JPS63248393A - Production of d-pipecolic acid by microorganism - Google Patents
Production of d-pipecolic acid by microorganismInfo
- Publication number
- JPS63248393A JPS63248393A JP8172487A JP8172487A JPS63248393A JP S63248393 A JPS63248393 A JP S63248393A JP 8172487 A JP8172487 A JP 8172487A JP 8172487 A JP8172487 A JP 8172487A JP S63248393 A JPS63248393 A JP S63248393A
- Authority
- JP
- Japan
- Prior art keywords
- pipecolic acid
- acid
- ferm
- pipecolic
- culturing
- 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.)
- Granted
Links
- HXEACLLIILLPRG-RXMQYKEDSA-N D-pipecolic acid Chemical compound OC(=O)[C@H]1CCCCN1 HXEACLLIILLPRG-RXMQYKEDSA-N 0.000 title claims abstract description 57
- 244000005700 microbiome Species 0.000 title claims abstract description 17
- 238000004519 manufacturing process Methods 0.000 title claims description 5
- HXEACLLIILLPRG-UHFFFAOYSA-N pipecolic acid Chemical compound OC(=O)C1CCCCN1 HXEACLLIILLPRG-UHFFFAOYSA-N 0.000 claims abstract description 23
- 241000192041 Micrococcus Species 0.000 claims abstract description 8
- 241000589516 Pseudomonas Species 0.000 claims abstract description 7
- 238000012258 culturing Methods 0.000 claims abstract description 7
- 230000002950 deficient Effects 0.000 claims abstract description 3
- 241000588986 Alcaligenes Species 0.000 claims abstract 2
- 239000007864 aqueous solution Substances 0.000 abstract description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 4
- 230000000813 microbial effect Effects 0.000 abstract description 4
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 2
- 241000186809 Kurthia Species 0.000 abstract 1
- 238000005273 aeration Methods 0.000 abstract 1
- 229910017053 inorganic salt Inorganic materials 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 16
- 230000001580 bacterial effect Effects 0.000 description 14
- 239000002609 medium Substances 0.000 description 12
- 238000000034 method Methods 0.000 description 10
- 239000002253 acid Substances 0.000 description 8
- 239000006916 nutrient agar Substances 0.000 description 8
- 239000006285 cell suspension Substances 0.000 description 7
- 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 5
- HXEACLLIILLPRG-YFKPBYRVSA-N L-pipecolic acid Chemical compound [O-]C(=O)[C@@H]1CCCC[NH2+]1 HXEACLLIILLPRG-YFKPBYRVSA-N 0.000 description 5
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 5
- 150000001342 alkaline earth metals Chemical class 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 230000003287 optical effect Effects 0.000 description 5
- 102000016938 Catalase Human genes 0.000 description 4
- 108010053835 Catalase Proteins 0.000 description 4
- 102000004316 Oxidoreductases Human genes 0.000 description 4
- 108090000854 Oxidoreductases Proteins 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000000049 pigment Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 238000003794 Gram staining Methods 0.000 description 3
- 241001148470 aerobic bacillus Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000001766 physiological effect Effects 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 210000004209 hair Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical class N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 238000009631 Broth culture Methods 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- 102000004674 D-amino-acid oxidase Human genes 0.000 description 1
- 108010003989 D-amino-acid oxidase Proteins 0.000 description 1
- 241000282596 Hylobatidae Species 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
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 159000000001 potassium salts Chemical class 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000005062 synaptic transmission Effects 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
（１）産業上の利用分野
本発明は、微生物を利用して光学的に活性なり一ピペコ
リン酸を調製する方法に関する。DETAILED DESCRIPTION OF THE INVENTION (1) Industrial Application Field The present invention relates to a method for preparing optically active monopipecolic acid using microorganisms.
Ｄ−ピペコリン酸は中枢神経系のシナプス伝達の調節な
どそれ自身も薬理活性を有する他、いくつかの抗生物質
やアルカロイド系薬剤の修飾用合成中間体としての利用
が期待されている。D-pipecolic acid itself has pharmacological activities such as regulating synaptic transmission in the central nervous system, and is also expected to be used as a synthetic intermediate for modifying several antibiotics and alkaloid drugs.
（２）従来の技術
しかしながら、Ｄ−ピペコリン酸を安価、且つ大量に供
する方法は未だ確立していない。光学分割剤を用いてＤ
−Ｌ−ピペコリン酸からＤ−ピペコリン酸を得る方法が
現在のところ採られているが、光学分割剤が高価であり
、且つその供給に難点がある為、この方法ではＤ−ピペ
コリン酸を安価、且つ大量に供することはできない。ち
なみに、この従来法で得たＤ−とベコリン酸の価格はＤ
・Ｌ−ピペコリン酸の１００倍にも及び、このような状
況はＤ−ピペコリン酸の工業的利用は言うまでもなく、
用途開発をも制約している。これに対しＤ−Ｌ−ピペコ
リン酸は安価であり、且つ大量に製造されているので、
このＤ−Ｌ−ピペコリン酸からＤ−ピペコリン酸を簡便
に分離することができれば、Ｄ−ピペコリン酸を安価、
且つ大量に製造することが可能になる。(2) Prior Art However, a method for providing D-pipecolic acid at low cost and in large quantities has not yet been established. D using optical resolving agent
-Currently, a method is used to obtain D-pipecolic acid from L-pipecolic acid, but the optical resolving agent is expensive and there are difficulties in supplying it. Moreover, it cannot be provided in large quantities. By the way, the price of D- and becolic acid obtained by this conventional method is D
・It is 100 times more expensive than L-pipecolic acid, and this situation makes it difficult to use D-pipecolic acid industrially.
It also restricts the development of applications. On the other hand, DL-pipecolic acid is inexpensive and produced in large quantities, so
If D-pipecolic acid can be easily separated from this DL-pipecolic acid, D-pipecolic acid can be produced at low cost.
Moreover, it becomes possible to manufacture in large quantities.
（３）発明が解決しようとする問題点
そこで、本発明者は、このような従来法に代わるＤ−と
ベコリン酸の製法を探索した結果、Ｄ−ピペコリン酸資
化能を実質的に欠損し、Ｌ−ピペコリン酸資化能を有す
る微生物を新たに自然界より分離し、このような微生物
をＤ−Ｌ−ピペコリン酸に作用させると、し−ピペコリ
ン酸はこれらの微生物により資化され消滅するのに対し
、Ｄ−ピペコリン酸は資化されることなく残在すること
を見出した。(3) Problems to be solved by the invention Therefore, as a result of searching for a method for producing D- and becolic acid in place of the conventional method, the present inventor found a method that substantially lacks the ability to assimilate D-pipecolic acid. If microorganisms that have the ability to assimilate L-pipecolic acid are newly isolated from the natural world and these microorganisms are allowed to act on D-L-pipecolic acid, the s-pipecolic acid will be assimilated by these microorganisms and disappear. On the other hand, it was found that D-pipecolic acid remained without being assimilated.
本発明は、この知見に基づき、完成されたものである。The present invention was completed based on this knowledge.
すなわち、本発明はミクロコツカス属、クルシア属、シ
ュードモナス属、アルカリ土類金属より選ばれ、且つＤ
−ピペコリン酸資化能が実質的に欠損し、Ｌ−ピペコリ
ン酸資化能を有する微生物をＤ−Ｌ−ピペコリン酸含有
培地で培養し、Ｄ−ピペコリン酸を分割・採取すること
を特徴とするＤ−ピペコリン酸の製造法に関するもので
ある。That is, the present invention is selected from the genus Micrococcus, the genus Crucia, the genus Pseudomonas, and alkaline earth metals, and
- Cultivating microorganisms that are substantially deficient in the ability to assimilate pipecolic acid and have the ability to assimilate L-pipecolic acid in a medium containing DL-pipecolic acid, and dividing and collecting D-pipecolic acid. The present invention relates to a method for producing D-pipecolic acid.
（４）問題点を解決するための手段
本発明に適用される微生物は、Ｌ−ピペコリン酸は活発
に資化するが、Ｄ−ピペコリン酸は全く資化しないか、
或いは極めて弱くしか資化しない微生物であれば、いず
れでも用いることができる。(4) Means for solving the problem The microorganisms applied to the present invention actively assimilate L-pipecolic acid but do not assimilate D-pipecolic acid at all, or
Alternatively, any microorganism that can be assimilated only extremely weakly can be used.
このような微生物を例示すれば本発明者らが自然界から
分離したミクロコツカスＮｏ、 ７Ｂ菌、クルシア属Ｎ
ｏ、１１３Ｂ菌、シュードモナスＮｏ、８５２０Ｂ菌。Examples of such microorganisms include Micrococcus No. 7B, which the present inventors isolated from the natural world, and Crucia genus N.
o, 113B bacteria, Pseudomonas No., 8520B bacteria.
アルカリ土類金属Ｎｏ、３０９８１菌が挙げられる。Examples include alkaline earth metal No. 30981 bacteria.
以下これらの微生物の菌学的性質は以下に示すとおりで
ある。The mycological properties of these microorganisms are shown below.
Ｏミクロコツカス属Ｎｏ、７Ｂ菌
（ａ）形態
一直径１μ霧程度の球菌、しばしは４個で１クラスター
を形成（栄養寒天上で生育した菌体）
・ ダラム染色：陽性
一運動性 ：無し
一芽胞形成能：無し
くｂ）栄養寒天培地における生育状態
−生育：良好
一生育状態は拡布状
− １屡の隆起ば偏平状
一光沢は純光
一色素生産性；無し
くｃ）生理学的性質
一好気性菌
一カタラーゼ：生産性有り
一オキシダーゼ：生産性無し
・ Ｏ−Ｆテスト、Ｍ比的
−グルコースより酸を生成する
以上の特徴はＲ，Ｅ、Ｂｕｃｈａｎａｕ＋ Ｎ、 ［
＋、Ｇｉｂｂｏｎｓ編パージエイズ マニュアル オブ
デタミネイティブ バクテリオロン４８版ｒＢｅｒｇ
ｅｙ・ｓ ｍａｎｕａｌｏｆ Ｄｅｒｔｅｒｍｉｎａｔ
ｉｖｅ Ｂａｃｔｅｒｉｏｌｏｇｙ ８ｔｈ Ｅｄｉｔ
ｉｏｎＪ及び長谷用武治編「微生物の分類と同定、第１
版」（学会出版センター、１９８１年）に記載のミクロ
コツカス属の特徴と一致している。O Micrococcus No. 7B Bacteria (a) Morphology - Cocci with a diameter of about 1 μm, often forming a cluster of 4 (bacterial bodies grown on nutrient agar) - Durham staining: positive - Motile: none - Spores Formation ability: None b) Growth condition on nutrient agar medium - Growth: Good - Growth condition is spread - 1 lot of ridges, flattened, glossy - Pure light - Pigment productivity; None c) Physiological properties - Aerobic Bacterial Catalase: Productive Oxidase: No Productivity O-F test, M ratio - Produces acid from glucose The above characteristics are R, E, Buchanau + N, [
+, Edited by Gibbons Purge Aids Manual of Determinative Bacteriolon 48th edition rBerg
ey・s manualof Derterminat
ive Bacteriology 8th Edit
ionJ and Takeharu Hase, “Classification and Identification of Microorganisms, Volume 1”
This is consistent with the characteristics of the genus Micrococcus described in "Hanban" (Gakkai Publishing Center, 1981).
本国をミクロコツカス属Ｎｏ 、 ７Ｂ菌と命名しＦＢ
Ｉ’１Ｍ Ｐ−Ｎｏ、９３１１として寄託されている。The home country was named Micrococcus genus No. 7B bacterium and posted on FB.
It has been deposited as I'1M P-No. 9311.
○ クルシア属Ｎｏ、 １１３８菌
（ａ）形態
−直径１μ閤、長さ２μ階程度の桿菌（栄養寒天培地上
で生育した菌体）
−グラム染色ｌ１性
一運動性であり、周ペン毛を存する
一芽胞形成能：無し
くｂ）栄養寒天培地上における生育状態・生育良好
一生育状態は拡布状
一菌層の隆起は偏平状
一光沢は純光
一色素生産性：無し
くｃ） 生理学的性質
一好気性菌
−カタラーゼ：生産性有り
−オキシダーゼ：生産性態し
・ グルコースから酸を生成しない
−０−Ｆテスト；グルコースを分解しない以上の特徴は
前記の２書に記載されているクルシア属（Ｋｕｒｔｈｉ
ａ属）の特徴と一致する。○ Crucia genus No. 1138 (a) Morphology - Bacillus with a diameter of 1 μm and length of about 2 μm (bacterium grown on a nutrient agar medium) – Gram staining, l1, monomotile, and has peristaltic hairs 1) Spore forming ability: None b) Growth condition on nutrient agar medium: Good growth 1) Growth condition is spread-like 1) Bacteria layer is flattened 1) Gloss is pure light 1) Pigment productivity: None c) Physiological properties 1 Aerobic bacteria - Catalase: Productive - Oxidase: Productive state - Does not produce acid from glucose - 0-F test; Does not decompose glucose The above characteristics are described in the above two books.
The characteristics match those of genus a).
本菌をクルシア属Ｎｏ、１１３Ｂ菌と命名し、ＦＥＲＭ
Ｐ−Ｎｏ。This bacterium was named Crucia genus No. 113B bacterium, and FERM
P-No.
９３１２として寄託されている。It has been deposited as No. 9312.
Ｏシュードモナス５２０Ｂ菌
（ａ）形態
一直径約１μ曙、長さ約２μ−程度の桿菌（栄養寒天上
で生育した菌体）
−グラム染色：陰性
−運動性であり、１本の極ベン毛を有する一芽胞形成能
：無し
くｂ）栄養寒天培地上での生育状態
−生育良好
一生育状態は拡布状
−菌屡の隆起は偏平状
一光沢は純光
一色素生産性：無し
くｃ）生理学的性質
一好気性菌
−カタラーゼ：生産性をり
・ オキシダーゼ：生産性有り
−グルコースより酸を生成する
− Ｏ−Ｆテスト：酸化的
以上の特徴は前記の２書に記載されているシュードモナ
ス属（Ｐｓｅｕｄｏ剛ｏｎａｓ属）の特徴と一致してい
る。Pseudomonas 520B bacterium (a) Morphology: A rod with a diameter of approximately 1 μm and a length of approximately 2 μm (bacterial body grown on nutrient agar) – Gram staining: negative – Motile, with one polar trichome. Spore-forming ability: None b) Growth condition on nutrient agar medium - Good growth - Growth condition is spread-like - Bacterial bulges are flat - Gloss is pure light - Pigment productivity: None c) Physiological Characteristics: Aerobic bacteria - Catalase: Productive - Oxidase: Productive - Produces acid from glucose - O-F test: Characteristics above oxidative This is consistent with the characteristics of the genus Goonas).
本国をシュードモナス５２０Ｂ菌と命名し、ＦＥＲＭ
Ｐ−Ｎｏ。The home country was named Pseudomonas 520B, and FERM
P-No.
９３１３として寄託されている。It has been deposited as No. 9313.
○ アルカリ土類金属Ｎｏ、３０９Ｂ１菌（ａ）形態
・ 直径約約１μｍ、長さ２μ蹟程度の桿菌（栄養寒天
上で生育した菌体）
−グラム染色：陰性
・ 運動性であり、周ペン毛を有する
一芽胞形成能：無し
くｂ）栄養寒天培地上での生育状態
−生育良好
一生育状態は拡布状
一菌屡の隆起は偏平状
・光沢は純光
・色素生産性：無し
くｃ）生理学的性質
一好気性菌
−カタラーゼ：生産性有り
−オキシダーゼ：生産性有り
−グルコースより酸を生成しない
−〇−Ｆテスト：グルコースを分解しない以上の特徴は
前記の２書に記載されているアルカリ土類金属（Ａｌｃ
ａｌｉｇｅｎｅｓ属）の特徴と一致している。○ Alkaline earth metal No. 309B1 bacteria (a) Morphology: Rod bacteria approximately 1 μm in diameter and 2 μm in length (bacteria grown on nutrient agar) - Gram staining: negative - Motile, with peripencil hairs Spore forming ability: No b) Growth condition on nutrient agar medium - Good growth; Growth condition is spread-like; protuberances of many bacteria are flat; gloss is pure light; Pigment productivity: no c) Physiological properties - Aerobic bacteria - Catalase: Productive - Oxidase: Productive - Does not produce acid from glucose - ○ - F test: Does not decompose glucose The above characteristics are described in the above two books. Earth metal (Alc
The characteristics are consistent with those of the genus Aligenes).
本国をアルカリ土類金属Ｎｏ、３０９８１と命名し、Ｆ
ＥＲＭＰ−９３１４として寄託されている。The home country is named alkaline earth metal No. 30981, and F
It has been deposited as ERMP-9314.
これらのいずれかの微生物をＤ−Ｌ−ピペコリン酸に作
用させることによりＤ−ピペコリン酸が得られるが、培
養及び回収は通常、次の方法が挙げられることができる
。D-pipecolic acid can be obtained by allowing any of these microorganisms to act on DL-pipecolic acid, and the following methods can usually be used for culturing and recovery.
Ｄ−Ｌ−ピペコリン酸の０．１〜３０％水溶液に、窒素
源としてペプトン、肉エキス、酵母エキス。A 0.1-30% aqueous solution of DL-pipecolic acid, peptone as a nitrogen source, meat extract, yeast extract.
コーンステイープリカー、アンモニア塩類、尿素等の有
機、および無機窒素含存物、その他カリウム塩類、リン
酸塩ｆｉ、’ｉ、−／グネシウム塩類、カルシウム塩類
等の無機塩類等を補い培地とし、この培地で前記の微生
物を培養する方法である。尚、この場合の培地の組成、
及び培養条件には何等限定はない。The medium is supplemented with organic and inorganic nitrogen-containing substances such as corn staple liquor, ammonia salts, urea, and other inorganic salts such as potassium salts, phosphates fi, 'i, -/gnesium salts, calcium salts, etc. This is a method of culturing the above-mentioned microorganisms in a medium. In addition, the composition of the medium in this case,
There are no limitations on the culture conditions.
Ｄ・Ｌ−ピペコリン酸の初期濃度と用いる菌株にもよる
が、通常、３０〜１００時間培養することにより、培地
中のし一ピペコリン酸は消費され尽くし、培養液中には
Ｄ−ピペコリン酸だけが残存するようになる。Depending on the initial concentration of D/L-pipecolic acid and the strain used, usually by culturing for 30 to 100 hours, the pipecolic acid in the medium is completely consumed, leaving only D-pipecolic acid in the culture solution. will remain.
もう一つの方法は肉汁培地など適当な培地で前記の微生
物を培養し、得られた菌体をｐＨ５〜８に調整した０、
１〜３０％のＤ−Ｌ−ピペコリン酸水溶液に懸濁し、通
気しつつ装置する方法である。ｐ１１調整に用いる試薬
には特に限定はない。また、このＤ−Ｌ−ピペコリン酸
水溶液に各種無機塩類やビタミン類等を添加しても差支
えない、この場合もＤ−Ｌ−ピペコリン酸の初期の濃度
と用いる微生物にもよるが、数時間から数日で菌体懸濁
中のし一ピペコリン酸は消滅し、Ｄ−ピペコリン酸だけ
が残存するようになる。Another method is to culture the above-mentioned microorganisms in a suitable medium such as a broth medium, and adjust the resulting bacterial cells to a pH of 5 to 8.
This is a method in which the product is suspended in a 1 to 30% aqueous solution of DL-pipecolic acid, and the device is placed under ventilation. There are no particular limitations on the reagent used for p11 adjustment. It is also possible to add various inorganic salts, vitamins, etc. to this aqueous solution of DL-pipecolic acid. In this case, it also depends on the initial concentration of DL-pipecolic acid and the microorganism used, but after several hours. In a few days, D-pipecolic acid in the suspension of bacterial cells disappears, and only D-pipecolic acid remains.
なお、いずれの場合も培養温度は通常２０〜５０℃、好
ましくは３０℃付近、ｐ）ｌは５〜８．好ましは７付近
により行われる。In any case, the culture temperature is usually 20 to 50°C, preferably around 30°C, and p)l is 5 to 8. Preferably, the value is around 7.
以上の操作により、Ｄ−とベコリン酸を含みＬ−ピペコ
リン酸を含まない培養液、或いは菌体懸濁液が得られる
ので次の回収工程でＤ−ピペコリン酸を回収する。By the above operations, a culture solution or bacterial cell suspension containing D- and becolic acid but not L-pipecolic acid is obtained, and D-pipecolic acid is recovered in the next recovery step.
培養液、或いは菌体懸濁液からのＤ−ピペコリン酸は通
常の任意の方法で回収できるが一例を挙げるならば、培
養液、或いは菌体懸濁液から遠心分離等により菌体を除
去し、次いでイオン交換等により脱塩した後、濃縮する
ことにより結晶として回収できる。D-pipecolic acid from a culture solution or bacterial cell suspension can be recovered by any conventional method, but for example, bacterial cells can be removed from the culture solution or bacterial cell suspension by centrifugation, etc. Then, after desalting by ion exchange or the like, it can be recovered as crystals by concentrating.
以下、本発明を実施例により更に詳細に説明する。Hereinafter, the present invention will be explained in more detail with reference to Examples.
（４）実施例
ミクロコツカス属Ｎｏ ７Ｂ菌（ＦＥＲＭ Ｐ−９３１
１）クルシア属Ｎｏ １１３Ｂ菌（ＦＥＲ？Ｉ Ｐ−９
３１２） 、シュードモナス属５２０Ｂ菌（ＦεＲＭＰ
−９３１３）。(4) Example Micrococcus No. 7B bacterium (FERM P-931
1) Crucia genus No. 113B bacterium (FER?I P-9
312), Pseudomonas 520B (FεRMP
-9313).
アルカリ土類金属３０９Ｂｉ石（ＦＥＲＭ Ｐ−９３１
４，）をそれぞれ１１の肉汁培地に接種し、３０℃で２
日間振盪培養した菌体を遠心分離により集め、ｐＨ７，
０に調整した２％のＤ・Ｌ−ピペコリン酸水溶液１００
ｍβに懸濁し、３０℃で４０時間振弔した。光学分割カ
ラム（ダイセル化学社製、商品名ＣＩＩＩＲＡＬＰＡＫ
匈［（）を用いた高速液体クロマトグラフィーで、この
菌体懸濁液上清を分析したところ、Ｌ−ピペリコリン酸
は検出されずＤ−ピペコリン酸だけが認められた。この
菌体懸濁液から遠心分離により菌体を除き、菌体懸濁液
上清を得た。この上清液を減圧濃縮し、水を完全に留去
し、得られた残留物よりＤ−ピペコリン酸をエタノール
で抽出した。この抽出液に対し、５容のジエチルエーテ
ルを加え、室温に１日放置しＤ−ピペコリン酸を析出さ
せた。Alkaline earth metal 309Bi stone (FERM P-931
4,) were inoculated into 11 broth cultures, respectively, and incubated at 30°C for 2
Bacterial cells cultured with shaking for days were collected by centrifugation, and the pH was adjusted to 7.
2% D/L-pipecolic acid aqueous solution adjusted to 0
It was suspended in mβ and shaken at 30°C for 40 hours. Optical resolution column (manufactured by Daicel Chemical Co., Ltd., trade name CIIIRALPAK)
When the supernatant of this bacterial cell suspension was analyzed by high-performance liquid chromatography using a high-performance liquid chromatography, L-pipecolic acid was not detected and only D-pipecolic acid was observed. The cells were removed from this cell suspension by centrifugation to obtain a supernatant of the cell suspension. The supernatant was concentrated under reduced pressure to completely remove water, and D-pipecolic acid was extracted from the resulting residue with ethanol. Five volumes of diethyl ether was added to this extract, and the mixture was left at room temperature for one day to precipitate D-pipecolic acid.
析出したＤ−ピペコリン酸の結晶を濾取することにより
回収した。得られたＤ−ピペコリン酸をアミノ酸分析シ
ステム（島津製作所製、商品名ＬＣ−６Ａ）で分析した
ところ、いずれの菌株を用いた場合もピペコリン酸だけ
が検出された。また、このＤ−ピペコリン酸を前記の光
学分割カラムを用いた高速液体クロマトグラフィーで分
析したところ、いずれの場合もＤ−ピペコリン酸だけが
検出された。 また、このＤ−ピペコリン酸の一定量を
水に溶かし、この水溶液中のピペコリン酸を前記の高速
液体クロマトグラフィー、アミノ酸分析システム、及び
Ｄ−アミノ酸だけに悪心するＤ−アミノ酸オキシダーゼ
法により定量したところ、この三つの方法とも同一の値
を与えた。以上の分析により菌体懸濁液から回収した化
合物がＤ−ピペコリン酸であることを確認した。The precipitated D-pipecolic acid crystals were collected by filtration. When the obtained D-pipecolic acid was analyzed using an amino acid analysis system (manufactured by Shimadzu Corporation, trade name LC-6A), only pipecolic acid was detected when any strain was used. Further, when this D-pipecolic acid was analyzed by high performance liquid chromatography using the optical resolution column described above, only D-pipecolic acid was detected in each case. In addition, a certain amount of this D-pipecolic acid was dissolved in water, and the pipecolic acid in this aqueous solution was quantified using the above-mentioned high-performance liquid chromatography, amino acid analysis system, and the D-amino acid oxidase method, which is used only for D-amino acids. , all three methods gave the same value. The above analysis confirmed that the compound recovered from the bacterial cell suspension was D-pipecolic acid.
〈以下余白〉
それぞれの菌株につい倍、、ｃｌｌ ＋、ｊピペコリン
酸の回収率と光学純度を下表に示す。(Left below) The table below shows the recovery rate and optical purity of pipecolic acid for each bacterial strain.
の総重量（１ｇ）
（５）発明の効果
本発明は、Ｄ−ピペコリン酸を安価、且つ大量に供給す
ることを可能にすることができる。Total weight (1 g) (5) Effects of the invention The present invention can make it possible to supply D-pipecolic acid at low cost and in large quantities.
特許出願人 工業技術院長 飯 塚 幸 三指定代
理人 工業技術院微生物工業技術研究所長２．−゛佐胚
昭ｉ−゛、“・：ハPatent applicant: Director of the Agency of Industrial Science and Technology Kozo Iizuka Designated agent: Director of the Institute of Microbial Technology, Agency of Industrial Science and Technology 2. −゛Sageaki −゛、“・:ha
Claims (1)
ルカリゲネス属より選ばれ、且つＤ−ピペコリン酸資化
能が実質的に欠損し、Ｌ−ピペコリン酸資化能を有する
微生物をＤ・Ｌピペコリン酸含有培地で培養し、Ｄ−ピ
ペコリン酸を分割・採取することを特徴とするＤ−ピペ
コリン酸の製造法A microorganism selected from the genus Micrococcus, Crucia, Pseudomonas, and Alcaligenes, which is substantially deficient in the ability to assimilate D-pipecolic acid and has the ability to assimilate L-pipecolic acid, is grown in a medium containing D.L-pipecolic acid. A method for producing D-pipecolic acid, which comprises culturing, dividing and collecting D-pipecolic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8172487A JPS63248393A (en) | 1987-04-02 | 1987-04-02 | Production of d-pipecolic acid by microorganism |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8172487A JPS63248393A (en) | 1987-04-02 | 1987-04-02 | Production of d-pipecolic acid by microorganism |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63248393A true JPS63248393A (en) | 1988-10-14 |
| JPH025398B2 JPH025398B2 (en) | 1990-02-01 |
Family
ID=13754360
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP8172487A Granted JPS63248393A (en) | 1987-04-02 | 1987-04-02 | Production of d-pipecolic acid by microorganism |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63248393A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995010604A1 (en) * | 1993-10-15 | 1995-04-20 | Chiroscience Limited | Enzyme and its use in preparing (s)-pipecolic acid |
| WO2000012745A1 (en) * | 1998-08-26 | 2000-03-09 | Lonza Ag | Method for preparing (2r)-piperidine derivatives |
-
1987
- 1987-04-02 JP JP8172487A patent/JPS63248393A/en active Granted
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995010604A1 (en) * | 1993-10-15 | 1995-04-20 | Chiroscience Limited | Enzyme and its use in preparing (s)-pipecolic acid |
| WO2000012745A1 (en) * | 1998-08-26 | 2000-03-09 | Lonza Ag | Method for preparing (2r)-piperidine derivatives |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH025398B2 (en) | 1990-02-01 |
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