JPH0353887A - Production of alpha-keto-beta-methylvaleric acid - Google Patents
Production of alpha-keto-beta-methylvaleric acidInfo
- Publication number
- JPH0353887A JPH0353887A JP18923689A JP18923689A JPH0353887A JP H0353887 A JPH0353887 A JP H0353887A JP 18923689 A JP18923689 A JP 18923689A JP 18923689 A JP18923689 A JP 18923689A JP H0353887 A JPH0353887 A JP H0353887A
- Authority
- JP
- Japan
- Prior art keywords
- keto
- isoleucine
- acid
- methylvaleric acid
- alpha
- 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
- JVQYSWDUAOAHFM-UHFFFAOYSA-N 3-methyl-2-oxovaleric acid Chemical compound CCC(C)C(=O)C(O)=O JVQYSWDUAOAHFM-UHFFFAOYSA-N 0.000 title claims abstract description 21
- 238000004519 manufacturing process Methods 0.000 title claims description 9
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims abstract description 24
- 229960000310 isoleucine Drugs 0.000 claims abstract description 23
- 230000003287 optical effect Effects 0.000 claims abstract description 21
- 229930182844 L-isoleucine Natural products 0.000 claims abstract description 16
- 239000007864 aqueous solution Substances 0.000 claims abstract description 10
- 244000005700 microbiome Species 0.000 claims abstract description 9
- 150000003839 salts Chemical class 0.000 claims abstract description 9
- 241000588769 Proteus <enterobacteria> Species 0.000 claims abstract description 3
- 210000004748 cultured cell Anatomy 0.000 claims abstract description 3
- 150000004716 alpha keto acids Chemical class 0.000 claims abstract 4
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims description 7
- 238000005273 aeration Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 241000588768 Providencia Species 0.000 claims description 3
- 241000219470 Mirabilis Species 0.000 claims description 2
- 241000221960 Neurospora Species 0.000 claims description 2
- OVBFMEVBMNZIBR-UHFFFAOYSA-N -2-Methylpentanoic acid Natural products CCCC(C)C(O)=O OVBFMEVBMNZIBR-UHFFFAOYSA-N 0.000 claims 2
- 210000004027 cell Anatomy 0.000 abstract description 11
- 239000003814 drug Substances 0.000 abstract description 5
- 229940079593 drug Drugs 0.000 abstract description 3
- 210000003734 kidney Anatomy 0.000 abstract description 3
- 238000012258 culturing Methods 0.000 abstract description 2
- 210000004185 liver Anatomy 0.000 abstract description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 17
- 238000006243 chemical reaction Methods 0.000 description 15
- 239000000243 solution Substances 0.000 description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000002253 acid Substances 0.000 description 9
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 8
- 239000013078 crystal Substances 0.000 description 7
- 230000001954 sterilising effect Effects 0.000 description 6
- 238000004659 sterilization and disinfection Methods 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 235000010469 Glycine max Nutrition 0.000 description 5
- 244000068988 Glycine max Species 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 5
- 235000019341 magnesium sulphate Nutrition 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 229910000160 potassium phosphate Inorganic materials 0.000 description 4
- 235000011009 potassium phosphates Nutrition 0.000 description 4
- SMDJDLCNOXJGKC-UHFFFAOYSA-M sodium;3-methyl-2-oxopentanoate Chemical compound [Na+].CCC(C)C(=O)C([O-])=O SMDJDLCNOXJGKC-UHFFFAOYSA-M 0.000 description 4
- AGPKZVBTJJNPAG-CRCLSJGQSA-N D-allo-isoleucine Chemical compound CC[C@H](C)[C@@H](N)C(O)=O AGPKZVBTJJNPAG-CRCLSJGQSA-N 0.000 description 3
- IGIDLTISMCAULB-UHFFFAOYSA-N 3-methylvaleric acid Chemical compound CCC(C)CC(O)=O IGIDLTISMCAULB-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- AGPKZVBTJJNPAG-RFZPGFLSSA-N D-Isoleucine Chemical compound CC[C@@H](C)[C@@H](N)C(O)=O AGPKZVBTJJNPAG-RFZPGFLSSA-N 0.000 description 2
- 229930182845 D-isoleucine Natural products 0.000 description 2
- 241000588767 Proteus vulgaris Species 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 159000000007 calcium salts Chemical class 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229940007042 proteus vulgaris Drugs 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- -1 D-iso Chemical compound 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
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 description 1
- 239000000920 calcium hydroxide Substances 0.000 description 1
- 229910001861 calcium hydroxide Inorganic materials 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000007796 conventional method Methods 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
- 230000000694 effects Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000007833 oxidative deamination reaction Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
Landscapes
- 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、微生物の作用によりＬ−イソロイシンより、
肝臓薬、腎臓薬として有用なα−ケトβ−メチル吉草酸
を製造する方法に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention is directed to the production of L-isoleucine by the action of microorganisms.
The present invention relates to a method for producing α-ketoβ-methylvaleric acid, which is useful as a liver drug and a kidney drug.
イソロイシンにはＬ−イソロイシン、Ｌ−アロイソロイ
シン、Ｄ−イソロイシン、Ｄ−アロイソロイシン４種の
光学異性体が存在する。それらを酵素反応を用いて酸化
的脱アミノさーυ−α−ケト酸に変換すると、Ｌ−イソ
ロイシン及びＤ−イソ、ロイシンからは、ｄ−α−ケト
−β−メチル吉草酸、Ｌ−アロイソロイシン及びＤ−ア
ロイソ口，イシンからは、Ｃ＝α−ゲ１・−β−メチル
吉草酸がζＬ成する。しかしながら、その光学活性は容
易に失われることが知られている。Isoleucine has four types of optical isomers: L-isoleucine, L-alloisoleucine, D-isoleucine, and D-alloisoleucine. When they are converted to υ-α-keto acids through oxidative deamination using enzymatic reactions, L-isoleucine, D-iso, and leucine are converted to d-α-keto-β-methylvalerate, L-allo From isoleucine, D-alloisomerase, and isine, C=α-ge1·-β-methylvaleric acid is formed as ζL. However, it is known that its optical activity is easily lost.
オールＩ・ン・マイスター（＾ｌｔｏｎ Ｍｅｉｓｔｅ
ｒ）の文献（Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．， １９０ ，
２６９ （１９５１）によって、α−ケト−β−メチ
ル吉草酸の光学活性の安定性の研究が行われており、生
戒したα−ケト−βメチル吉草酸の光学活性は、ｐｕｓ
．４を越えると容易に失われ、８．４以下では安定であ
ることを見いだされている。そしてＤ−イソロイシンお
よび、Ｄ−アロイソロイシンに豚の腎臓（Ｈｏｇ Ｋｉ
ｄｎｅｙ）由来のＤ−アくノ酸オキシダーゼをｐＨ８．
２で作用させ、光学活性を有するα−ケト−β−メチル
吉草酸を得ていた。All I-n-Meister (^lton Meiste)
r) (J. Biol. Chem., 190,
269 (1951) conducted a study on the stability of the optical activity of α-keto-β-methylvaleric acid, and the optical activity of α-keto-β-methylvaleric acid was
．． It has been found that above 4 it is easily lost and below 8.4 it is stable. D-isoleucine and D-alloisoleucine were added to pig kidney (Hog Ki).
D-acnoic acid oxidase derived from Dney) at pH 8.
2 to obtain optically active α-keto-β-methylvaleric acid.
本発明者らは、詳細にα−ケト−β−メチル吉草酸の光
学活性の安定性を検討した結果、光学活性の安定のため
には、ｐｌ＋７．２よりもより低いｐｌ＋反応条件が必
要であり、オールトン・マイスターが得たα−ケ１・一
β−メチル吉ｖｌ′（酸は、その比旋光度から既に一部
ラセミ化しているものと考えられた。また、イソロイシ
ンの４種の光学活性体のうち、オールトン・マイスター
が用いた上記２種のイソロイシン等は非常に高価であり
、それらを利用することは産業上において不利にならざ
るを得ない。さらに、Ｌ−イソ口イシンから得られるα
ケ１へ酸はｄ−α−ケト−β−メチル吉草酸（以下、α
−ケト−β−メチル吉草酸と略記する）てあり、天然型
の構造を有するので、医薬として使用する場合望ましい
と考えられる。The present inventors investigated the stability of the optical activity of α-keto-β-methylvaleric acid in detail, and found that a lower pl+ reaction condition than pl+7.2 is required to stabilize the optical activity. Alton-Meister obtained α-ke1,-beta-methyl-l' (acid was thought to have already been partially racemized based on its specific optical rotation. Also, four types of isoleucine Among the optically active substances, the above-mentioned two types of isoleucine used by Oulton Meister are very expensive, and their use is inevitably disadvantageous in industry. α obtained from
The hemoacid is d-α-keto-β-methylvaleric acid (hereinafter referred to as α
-keto-β-methylvaleric acid) and has a natural structure, so it is considered desirable for use as a medicine.
人手容易なＬ−イソロイシンを原料として、医薬として
有用な天然型α−ケト−β−メチル吉草酸を、安価によ
り高い光学純度を有して安定に製造できる方法の開発が
要請されている。There is a need for the development of a method that can stably produce natural α-keto-β-methylvaleric acid, which is useful as a medicine, at low cost and with high optical purity using L-isoleucine, which is easy to handle, as a raw material.
前記の課題は、請求項１に記載の方法により達威される
。The above object is achieved by a method according to claim 1.
ずなわら、本発明のα−ケト−β−メチル六４！酸の製
造方法は、■７−イソｌコイシンをα−ケ１・酸に変換
する能力を有する微生物の培養菌体またはそれらの処理
物と、Ｌ−イソロイシンまたはその塩とをｐＨ７．２以
下の水溶液中で反応させ、光学活性を有するα−ケ１・
−β−メチル店ｉｔ，！，：酸を生成・已しめることを
ｑ、＋ｉｍとする。Of course, the α-keto-β-methyl 64 of the present invention! The method for producing acid is as follows: (1) Cultured cells of microorganisms having the ability to convert 7-iso-l-coicine into α-ke-1-acid or their processed products, and L-isoleucine or its salt at a pH of 7.2 or less. React in aqueous solution to obtain optically active α-Ke1.
-β-methyl store it,! , : Generate and increase acid as q, +im.
本発明によれば、Ｌ−イソロイシンをα−ケＩ・β−メ
チル吉草酸に変換する能力のある微生物を適当な培地で
培養し、得られる培養物、これより分離した菌体または
菌体の処理物をＬ−イソロイシンまたはその塩と水中で
反応させることにより、光学活性を有するα−ケト−β
−メチル吉草酸を生成蓄積させることができる。さらに
、反応液中から光学活性を失せずにα−ケト−β−メチ
ル吉草酸またはその塩を採取することができる。According to the present invention, a microorganism capable of converting L-isoleucine into α-keI·β-methylvaleric acid is cultured in an appropriate medium, and the resulting culture, bacterial cells isolated from this, or bacterial cells By reacting the treated product with L-isoleucine or its salt in water, optically active α-keto-β
- Methylvaleric acid can be produced and accumulated. Furthermore, α-keto-β-methylvaleric acid or its salt can be collected from the reaction solution without losing its optical activity.
使用できる菌株の例としては、プロテウス属、プロビデ
ンシア属、ミラビリス属、ノイロスポラ属が挙げられる
が、その他の微生物も有用であり、Ｌ−イソロイシンか
らα−ケト−β−メチル吉草酸を生或する能力のあるも
のであれば、いずれもが用いられる。Examples of strains that can be used include Proteus, Providencia, Mirabilis, and Neurospora, although other microorganisms are also useful and have the ability to produce α-keto-β-methylvaleric acid from L-isoleucine. Any one can be used as long as there is one.
微生物を培養する培地は、菌株により異なるが、目的を
達する限り、何ら１、？別の制限はなく、炭素源、窒素
源、無機物、さらに微量の栄養素等を適当に含有する天
然培地及び合或培地が好ましい。The medium for culturing microorganisms differs depending on the strain, but as long as it achieves the purpose, it can be used as any medium. There are no other restrictions, and natural and combined media containing appropriate amounts of carbon sources, nitrogen sources, inorganic substances, trace nutrients, etc. are preferred.
培養条件としては、菌種、培地によって異なるが、通常
温度は２５℃〜４０℃、ｐＨは４〜９で調整される。Culture conditions vary depending on the bacterial species and medium, but the temperature is usually adjusted to 25°C to 40°C and the pH to 4 to 9.
菌体処理物としては、培養物の濃縮物、乾燥物、菌体の
アセトン処理物、界面活性剤処理物、乾燥物、酵素処理
物、また、菌体からの抽出酵素および適当な固定化担体
に固定化したもの等が挙げられる。Examples of bacterial cell-treated products include culture concentrates, dried products, acetone-treated bacterial cells, surfactant-treated products, dried products, enzyme-treated products, enzymes extracted from bacterial cells, and suitable immobilization carriers. Examples include those fixed to.
原料は、Ｌ−イソロイシン又はその塩が用いられる。Ｌ
−イソロイシンの塩としては、塩酸塩等が好ましい。The raw material used is L-isoleucine or a salt thereof. L
- As the salt of isoleucine, hydrochloride and the like are preferred.
反応条件は用いる菌種、またその処理方法により異なる
が、ｐＨは７．２以下で行うことが、光学活性を有する
α−ケト−β−メチル吉草酸を反応液中に得るのに必要
である。好ましいｐＨ範囲は６．５〜７．２である。反
応は２５℃〜３５℃、６〜２４時間、通気攪拌して行う
。The reaction conditions vary depending on the bacterial species used and the treatment method used, but it is necessary to conduct the reaction at a pH of 7.2 or less in order to obtain optically active α-keto-β-methylvaleric acid in the reaction solution. . The preferred pH range is 6.5-7.2. The reaction is carried out at 25°C to 35°C for 6 to 24 hours with aeration and stirring.
反応液から、光学活性をイＪしたα−ゲ１・ βメチル
吉草酸を採取する方法としては、常法が用いられる。そ
の場合も光学活性を失わないために、ｐＨと温度を制御
する必要がある。すなわち反応終了後、直ちにｐＨを塩
酸にて４〜５に調整する。そして、反応液から、酵素源
を遠心分離、膜分離等を用いて除去する。その後、４０
℃以下にて、減圧濃縮を行い塩化カルシウム等のカルシ
ウム塩、得られた結晶を真空乾燥にて採取する。A conventional method can be used to collect optically active α-ge1.β-methylvaleric acid from the reaction solution. In that case as well, it is necessary to control the pH and temperature in order not to lose optical activity. That is, immediately after the reaction is completed, the pH is adjusted to 4 to 5 with hydrochloric acid. Then, the enzyme source is removed from the reaction solution using centrifugation, membrane separation, or the like. After that, 40
Concentration is carried out under reduced pressure at a temperature below 0.degree. C., and calcium salts such as calcium chloride and the resulting crystals are collected by vacuum drying.
また、酵素源を除いた後、塩酸、硫酸、硝酸等の鉱酸で
ｐＨを１．０以下に調整し、直ちに酢酸エチル等の溶剤
を用いて抽出後、減圧下にて溶剤を蒸発し、α−ケト−
β−メチル吉草酸を得ることもできる。塩の結晶が必要
な場合は水酸化カルシウムまたは水酸化ナトリウム等を
加え、得られる結晶を真空乾燥して採取する。In addition, after removing the enzyme source, the pH is adjusted to 1.0 or less with a mineral acid such as hydrochloric acid, sulfuric acid, or nitric acid, and immediately after extraction using a solvent such as ethyl acetate, the solvent is evaporated under reduced pressure. α-keto-
β-Methylvaleric acid can also be obtained. If salt crystals are required, add calcium hydroxide or sodium hydroxide, and collect the resulting crystals by vacuum drying.
以上のような操作にて採取されたα−ケト−βメチル吉
草酸ナトリウム塩の比は、〔α〕Ｄ′。The ratio of α-keto-β methylvaleric acid sodium salt collected by the above procedure is [α]D'.
＋ ３ ６． ３゜ （１％水溶液）であり、前出のオ
ールトン・マイスターが報告しているα−ケト−βメチ
ル吉草酸ナトリウム塩の比旋光度の〔α〕Ｄ′。+ 3 6. 3° (1% aqueous solution), and the specific optical rotation [α]D' of α-keto-β methylvaleric acid sodium salt reported by Alton Meister mentioned above.
＋３ ２． ６゜ （１％水溶液〉よりもより光学純度
が高いと判断される。+3 2. It is judged that the optical purity is higher than that of 6° (1% aqueous solution).
以下、実施例により本発明を詳細に述べる。 Hereinafter, the present invention will be described in detail with reference to Examples.
実施例１
プロテウスブルガリス Ｉ Ｆ Ｏ ３０４５を接種し
た、グルコース０．５％、大豆加水分解物（全窒素）１
５０■／ｄｌ、リン酸第−カリウム０．１％、硫酸マグ
ネシウム０．０４％を含む培地（殺菌前ｐＨ７．０）５
００ｍｊ！を５ ０ ０ Ｏｎ＋４！容フラスコに仕込
み、３０℃で１６時間振盪培養した。得られた培養液を
遠心分離して集菌し、その菌に２ｇ／ｄｊｌ！のＩ、−
イソロイシン溶液を５ ０ ０ｍＩｌを加え、ｐｌ＋７
．０に水酸化ナトリウム溶液または塩酸で調整し、５
０ ０ ０ｍ７！容坂口フラスコにて３５℃、６時間振
盪反応させ、転換率９０％でα−ケト−β−メチル吉草
酸の生成が認められた。次いで、塩酸でｐＨを４〜５に
調整後、反応液から菌体を除去し、さらに塩酸でｐｔｔ
ｔ．ｏ以下に調整した後、直ちに等量の酢酸エチルで抽
出した。抽出液中の酢酸エチルを減圧下にて蒸発させ、
冷水酸化ナトリウムをα−ケ１・酸に対して等モル量加
えて、５℃でｌ２時間静置後、得られた結晶を濾紙にて
回収し、２０℃で真空乾燥して、２．４ｇのα−ケ１・
−βメチル吉草酸ナトリウム塩を得た。得られた結晶の
比旋光度を測定したところ、［α］ ｎ” ＋ ３６．
３゜（１％水溶液）であった。Example 1 Proteus vulgaris IFO 3045 inoculated, glucose 0.5%, soybean hydrolyzate (total nitrogen) 1
50 ■/dl, medium containing potassium phosphate 0.1%, magnesium sulfate 0.04% (pH 7.0 before sterilization)5
00mj! 5 0 0 On+4! The mixture was placed in a volumetric flask and cultured with shaking at 30°C for 16 hours. The resulting culture solution was centrifuged to collect the bacteria, and the bacteria were exposed to 2 g/djl! I, -
Add 500 ml of isoleucine solution, pl+7
．． Adjust to 0 with sodium hydroxide solution or hydrochloric acid, 5
0 0 0m7! A shaking reaction was carried out in a Sakaguchi flask at 35° C. for 6 hours, and the production of α-keto-β-methylvaleric acid was observed at a conversion rate of 90%. Next, after adjusting the pH to 4 to 5 with hydrochloric acid, the bacterial cells were removed from the reaction solution, and further ptt was added with hydrochloric acid.
t. After adjusting the concentration to below 0.0, the mixture was immediately extracted with an equal amount of ethyl acetate. Ethyl acetate in the extract was evaporated under reduced pressure,
Add cold sodium hydroxide in an equimolar amount to α-Ke1 acid, leave it at 5°C for 12 hours, collect the obtained crystals with a filter paper, vacuum dry at 20°C, and obtain 2.4 g. α-ke1・
-β-methylvaleric acid sodium salt was obtained. When the specific optical rotation of the obtained crystal was measured, it was found to be [α] n” + 36.
3° (1% aqueous solution).
実施例２
実施例１と同様の方法でスケールアンプすることにより
、採取した旋光度〔α〕１°＋ ３ ６． ３゜（１％
水溶液）のα−ケト−β−メチル吉草酸２０ｇを少量の
水に溶解し、４０℃で減圧濃縮して、得られた結晶の旋
光度を測定したところ変化は見られなかった。同様の操
作をさらに４回繰り返したが旋光度の変化はなかった。Example 2 Optical rotation [α] 1° + 3 6. was collected by scale amplification in the same manner as in Example 1. 3゜(1%
When 20 g of α-keto-β-methylvaleric acid (aqueous solution) was dissolved in a small amount of water and concentrated under reduced pressure at 40°C, the optical rotation of the obtained crystal was measured, and no change was observed. The same operation was repeated four more times, but there was no change in the optical rotation.
このことから、光学純度１００％のα−ケト−β−メチ
ル吉草酸ナトリウム塩の旋光度は、（α） ｎ”＋３
６． ３゜（１％水溶液）であると考えられた。From this, the optical rotation of α-keto-β-methylvaleric acid sodium salt with 100% optical purity is (α) n”+3
6. 3° (1% aqueous solution).
実施例３
プロテウスブルガリス ＩＦＯ３０４５を接種した、グ
ルコース０．５％、大豆加水分解物（全窒素）１５０■
／ｄ７！、リン酸第一カリウム０． １％、硫酸マグネ
シウム０．０４％を含む培地（殺菌前ｐｌ＋７．０）５
０ｍ６を５ ０ ０ｗｌ容坂口フラスコに仕９
込み、３０℃で１６時間振盪培養した。得られた培養液
１ｍＩｌを、グルコース２．０％、大豆加水分解物（全
窒素）２２０■／ｄ７！、リン酸第一・カリウム０．
１％、硫酸マグネシウム０．０４％を含むｌａ地（殺菌
前ｐＨ７．０） ３ ０ ０ｍＡを仕込んだ６基の５
０ ０ｍｊ！容ジャーそれぞれに添加し、３０℃で２０
時間通気攪拌して、培養した。得られた培養液をそれぞ
れ遠心分離して、得られた菌体を３００ｔｎｌ２の０．
９％食塩水に懸濁し、１５ｇのＬ−イソロイシンを添加
した。そして５ ０ ０ｍ＃容のジャーを用いて、３５
℃で６種のｐＨ条件下、６．５、６．８、７．０、７．
２、７．５、８．０、８．５にて２４時間、通気攪拌反
応した。尚ｐｉは、アンモニアまたは塩酸にて調整した
。得られたα−ケｌ・−β−メチル吉草酸への転換収率
は、６３、８４、９０、９３、９５、９８、９８％であ
った。実施例１の方法に従って、α−ケト−β−メチル
吉草酸ナトリウム塩を得た。得られた比旋光度はそれぞ
れ〔α〕１°ｌ− ３ ６． ３゜，＋３６．４゜，ｌ
３６．３゜＋ ３ ６． ２゜ →−２８．３゜，＋
２３．２゜，＋７．／Ｉ゜１０
（各１％水溶液）であった。Example 3 Inoculated with Proteus vulgaris IFO3045, glucose 0.5%, soybean hydrolyzate (total nitrogen) 150 ■
/d7! , potassium phosphate 0. 1%, magnesium sulfate 0.04% (pl + 7.0 before sterilization) 5
0m6 was placed in a 500wl capacity Sakaguchi flask and cultured with shaking at 30°C for 16 hours. 1 ml of the obtained culture solution was mixed with 2.0% glucose and 220 μ/d7! of soybean hydrolyzate (total nitrogen). , potassium potassium phosphate 0.
1%, magnesium sulfate 0.04% (pH 7.0 before sterilization) 6 units of 5 charged with 300mA
0 0mj! 20 minutes at 30°C.
The cells were cultured with aeration and stirring for a period of time. The obtained culture solutions were each centrifuged, and the obtained bacterial cells were separated into 300 tnl2 of 0.000 g.
It was suspended in 9% saline and 15 g of L-isoleucine was added. Then, using a 500 m # capacity jar, 35
℃ under six pH conditions: 6.5, 6.8, 7.0, 7.
2, 7.5, 8.0, and 8.5 for 24 hours with aeration and stirring. Note that pi was adjusted with ammonia or hydrochloric acid. The conversion yields to α-kel·-β-methylvaleric acid obtained were 63, 84, 90, 93, 95, 98, and 98%. According to the method of Example 1, α-keto-β-methylvaleric acid sodium salt was obtained. The specific rotations obtained are [α] 1°l-36. 3゜, +36.4゜, l
36.3°+3 6. 2゜ → -28.3゜, +
23.2°, +7. /I°10 (each 1% aqueous solution).
実施例４
プロビデンシア レソトゲリ ＡＴＣＣ ９２５０を接
種した、グルコース０．５％、大豆加水分解物（全窒素
）１５０■／ｄＮ、リン酸第一カリウム０． １％、硫
酸マグネシウム０．０４％を含む培地（殺菌前ｐｌ＋７
．０）５０ｍｊ！を５ ０ ０ｍｌｌ容坂口フラスコに
仕込み、３０℃で１６時間振盪培養した。得られた培養
液１ ０ｎ＋ｎを、グルコース２．０％、大豆加水分解
物２２０■／ｄ１、リン酸第一カリウム０． １％、硫
酸マグネシウム０．０４％を含む培地（殺菌前ｐｌｌ７
．０） ３ ０ ０ ０ｍ６を仕込んだ５ ０ ０ ０
ｍｊ２容ジャーに添加し、３２℃で１５時間通気攪拌し
て、培養した。得られた培養液をそれぞれ遠心分離して
、得られた菌体を３ ０ ０ ０ｍＡの０．９％ＮａＣ
Ｉｌ液に懸濁し、１２０ｇのＬ−イソロイシンを添加
した。Example 4 Inoculated with Providencia lesotrogeri ATCC 9250, glucose 0.5%, soybean hydrolyzate (total nitrogen) 150 μ/dN, potassium phosphate 0.5%. 1%, magnesium sulfate 0.04% (before sterilization pl + 7
．． 0) 50mj! was placed in a 500 ml Sakaguchi flask and cultured with shaking at 30°C for 16 hours. The obtained culture solution 10n+n was mixed with 2.0% glucose, 220 μm/d1 of soybean hydrolyzate, and 0.0% potassium phosphate. 1%, magnesium sulfate 0.04% (pll7 before sterilization)
．． 0) 5000 filled with 3000m6
The mixture was added to a 2-volume mj jar and cultured at 32° C. with aeration for 15 hours. The obtained culture solution was centrifuged, and the obtained bacterial cells were heated with 0.9% NaC at 3000 mA.
The suspension was suspended in Il solution, and 120 g of L-isoleucine was added thereto.
５ ０ ０ ０ｍｆｆ容のジャーを用いて３５℃、ｐＨ
７．０で通気攪拌して反応させた。ｐＨは、塩酸および
アンモニアで調整した。８時間後には転換率９２％で、
α−ケ１・−β−メチル吉草酸が得られた。反ｌ１
の限外濾過膜を用いて除菌、除蛋白をした後、４０゜で
減圧濃縮し、α−ケ１・一β−メチル吉草酸に対して、
１／２モルの塩化カルシウムを攪拌しながら徐々に添加
して、５℃で１晩静置した。Using a jar with a capacity of 5000 mff, at 35°C, pH
The reaction was carried out with aeration and stirring at 7.0°C. pH was adjusted with hydrochloric acid and ammonia. After 8 hours, the conversion rate was 92%,
α-Ke1·-β-methylvaleric acid was obtained. After sterilization and protein removal using an anti-l1 ultrafiltration membrane, it was concentrated under reduced pressure at 40°, and the
1/2 mol of calcium chloride was gradually added with stirring, and the mixture was left standing at 5° C. overnight.
得られた結晶を濾紙で回収し、２０℃で真空乾燥した。The obtained crystals were collected using filter paper and vacuum dried at 20°C.
得られたα−ケト−β−メヂル吉草酸カルシウム塩はＩ
Ｉ７ｇであった。また比旋光度は〔α）ｎ”＋３５．６
゜ （６Ｎ塩酸１％？容液）であった。The obtained α-keto-β-medylvaleric acid calcium salt was
It was 7g. Also, the specific optical rotation is [α)n”+35.6
(6N hydrochloric acid 1%? solution).
本発明によれば、α−ケト−β−メチル吉草酸の天然型
の光学活性体が容易に取得でき、α−ゲト酸を用いた治
療方法の検討が容易になることがＩＵ１持される。According to the present invention, it is possible to easily obtain a naturally occurring optically active form of α-keto-β-methylvaleric acid, and it is possible to easily examine a therapeutic method using α-getoic acid.
Claims (4)
有する微生物の培養菌体またはそれらの処理物と、Ｌ−
イソロイシンまたはその塩とをｐＨ７．２以下の水溶液
中で反応させ、光学活性を有するα−ケト−β−メチル
吉草酸を生成せしめることを特徴とするα−ケト−β−
メチル吉草酸の製造方法。(1) Cultured cells of microorganisms having the ability to convert L-isoleucine into α-keto acids or processed products thereof, and L-
α-keto-β-, which is characterized by reacting isoleucine or a salt thereof in an aqueous solution with a pH of 7.2 or lower to produce optically active α-keto-β-methylvaleric acid.
Method for producing methylvaleric acid.
有する微生物が、プロテウス属、プロビデンシア属、ミ
ラビリス属およびノイロスポラ属よりなる群から選ばれ
たものである請求項１に記載の製造方法。(2) The production method according to claim 1, wherein the microorganism having the ability to convert L-isoleucine into α-keto acid is selected from the group consisting of the genus Proteus, Providencia, Mirabilis, and Neurospora.
の変換反応温度が通気攪拌下２５〜３５℃の温度範囲で
ある請求項１に記載の製造方法。(3) The production method according to claim 1, wherein the conversion reaction temperature to optically active α-keto-β-methylvaleric acid is in the temperature range of 25 to 35°C under aeration and stirring.
の変換反応をする際のｐＨが６．５〜７．２である請求
項１に記載の製造方法。(4) The manufacturing method according to claim 1, wherein the pH during the conversion reaction to α-keto-β-methylvaleric acid having optical activity is 6.5 to 7.2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18923689A JPH0353887A (en) | 1989-07-22 | 1989-07-22 | Production of alpha-keto-beta-methylvaleric acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18923689A JPH0353887A (en) | 1989-07-22 | 1989-07-22 | Production of alpha-keto-beta-methylvaleric acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0353887A true JPH0353887A (en) | 1991-03-07 |
Family
ID=16237888
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP18923689A Pending JPH0353887A (en) | 1989-07-22 | 1989-07-22 | Production of alpha-keto-beta-methylvaleric acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0353887A (en) |
-
1989
- 1989-07-22 JP JP18923689A patent/JPH0353887A/en active Pending
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