物質に耐性を示す微生物を用いる方法(
特公昭52−48195号公報)、コリネバクテリウム
属に属し、α−アミノ−β−ヒドロキシ吉草酸およびS
−(2−アミノエチル)−L−システィンに耐性で、か
つメチオニンの要求性を有する微生物を用いる方法(特
開昭47−19087号公報)、ブレビバクテリウム属
に属し、α−アミノ−β−ヒドロキシ吉草酸および5−
(2−アミノエチル)−L−システィンに耐性で、かつ
ロイシンの要求性を有する微生物を用いる方法(特開昭
50−31093号公報)、ブレビバクテリウム属に属
し、α−アミノ−β−ヒドロキシ吉草酸および5−(2
−アミノエチル)−L−システィンに耐性で、かつL−
イソロイシンおよびリジンの要求性ををする微生物を用
いる方法(特開昭58−224684号公報)、L−ス
レオニン生産能を有する微生物をフマール酸、マレイン
酸およびリンゴ酸より選ばれる有機酸の1つ以上と糖類
を有機酸1モルに対し糖類1モルから8モルの比率で含
有する培地中に培養する方法(特開昭58−14969
0号公報)などが知られている。Conventional technology Conventionally, the method for producing L-threonine by fermentation method is as follows:
A method using a microorganism belonging to the genus Escherichia that is sensitive to borrelidin (Japanese Patent Publication No. 51-6752); a microorganism that belongs to the genus Escherichia and exhibits a requirement for diaminopimelic acid and methionine; (Japanese Patent Publication No. 56-10037), a method using a microorganism belonging to the genus Serratia that is deficient in threonine dehydrogenase, and is resistant to threonine antimetabolites (
(Japanese Patent Publication No. 52-48195), belongs to the genus Corynebacterium, and contains α-amino-β-hydroxyvaleric acid and S
A method using a microorganism resistant to -(2-aminoethyl)-L-cysteine and auxotrophic for methionine (Japanese Unexamined Patent Publication No. 19087/1987), which belongs to the genus Brevibacterium and has α-amino-β- Hydroxyvaleric acid and 5-
A method using a microorganism resistant to (2-aminoethyl)-L-cysteine and auxotrophic for leucine (Japanese Unexamined Patent Publication No. 50-31093), which belongs to the genus Brevibacterium and is an α-amino-β-hydroxy Valeric acid and 5-(2
-aminoethyl)-L-cysteine and L-
A method using microorganisms that require isoleucine and lysine (Japanese Unexamined Patent Publication No. 58-224684), a method using microorganisms capable of producing L-threonine with one or more organic acids selected from fumaric acid, maleic acid, and malic acid. A method of culturing in a medium containing 1 mol of organic acid and saccharides at a ratio of 1 to 8 mol of saccharides (Japanese Patent Laid-Open No. 14969/1983)
Publication No. 0) and the like are known.
発明が解決I〜ようとする問題点
アミノ酸製剤などとして有用なL−スレオニンを、より
収率よく安価に製造する方法が求められている。Problems to be Solved by the Invention There is a need for a method for producing L-threonine, which is useful as an amino acid preparation, at a higher yield and at a lower cost.
問題点を解決するための手段
本発明者は、L−スレオニンを高収率で得るためにより
優れたL−スレオニン生産条件の研究を行った。その結
果L−スレオニン生産能を有する微生物を糖類を主炭素
源とする培地に酢酸を添加した培地で培養することによ
り、L−スレオニンの生産性を向上させることができる
ことを見出し本発明を完成した。Means for Solving the Problems The present inventor conducted research on better L-threonine production conditions in order to obtain L-threonine in high yield. As a result, they discovered that L-threonine productivity could be improved by culturing microorganisms capable of producing L-threonine in a medium containing sugar as the main carbon source and acetic acid added, and completed the present invention. .
以下に本発明の詳細な説明する。The present invention will be explained in detail below.
本発明は、L−スレオニン生産能を有する微生物を、酢
酸またはその塩を0.2〜5g/l添加した培地に培養
し、培養物中にL−スレオニンを生成蓄積させ、該培養
物よりL−スレオニンを採取することを特徴とする発酵
法によるL−スレオニンの製造法を提供する。The present invention involves culturing a microorganism capable of producing L-threonine in a medium supplemented with 0.2 to 5 g/l of acetic acid or its salt, producing and accumulating L-threonine in the culture, and producing L-threonine from the culture. - Provides a method for producing L-threonine by a fermentation method characterized by collecting threonine.
本発明に使用する微生物の例としては、L−スレオニン
生産能を有する微生物であればいずれも用いることがで
きる。具体的には、エッシェリヒア・コ’JATCC2
1530などがあげられる。As an example of the microorganism used in the present invention, any microorganism can be used as long as it has the ability to produce L-threonine. Specifically, Escherichia Co'JATCC2
Examples include 1530.
エッシェリヒア属のスレオニン生産菌においては、副生
のアミノ酸が少ないという利点がある。Threonine-producing bacteria of the genus Escherichia have the advantage of having a small amount of by-product amino acids.
L−スレオニン生産菌を炭素源、窒素源、無機塩類、生
育因子などを含有する合成培地または天然培地に酢酸ま
たはその塩を添加した培地を用いて培養することにより
、L−スレオニンを培養物中にM積させ、これを採取す
ることにより■7−スレオニンを製造することができる
。By culturing L-threonine-producing bacteria in a synthetic medium or natural medium containing carbon sources, nitrogen sources, inorganic salts, growth factors, etc., and acetic acid or its salts added, L-threonine can be produced in the culture. (1) 7-threonine can be produced by accumulating M and collecting this.
炭素源としては、グルコース、フラクトース、糖蜜、澱
粉加水分解物などの糖類を主な炭素源として用いる。As the carbon source, sugars such as glucose, fructose, molasses, and starch hydrolyzate are used as the main carbon source.
窒素源としては、アンモニア、塩化アンモニウム、硫酸
アンモニウム、燐酸アンモニウムなどの各種無機塩類や
有機酸のアンモニウム塩、アミン類、その他の含窒素化
合物、ならびにペプトン、肉エキス、コーン・ステイー
プ・リカー、カゼイン加水分解物、大豆粕加水分解物、
各種発酵菌体およびその消化物などが用いられる。Nitrogen sources include various inorganic salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate, ammonium salts of organic acids, amines, and other nitrogen-containing compounds, as well as peptone, meat extract, corn steep liquor, and casein hydrolysis. Soybean meal hydrolyzate,
Various fermented microbial cells and their digested products are used.
無機塩類としては、燐酸第一カリウム、燐酸第二カリウ
ム、燐酸マグネシウム、硫酸マグネシウム、塩化ナトリ
ウム、硫酸第一鉄、硫酸マンガン、硫酸銅、炭酸カルシ
ウムなどが用いられる。As the inorganic salts, potassium phosphate, dibasic potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc. are used.
培地に加える酢酸の添加濃度は0.2〜5g/βが好ま
しい。また酢酸アンモニウム、酢酸ナトリウム、酢酸カ
リウムなどの塩として添加することもできる。酢酸は合
成品、天然物いずれも使用でき、純品でなくても酢酸を
含有している物質であればいずれも使用できる。The concentration of acetic acid added to the medium is preferably 0.2 to 5 g/β. It can also be added as a salt such as ammonium acetate, sodium acetate, potassium acetate, or the like. Both synthetic and natural acetic acids can be used, and any substance containing acetic acid can be used even if it is not a pure product.
培養は振盪培養または深部通気撹拌培養などの好気的条
件下で行う。培養温度は20=40℃、好ましくは28
〜38℃の範囲である。培地のpHは5〜9の範囲で、
好ましくは中性付近に保持する。培地のpH調整は炭酸
カルシウム、無機または有機の酸、アルカリ溶液、アン
モニア、pH緩衝液などによって行う。Cultivation is performed under aerobic conditions such as shaking culture or deep aeration agitation culture. Culture temperature is 20=40℃, preferably 28℃
-38°C. The pH of the medium is in the range of 5 to 9,
Preferably, it is maintained near neutrality. The pH of the medium is adjusted using calcium carbonate, inorganic or organic acids, alkaline solutions, ammonia, pH buffers, and the like.
培養期間は通常2〜7日間で培養物中にL−スレオニン
が生成蓄積する。The culture period is usually 2 to 7 days, and L-threonine is produced and accumulated in the culture.
培養終了後、培養液から菌体などの沈澱物を除去し、イ
オン交換処理法、濃縮法、塩析法などを併用することに
より、培養液からL−スレオニンを回収することができ
る。After completion of the culture, L-threonine can be recovered from the culture solution by removing precipitates such as bacterial cells from the culture solution and using an ion exchange treatment method, a concentration method, a salting out method, etc. in combination.
従来、炭素源として酢酸を、窒素源として酢酸アンモニ
ウムなどの塩類を使用できることが知られている。しか
し、この場合の酢酸の添加濃度は、10g/β以上であ
る。It has been known that acetic acid can be used as a carbon source and salts such as ammonium acetate can be used as a nitrogen source. However, the concentration of acetic acid added in this case is 10 g/β or more.
本発明は、酢酸または酢酸アンモニウムを炭素源または
窒素源として用いるのではなく、L−スレオニン生産菌
を培養するのに用いる合成培地または天然培地(ただし
、酢酸は含有しない培地)に酢酸を0.2〜5g/βに
なるように添加することにより、より収率よくL−スレ
オニンを製造できることを示している。The present invention does not use acetic acid or ammonium acetate as a carbon source or nitrogen source, but instead uses 0.0% acetic acid in a synthetic medium or a natural medium (but not containing acetic acid) used for culturing L-threonine producing bacteria. It is shown that L-threonine can be produced with higher yield by adding 2 to 5 g/β.
以下に実施例を示す。Examples are shown below.
実施例1
種菌としてエッシェリヒア・コリATCC21530株
を用いり、−スレオニン生産試験を行った。ATCC2
1530株をグルコース2%、ペプトン1%、酵母エキ
ス1%、Na(1! 0.25%、ジアミノピメリン
酸0.1g/j!の組成の種培地(pH7,4)で30
℃、16時間振盪培養した。得られた種培養液2mlを
、下記の組成の生産培地に第1表に示した濃度になるよ
うに酢酸を添加した培地23m1を含む25 Qmlの
三角フラスコにそれぞれ植菌し、30℃で72時間振盪
培養した。Example 1 A -threonine production test was conducted using Escherichia coli ATCC 21530 strain as the inoculum. ATCC2
1530 strain was grown in a seed medium (pH 7.4) with a composition of 2% glucose, 1% peptone, 1% yeast extract, Na (1! 0.25%, and diaminopimelic acid 0.1 g/j!) for 30 minutes.
The cells were cultured with shaking at ℃ for 16 hours. 2 ml of the obtained seed culture solution was inoculated into 25 Qml Erlenmeyer flasks each containing 23 ml of a production medium with the following composition and acetic acid added to the concentration shown in Table 1, and incubated at 30°C for 72 ml. Cultured with shaking for hours.
そのときのL−スレオニン蓄積量を第1表に示す。Table 1 shows the amount of L-threonine accumulated at that time.
第 1 表 生産培地の組成は次のとおりである。Chapter 1 Table The composition of the production medium is as follows.
グルコース7%、(N H4) a 3041.4%、
KH,PO40,2%、M g S 04・7H,OO
,1%、ジアミノピメリン酸0.3g/l、DL−,1
オニン0.1g/Cコーン・ステイープ・リカー〇、2
%、CaC0a 3%(pH7,4)酢酸1 g/f
を含む生産培地を用いて上記のようにして得たL−スレ
オニン含有培養液20 Qmlを遠心分離(3000r
pm、10分間)にかけ、菌体その他の不純物を除去し
た。得られた上澄液を強酸性イオン交換樹脂ダイヤイオ
ン5KI(H”型)(三菱化成工業社製)のカラムに通
し、L−スレオニンを吸着させ、水洗後0.5規定のア
ンモニア水で溶出して、L−スレオニン画分を集めた。Glucose 7%, (NH4) a 3041.4%,
KH, PO40, 2%, M g S 04.7H, OO
,1%, diaminopimelic acid 0.3g/l, DL-,1
Onin 0.1g/C Corn Steep Liquor 〇, 2
%, CaC0a 3% (pH 7,4) Acetic acid 1 g/f
20 Qml of the L-threonine-containing culture solution obtained as above using the production medium containing
pm for 10 minutes) to remove bacterial cells and other impurities. The obtained supernatant liquid was passed through a column of strongly acidic ion exchange resin Diaion 5KI (H” type) (manufactured by Mitsubishi Chemical Industries, Ltd.) to adsorb L-threonine, and after washing with water, it was eluted with 0.5N ammonia water. The L-threonine fraction was collected.
集めた両分を濃縮し、エタノールを加えて冷却下で保存
することにより、純度98%以上のL−スレオニンの結
晶が1.9g得られた。Both collected fractions were concentrated, ethanol was added, and the mixture was stored under cooling to obtain 1.9 g of L-threonine crystals with a purity of 98% or higher.
実施例2
酢酸の代わりに、酢酸ナトリウムまたは酢酸アンモニウ
ムを生産培地に添加する以外は、実施例1と同様にして
L−スレオニン生産試験を行った。Example 2 An L-threonine production test was conducted in the same manner as in Example 1, except that sodium acetate or ammonium acetate was added to the production medium instead of acetic acid.
酢酸す) IJウムおよび酢酸アンモニウムは、第2表
、第3表に示したような濃度になるように、酢酸に換算
して生産培地に添加した。IJ and ammonium acetate were added to the production medium in terms of acetic acid so that the concentrations were as shown in Tables 2 and 3.
L−スレオニン蓄積量を第2表および第3表に示す。The amount of L-threonine accumulated is shown in Tables 2 and 3.
第 2 表
第 3 表
発明の効果
本発明により、収率よくL−スレオニンを得ることがで
きる。Table 2 Table 3 Effects of the Invention According to the present invention, L-threonine can be obtained in good yield.
Claims (1)
の塩を0.2〜5g/l添加した培地に培養し、培養物
中にL−スレオニンを生成蓄積させ、該培養物よりL−
スレオニンを採取することを特徴とする発酵法によるL
−スレオニンの製造法。A microorganism capable of producing L-threonine is cultured in a medium supplemented with 0.2 to 5 g/l of acetic acid or its salt, L-threonine is produced and accumulated in the culture, and L-threonine is produced and accumulated in the culture.
L produced by a fermentation method characterized by collecting threonine
- A method for producing threonine.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP4233687A JPS63209593A (en) | 1987-02-25 | 1987-02-25 | Production of l-threonine by fermentation method |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP4233687A JPS63209593A (en) | 1987-02-25 | 1987-02-25 | Production of l-threonine by fermentation method |
Publications (1)
Publication Number | Publication Date |
---|---|
JPS63209593A true JPS63209593A (en) | 1988-08-31 |
Family
ID=12633169
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP4233687A Pending JPS63209593A (en) | 1987-02-25 | 1987-02-25 | Production of l-threonine by fermentation method |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS63209593A (en) |
-
1987
- 1987-02-25 JP JP4233687A patent/JPS63209593A/en active Pending
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