JPH0429354B2 - - Google Patents
Info
- Publication number
- JPH0429354B2 JPH0429354B2 JP25004184A JP25004184A JPH0429354B2 JP H0429354 B2 JPH0429354 B2 JP H0429354B2 JP 25004184 A JP25004184 A JP 25004184A JP 25004184 A JP25004184 A JP 25004184A JP H0429354 B2 JPH0429354 B2 JP H0429354B2
- Authority
- JP
- Japan
- Prior art keywords
- phenylalanine
- pyruvate kinase
- strain
- kinase activity
- fluorophenylalanine
- 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.)
- Expired
Links
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 30
- 229960005190 phenylalanine Drugs 0.000 claims description 27
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 24
- 102000013009 Pyruvate Kinase Human genes 0.000 claims description 14
- 108020005115 Pyruvate Kinase Proteins 0.000 claims description 14
- 230000000694 effects Effects 0.000 claims description 11
- 241000186146 Brevibacterium Species 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 3
- 230000003247 decreasing effect Effects 0.000 claims 1
- VWHRYODZTDMVSS-QMMMGPOBSA-N m-fluoro-L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC(F)=C1 VWHRYODZTDMVSS-QMMMGPOBSA-N 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 238000000034 method Methods 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 4
- 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 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 4
- 235000011130 ammonium sulphate Nutrition 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 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 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 241000319304 [Brevibacterium] flavum Species 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 239000011790 ferrous sulphate Substances 0.000 description 3
- 235000003891 ferrous sulphate Nutrition 0.000 description 3
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 3
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 235000019341 magnesium sulphate Nutrition 0.000 description 3
- 229940099596 manganese sulfate Drugs 0.000 description 3
- 239000011702 manganese sulphate Substances 0.000 description 3
- 235000007079 manganese sulphate Nutrition 0.000 description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 description 3
- 150000007524 organic acids Chemical class 0.000 description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- JZRWCGZRTZMZEH-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 3
- VWHRYODZTDMVSS-UHFFFAOYSA-N 2-azaniumyl-3-(3-fluorophenyl)propanoate Chemical compound OC(=O)C(N)CC1=CC=CC(F)=C1 VWHRYODZTDMVSS-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 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
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 229940068840 d-biotin Drugs 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000011785 micronutrient Substances 0.000 description 2
- 235000013369 micronutrients Nutrition 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 150000002993 phenylalanine derivatives Chemical class 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 239000013587 production medium Substances 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- -1 Alternatively Chemical class 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- HUNCSWANZMJLPM-UHFFFAOYSA-N 5-methyltryptophan Chemical compound CC1=CC=C2NC=C(CC(N)C(O)=O)C2=C1 HUNCSWANZMJLPM-UHFFFAOYSA-N 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
- 108010011485 Aspartame Proteins 0.000 description 1
- 241000807905 Corynebacterium glutamicum ATCC 14067 Species 0.000 description 1
- 229930091371 Fructose Natural products 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
- 239000005715 Fructose Substances 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 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
- 241000192130 Leuconostoc mesenteroides Species 0.000 description 1
- 241000192041 Micrococcus Species 0.000 description 1
- VZUNGTLZRAYYDE-UHFFFAOYSA-N N-methyl-N'-nitro-N-nitrosoguanidine Chemical compound O=NN(C)C(=N)N[N+]([O-])=O VZUNGTLZRAYYDE-UHFFFAOYSA-N 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-N Nitrous acid Chemical compound ON=O IOVCWXUNBOPUCH-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 239000000605 aspartame Substances 0.000 description 1
- IAOZJIPTCAWIRG-QWRGUYRKSA-N aspartame Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 IAOZJIPTCAWIRG-QWRGUYRKSA-N 0.000 description 1
- 235000010357 aspartame Nutrition 0.000 description 1
- 229960003438 aspartame Drugs 0.000 description 1
- 230000001580 bacterial effect Effects 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
- 230000004071 biological effect Effects 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007423 decrease 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
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 159000000014 iron salts Chemical class 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 150000002696 manganese Chemical class 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 229910021654 trace metal Chemical class 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 229960004441 tyrosine Drugs 0.000 description 1
- 150000003668 tyrosines Chemical class 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
〔発明の目的〕
〈産業上の利用分野〉
本発明は発酵法によるＬ−フエニルアラニン
（以下、フエニルアラニンと記す。）の製造法に関
する。フエニルアラニンはペプチド系甘味料であ
るアスパルテームの原料として重要である。
〈従来の技術〉
従来発酵法によるフエニルアラニンの製造法と
してはブレビバクテリウム属又はミクロコツカス
属細菌のＬ−チロシン要求菌を使用する方法（特
公昭37−6345）、フエニルアラニンアナログに耐
性を有する変異株を使用する方法（特公昭551−
28712）などが知られている。
〈本発明が解決しようとする問題点〉
本発明が解決しようとする問題点は更に安価に
フエニルアラニンを製造することにある。
〔発明の構成〕
〈問題点を解決するための手段〉
本発明者等は発酵法により更に安価にフエニル
アラニンを製造する方法を開発すべく研究を行な
つた結果、ブレビバクテリウム属細菌にフエニル
アラニン生産能とともにピルビン酸キナーゼ活性
の低下という性質を併せ持たせたところ、従来の
ピルビン酸キナーゼ活性の低下を持たないフエニ
ルアラニン生産菌より更に大量にフエニルアラニ
ンを生産することを見い出した。この発明はこの
知見に基づいて更に研究の結果完成されたもので
ある。
本発明のフエニルアラニン製造法において用い
られる微生物は、ブレビバクテリウム属に属しピ
ルビン酸キナーゼ活性が低下していてかつフエニ
ルアラニン生産能に必要な性質、例えばｍ−フロ
ロフエニルアラニン耐性を有する変異株である。
これらの性質の他に更にＬ−チロシン要求性、他
のフエニルアラニンアナログ耐性、チロシンアナ
ログ耐性、５−メチルトリプトフアン耐性、デコ
イニン感受性などを付与すると生産能を更に向上
させることができる。
本発明の変異株の親株は、いわゆるＬ−グルタ
ミン酸生産菌として知られているブレビバクテリ
ウム属の微生物であり、例えば次のような菌株を
あげることができる。
ブレビバクテリウム フラバム ATCC14067
ブレビバクテリウム デイバリカタム ATCC
14020
ブレビバクテリウム ラクトフアーメンタム
ATCC13869
ブレビバクテリウム ロゼウム ATCC 13825
本発明で用いる変異株はこれら上述の菌株を親
株として変異操作を施して、ピルビン酸キナーゼ
活性の低下及びフエニルアラニン生産能を付与す
ることによつて得られる。なお変異操作は通常の
方法、例えば紫外線照射或いはＮ−メチル−
N′−ニトロ−Ｎ−ニトロソグアニジン（以下、
NGと略す）、亜硝酸等の化学薬剤処理によつて
行なうことができる。
なおピルビン酸キナーゼ活性の低下した変異株
は親株を変異処理し変異処理した菌体のピルビン
酸キナーゼ活性を測定し、選択することによつて
得ることができる（特開昭58−170487）。
以下に本発明の使用菌株の一例ブレビバクテリ
ウムフラバムAJ−12179（FERM−P7913）の具
体的誘導方法を示す。即ち特願昭58−170487記載
のピルビン酸キナーゼ活性低下株ブレビバクテリ
ウムフラバムAJ−11955（FERM−P6665、
FERMBP−2178）を親株として、これよりフエ
ニルアラニンアナログ耐性株を誘導した。ブレビ
バクテリウムフラバムAJ−11955を150μg／mlの
NGで30℃15分間処理した（生残率2.0％）。つい
で第一表に示す合成培地に親株が生育できない濃
度の800μg／mlになるようにフエニルアラニンの
アナログの一つであるDL−ｍ−フロロフエニル
アラニンを添加して平板培地を作製し、これに変
異処理したAJ−11955株を塗布し、30℃10日間放
置後、コロニーとして生育する菌株即ちｍ−フロ
ロフエニルアラニン耐性変異株を採取し、このう
ちフエニルアラニン生産能のすぐれた変異株AJ
−12179（FERM−P7913）（ピルビン酸キナーゼ
低下、ｍ−フロロフエニルアラニン耐性、Ｓ−
（２−アミノエチル）−Ｌ−システイン耐性）を採
取した。この変異株は実施例に示すようにピルビ
ン酸キナーゼ活性を有するｍ−フロロフエニルア
ラニン耐性フエニルアラニン生産変異株AJ−
12180（FERM−P7914）よりフエニルアラニンを
1.8倍も多く生成した。
次にこの操作によつて得られた変異株AJ−
12179のピルビン酸キナーゼ活性及びｍ−フロロ
フエニルアラニンに対する耐性の度合を親株AJ
−11955、野生株ATCC−14067、ピルビン酸キナ
ーゼ活性を有するフエニルアラニン生産株AJ−
12180と比較した結果をそれぞれ第二表、第三表
に示した。
なお、ピルビン酸キナーゼ活性の測定はAgric.
Biol.Chem.、48、(5)1189〜1197（1984）に従つて
行なつた。またｍ−フロロフエニルアラニンに対
する耐性の度合は次のように調べた。第一表に示
す合成培地にDL−ｍ−フロロフエニルアラニン
を第三表に示した濃度になるように溶解して、平
板培地（直径8.5cm）を作り、各々の培地に第四
表に示した培地に生育した菌をおよそ107コ接種
した後30℃で４日間培養を行ない生成したコロニ
ー数を調べた。
第二表及び第三表に示すようにAJ−12179株は
ピルビン酸キナーゼの低下及びｍ−フロロフエニ
ルアラニンに対して耐性の両性質を有しているこ
とが判明した。なお本発明で言うｍ−フロロフエ
ニルアラニン耐性とは上記培養条件下でDL−ｍ
−フロロフエニルアラニンを3000μg／ml含む上
記培地に微生物をおよそ107コ接種し30℃で４日
間培養後1000コ以上のコロニーを生成する場合を
言う。
フエニルアラニン生産用の培養培地は特に制限
するところはなく、炭素源、窒素源、無機塩及び
必要ならば有機微量栄養素を含有する通常の培地
である。炭素源としては炭水化物（グルコース、
フラクトース或いはデンプン、セルロース等の加
水分解物、糖蜜等）、有機酸（酢酸、クエン酸
等）、アルコール（グリセリン、エタノール等）、
或いは炭化水素（ノルマルパラフイン等）が使用
できる。窒素源としては硫酸アンモニウム、尿
素、硝酸アンモニウム、リン酸アンモニウム、塩
化アンモニウム、アンモニアガス、その他を、無
機塩としてはリン酸塩、マグネシウム塩、カルシ
ウム塩、鉄塩、マンガン塩、その他微量金属塩等
を必要に応じて使用する。有機微量栄養素として
は、栄養要求性のある場合には該当するアミノ
酸、ビタミン、脂肪酸類、有機塩基物質等を適量
添加し、必要に応じて更に生育促進物質としてア
ミノ酸、ビタミン、味液（登録商標、大豆加水分
解物）、酵母エキス、ペプトン、カザミノ酸等が
使用できる。
培養条件は通常の方法でPH５ないし９、温度は
20ないし40℃で好気的条件下に24ないし72時間培
養すれば良い。培養中にPHが下がる場合には炭酸
カルシウムを別殺菌して加えるか又はアンモニア
水、アンモニアガス等のアルカリで中和する。又
有機酸を炭素源とする場合はPHの上昇を鉱酸又は
有機酸で中和する。
フエニルアラニンの単離採取は常法によつて行
いうる。得られたものはペーパークロマトグラム
のRf値及び微生物定量法による生物活性値によ
り、フエニルアラニン標品のそれらと一致するこ
とを確めフエニルアラニンと同定した。
フエニルアラニンの定量はロイコノストツクメ
センテロイデス（ATCC8042）を用いる微生物定
量法に従つて行なつた。
以下、実施例にて説明する。
実施例 １
下記第五表に示した組成のフエニルアラニン生
産用培地20mlを500mlのフラスコに分注し、これ
に第六表に示す微生物をそれぞれ１白金耳植えつ
け30℃で72時間振盪培養した。それぞれの培養液
中のフエニルアラニン生成量は第六表の如くであ
つた。
第一表 合成培地組成
グルコース 20g／
硫酸アンモニウム ５ 〃
尿 素 ２〃
リン酸１カリウム １ 〃
硫酸マグネシウム １ 〃
硫酸第１鉄 10mg／
硫酸マンガン ８ 〃
ｄ−ビオチン 50μg／
ビタミンB1・塩酸塩 2000μg／ 寒 天 20g／
PH6.6
[Object of the Invention] <Industrial Application Field> The present invention relates to a method for producing L-phenylalanine (hereinafter referred to as phenylalanine) by a fermentation method. Phenylalanine is an important raw material for aspartame, a peptide sweetener. <Prior art> Conventional fermentation methods for producing phenylalanine include a method using L-tyrosine-requiring bacteria of the genus Brevibacterium or Micrococcus (Japanese Patent Publication No. 37-6345); Method of using mutant strains with
28712) are known. <Problems to be Solved by the Present Invention> The problems to be solved by the present invention are to produce phenylalanine at a lower cost. [Structure of the Invention] [Means for Solving the Problems] The present inventors conducted research to develop a method for producing phenylalanine at a lower cost by a fermentation method, and as a result, they discovered that Brevibacterium bacteria By combining the ability to produce phenylalanine with the property of reducing pyruvate kinase activity, we discovered that it can produce even more phenylalanine than conventional phenylalanine-producing bacteria that do not have the ability to reduce pyruvate kinase activity. Ta. This invention was completed as a result of further research based on this knowledge. The microorganism used in the method for producing phenylalanine of the present invention belongs to the genus Brevibacterium, has reduced pyruvate kinase activity, and has properties necessary for the ability to produce phenylalanine, such as resistance to m-fluorophenylalanine. It is a mutant strain.
In addition to these properties, production ability can be further improved by imparting L-tyrosine requirement, resistance to other phenylalanine analogs, resistance to tyrosine analogs, resistance to 5-methyltryptophan, sensitivity to decoinine, etc. The parent strain of the mutant strain of the present invention is a microorganism of the genus Brevibacterium known as a so-called L-glutamic acid producing bacterium, and examples thereof include the following strains. Brevibacterium flavum ATCC14067 Brevibacterium deivaricata ATCC
14020 Brevibacterium lactofamentum
ATCC13869 Brevibacterium roseum ATCC 13825 The mutant strains used in the present invention can be obtained by mutating the above-mentioned bacterial strains as parent strains to impart a reduction in pyruvate kinase activity and the ability to produce phenylalanine. The mutation operation can be carried out using conventional methods, such as ultraviolet irradiation or N-methyl-
N'-nitro-N-nitrosoguanidine (hereinafter referred to as
(abbreviated as NG), can be carried out by chemical treatment such as nitrous acid. Mutant strains with reduced pyruvate kinase activity can be obtained by mutating the parent strain, measuring the pyruvate kinase activity of the mutated cells, and selecting (Japanese Patent Application Laid-Open No. 170487-1987). A specific method for inducing Brevibacterium flavum AJ-12179 (FERM-P7913), which is an example of the strain used in the present invention, is shown below. Specifically, the pyruvate kinase activity-decreasing strain Brevibacterium flavum AJ-11955 (FERM-P6665,
FERMBP-2178) was used as the parent strain, from which a phenylalanine analog-resistant strain was derived. Brevibacterium flavum AJ-11955 at 150μg/ml
Treated with NG at 30°C for 15 minutes (survival rate 2.0%). Next, DL-m-fluorophenylalanine, which is an analog of phenylalanine, was added to the synthetic medium shown in Table 1 at a concentration of 800 μg/ml, which is a concentration at which the parent strain cannot grow, to prepare a plate medium. The mutation-treated strain AJ-11955 was applied to this, and after it was left at 30°C for 10 days, the strains that grew as colonies, that is, m-fluorophenylalanine-resistant mutants, were collected. Stock AJ
-12179 (FERM-P7913) (low pyruvate kinase, m-fluorophenylalanine resistance, S-
(2-aminoethyl)-L-cysteine resistance) was collected. This mutant strain is a m-fluorophenylalanine-resistant phenylalanine-producing mutant strain AJ- having pyruvate kinase activity, as shown in Examples.
Phenylalanine from 12180 (FERM-P7914)
It produced 1.8 times more. Next, the mutant strain AJ− obtained by this operation
The pyruvate kinase activity of 12179 and the degree of resistance to m-fluorophenylalanine were compared with the parent strain AJ.
-11955, wild strain ATCC-14067, phenylalanine producing strain AJ with pyruvate kinase activity
The results of comparison with 12180 are shown in Tables 2 and 3, respectively. Please note that pyruvate kinase activity was measured by Agric.
Biol. Chem., 48, (5) 1189-1197 (1984). Furthermore, the degree of resistance to m-fluorophenylalanine was examined as follows. Dissolve DL-m-fluorophenylalanine in the synthetic medium shown in Table 1 to the concentration shown in Table 3 to make a plate medium (diameter 8.5 cm), and add the contents shown in Table 4 to each medium. Approximately 10 7 of the grown bacteria were inoculated onto the indicated medium and cultured at 30°C for 4 days, and the number of colonies produced was determined. As shown in Tables 2 and 3, strain AJ-12179 was found to have both the properties of reduced pyruvate kinase and resistance to m-fluorophenylalanine. In the present invention, m-fluorophenylalanine resistance refers to DL-m resistance under the above culture conditions.
- This refers to the case where approximately 10 7 microorganisms are inoculated into the above medium containing 3000 μg/ml of fluorophenylalanine and more than 1000 colonies are produced after culturing at 30°C for 4 days. The culture medium for phenylalanine production is not particularly limited, and is a conventional medium containing a carbon source, a nitrogen source, inorganic salts and, if necessary, organic micronutrients. Carbohydrates (glucose,
fructose or starch, hydrolysates of cellulose, molasses, etc.), organic acids (acetic acid, citric acid, etc.), alcohols (glycerin, ethanol, etc.),
Alternatively, hydrocarbons (normal paraffin, etc.) can be used. Nitrogen sources include ammonium sulfate, urea, ammonium nitrate, ammonium phosphate, ammonium chloride, ammonia gas, and others; inorganic salts include phosphates, magnesium salts, calcium salts, iron salts, manganese salts, and other trace metal salts. Use accordingly. As organic micronutrients, appropriate amounts of amino acids, vitamins, fatty acids, organic basic substances, etc. are added if there is a nutritional requirement, and if necessary, amino acids, vitamins, and flavor liquid (registered trademark) are added as growth-promoting substances. , soybean hydrolyzate), yeast extract, peptone, casamino acids, etc. can be used. Culture conditions are normal, pH 5 to 9, temperature.
It may be cultured under aerobic conditions at 20 to 40°C for 24 to 72 hours. If the pH decreases during culture, add calcium carbonate after sterilization or neutralize with alkali such as aqueous ammonia or ammonia gas. In addition, when an organic acid is used as a carbon source, the increase in pH is neutralized with a mineral acid or an organic acid. Phenylalanine can be isolated and collected by conventional methods. The obtained product was confirmed to be phenylalanine based on the Rf value of the paper chromatogram and the biological activity value determined by the microbial quantitative method, and it was confirmed that it matched that of the phenylalanine standard. Phenylalanine was quantified according to a microbial quantification method using Leuconostocmesenteroides (ATCC8042). Examples will be described below. Example 1 20 ml of a phenylalanine production medium having the composition shown in Table 5 below was dispensed into 500 ml flasks, and one platinum loopful of each of the microorganisms shown in Table 6 was inoculated into each flask and cultured with shaking at 30°C for 72 hours. did. The amount of phenylalanine produced in each culture solution was as shown in Table 6. Table 1 Synthetic medium composition Glucose 20g / Ammonium sulfate 5 Urea 2 Monopotassium phosphate 1 Magnesium sulfate 1 Ferrous sulfate 10mg / Manganese sulfate 8 d-biotin 50μg / Vitamin B 1 , hydrochloride 2000μg / Cold Heaven 20g/ PH6.6
【表】【table】
【表】 第四表 シード培養培地 グルコース 20g／ 硫酸アンモニウム 10g／ 尿 素 3g／ リン酸１カリウム 1g／ 硫酸マグネシウム 0.4g／ 硫酸第１鉄 10mg／ 硫酸マンガン ８mg／ ｄ−ビオチン 300μg／ ビタミンB1・塩酸塩 100μg／ PH7.3 第五表 フエニルアラニン生産用培地 グルコース 130g／ 硫酸アンモニウム 25g／ フマル酸 12g／ 酢 酸 ３ml／ リン酸１カリウム 1g／ 硫酸マグネシウム 1g／ 硫酸第１鉄 10mg／ 硫酸マンガン ８mg／ ｄ−ビオチン 200μg／ ビタミンB1・塩酸塩 2000μg／ 「味液」 50ml／ 炭酸カルシウム（別殺菌） 50g／ PH6.5[Table] Table 4 Seed culture medium Glucose 20g / Ammonium sulfate 10g / Urea 3g / Monopotassium phosphate 1g / Magnesium sulfate 0.4g / Ferrous sulfate 10mg / Manganese sulfate 8mg / d-biotin 300μg / Vitamin B 1 , hydrochloric acid Salt 100μg / PH7.3 Table 5 Phenylalanine production medium Glucose 130g / Ammonium sulfate 25g / Fumaric acid 12g / Acetic acid 3ml / Monopotassium phosphate 1g / Magnesium sulfate 1g / Ferrous sulfate 10mg / Manganese sulfate 8mg / d -Biotin 200μg / Vitamin B 1 / Hydrochloride 2000μg / “Taste liquid” 50ml / Calcium carbonate (separately sterilized) 50g / PH6.5
Claims (1)
ナーゼ活性が低下し、かつＬ−フエニルアラニン
生産能を有する微生物を液体培地中に好気的に培
養し、培養液中にＬ−フエニルアラニンを生成蓄
積せしめ、これを採取することを特徴とするＬ−
フエニルアラニンの製造法。1. A microorganism that belongs to the genus Brevibacterium and has a decreased pyruvate kinase activity and has the ability to produce L-phenylalanine is cultivated aerobically in a liquid medium to produce L-phenylalanine in the culture solution. L- characterized by accumulating and collecting this
Method for producing phenylalanine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP25004184A JPS61128897A (en) | 1984-11-27 | 1984-11-27 | Production of l-phenylalanine by fermentation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP25004184A JPS61128897A (en) | 1984-11-27 | 1984-11-27 | Production of l-phenylalanine by fermentation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS61128897A JPS61128897A (en) | 1986-06-16 |
| JPH0429354B2 true JPH0429354B2 (en) | 1992-05-18 |
Family
ID=17201942
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP25004184A Granted JPS61128897A (en) | 1984-11-27 | 1984-11-27 | Production of l-phenylalanine by fermentation method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS61128897A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2202607C2 (en) | 2000-03-03 | 2003-04-20 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" | Strain of bacterium methylophilus methylotrophus as producer of l-phenylalanine (variants), method of l-phenylalanine preparing |
| CN103319349A (en) * | 2013-07-02 | 2013-09-25 | 南京大学 | Production process of m-nitrophenylamine |
-
1984
- 1984-11-27 JP JP25004184A patent/JPS61128897A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS61128897A (en) | 1986-06-16 |
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