JP2011036135A - METHOD FOR PRODUCING OPTICALLY ACTIVE tert-LEUCINE - Google Patents
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本発明は、医農薬中間体として重要な光学活性ｔｅｒｔ−ロイシンの、操作性に優れた工業的に実施可能な製造方法に関する。詳しくは、原料基質のＤＬ−ｔｅｒｔ−ロイシンアミドを立体選択性を有する生体触媒を用いて加水分解した後、貧溶媒に置換して光学活性ｔｅｒｔ−ロイシンを析出させる方法に関する。 The present invention relates to an industrially feasible production method of optically active tert-leucine, which is important as an intermediate for medicines and agrochemicals, with excellent operability. More specifically, the present invention relates to a method for precipitating optically active tert-leucine by hydrolyzing DL-tert-leucine amide as a raw material substrate using a biocatalyst having stereoselectivity and then substituting with a poor solvent.
本発明の方法によって得られる光学活性ｔｅｒｔ−ロイシンは、ＨＩＶやＨＣＶのプロテアーゼ阻害剤等の医薬品原料として重要な中間体である（例えば、特許文献１、２参照）。従来、立体選択性を有する生体触媒、即ち、酵素または該酵素を有する微生物もしくはその処理物をＤＬ−ｔｅｒｔ−ロイシンアミドに作用させてＬ体またはＤ体選択的加水分解を行うことにより、光学活性Ｌ−またはＤ−ｔｅｒｔ−ロイシンを生成させた後、反応液の溶媒を水から有機溶媒へ置換することにより未反応のＤ−またはＬ−ｔｅｒｔ−ロイシンアミドを有機溶媒に溶解させ、光学活性Ｌ−またはＤ−ｔｅｒｔ−ロイシンを有機溶媒から析出させる方法が知られている（例えば、特許文献３，４，５参照）。 Optically active tert-leucine obtained by the method of the present invention is an important intermediate as a raw material for pharmaceuticals such as HIV and HCV protease inhibitors (see, for example, Patent Documents 1 and 2). Conventionally, a biocatalyst having stereoselectivity, that is, an optical activity is obtained by allowing an enzyme, a microorganism having the enzyme, or a processed product thereof to act on DL-tert-leucinamide to perform L-form or D-form selective hydrolysis. After producing L- or D-tert-leucine, unreacted D- or L-tert-leucine amide is dissolved in the organic solvent by substituting the solvent of the reaction solution from water to an organic solvent. A method for precipitating-or D-tert-leucine from an organic solvent is known (see, for example, Patent Documents 3, 4, and 5).
一般に、生体触媒反応の際、生体触媒の至適ｐＨにおいて行われることが望ましい。ｔｅｒｔ−ロイシンアミドの立体選択的加水分解に適したｐＨは、使用する生体触媒により異なり一概に規定することはできないが、例えばＬ体選択的加水分解活性を有する遺伝子組換菌株であるｐＭＣＡ１／ＪＭ１０９（ＦＥＲＭ ＡＰ−２００５６）由来の酵素を用いた場合はｐＨ６〜１０、より好ましくはｐＨ６〜９において好適に反応が進行する。遊離のｔｅｒｔ−ロイシンアミドの水溶液のｐＨは１０．５程度となるため、立体選択的加水分解を至適ｐＨ条件で実施するためには酸を添加してｐＨを調整しなければならない。至適ｐＨに合わせるために塩酸などの無機酸を加えるのが一般的であるが、加えられた酸は未反応のｔｅｒｔ−ロイシンアミドに作用して有機溶媒に対して溶解性の低い酸塩を形成してしまうため、有機溶媒中に析出した光学活性ｔｅｒｔ−ロイシンを分離取得する際に、光学活性Ｌ−またはＤ−ｔｅｒｔ−ロイシンにその対掌体のＤ−またはＬ−ｔｅｒｔ−ロイシンアミドの酸塩が混入してしまう。対掌体のｔｅｒｔ−ロイシンアミドの酸塩が混入した光学活性Ｌ−またはＤ−ｔｅｒｔ−ロイシンを原料として誘導体を製造した場合、ｔｅｒｔ−ロイシンの光学純度が高いにもかかわらずｔｅｒｔ−ロイシンアミドがｔｅｒｔ−ロイシンと類似の反応性を示すことによりｔｅｒｔ−ロイシン誘導体の光学純度を低下させる場合がある。従って、Ｄ−またはＬ−ｔｅｒｔ−ロイシンの対掌体であるｔｅｒｔ−ロイシンアミドの酸塩の混入を防ぐ必要があり、生体触媒反応後にｔｅｒｔ−ロイシンアミドより強い塩基を加えてアミドを遊離化する操作が必要になる。 In general, it is desirable that the biocatalytic reaction is performed at an optimum pH of the biocatalyst. The pH suitable for the stereoselective hydrolysis of tert-leucine amide differs depending on the biocatalyst used and cannot be defined unconditionally. For example, pMCA1 / JM109, which is a recombinant strain having L-form selective hydrolysis activity, is available. When an enzyme derived from (FERM AP-20056) is used, the reaction suitably proceeds at pH 6 to 10, more preferably pH 6 to 9. Since the pH of the aqueous solution of free tert-leucinamide is about 10.5, it is necessary to adjust the pH by adding an acid in order to carry out stereoselective hydrolysis under optimum pH conditions. In general, an inorganic acid such as hydrochloric acid is added to adjust to an optimum pH, but the added acid acts on an unreacted tert-leucinamide to form an acid salt having low solubility in an organic solvent. Therefore, when the optically active tert-leucine precipitated in the organic solvent is separated and obtained, the optically active L- or D-tert-leucine is converted into the enantiomer of D- or L-tert-leucine amide. Acid salts are mixed in. When a derivative is produced using optically active L- or D-tert-leucine mixed with an enantiomer of tert-leucine amide as a raw material, the tert-leucine amide is obtained despite the high optical purity of tert-leucine. In some cases, the optical purity of the tert-leucine derivative is lowered by showing a reactivity similar to that of tert-leucine. Therefore, it is necessary to prevent the inclusion of the acid salt of tert-leucine amide, which is the enantiomer of D- or L-tert-leucine, and after the biocatalytic reaction, a stronger base than tert-leucine amide is added to liberate the amide. Operation is required.
ところが、酵素反応後にアルカリ金属水酸化物などの無機塩基を加えてアミドを遊離化した後、反応液の溶媒を水から有機溶媒へ置換することにより光学活性ｔｅｒｔ−ロイシンを有機溶媒から析出させる操作を行うと、有機溶媒のゲル化が生じ、流動性が失われてしまう問題が発生した。 However, after the enzymatic reaction, an inorganic base such as an alkali metal hydroxide is added to liberate the amide, and then the optically active tert-leucine is precipitated from the organic solvent by substituting the solvent in the reaction solution from water to an organic solvent. When this was performed, gelation of the organic solvent occurred, resulting in a problem of loss of fluidity.
また、原料としてｔｅｒｔ−ロイシンアミド塩酸塩などの酸塩を使用し、アルカリ金属水酸化物などの無機塩基でｐＨ調整を行って生体触媒反応を行った場合も、溶媒を水から有機溶媒へ置換する操作に伴って同様に溶媒のゲル化が生じた。 In addition, when an acid salt such as tert-leucinamide hydrochloride is used as a raw material and a biocatalytic reaction is performed by adjusting the pH with an inorganic base such as an alkali metal hydroxide, the solvent is replaced from water to an organic solvent. In the same way, gelation of the solvent occurred.
その際、光学活性ｔｅｒｔ−ロイシンは、溶媒の流動性が失われたことにより回収することが困難であった。また、流動性が失われたことによりｔｅｒｔ−ロイシンアミドを分離することができなくなり、光学活性ｔｅｒｔ−ロイシンの純度を著しく低下させる問題が生じた。反応液のゲル化には塩の存在が関係しており、ゲル化を回避するために有効と考えられる脱塩操作を行った場合には、工程数の増加に伴い収率の低下を免れない。さらに、流動性を回復するために大量の有機溶媒を添加した場合には、ｔｅｒｔ−ロイシンアミドは除去することができるが、光学活性ｔｅｒｔ−ロイシンの回収率の低下を招く結果となった。工業的な面からも大量の有機溶媒の使用は好ましくない。文献にはゲル化については記載されていないが、文献記載の方法ではこのように、光学活性ｔｅｒｔ−ロイシンの製造方法として工業的に実施することが困難であることが判明した。 At that time, the optically active tert-leucine was difficult to recover due to the loss of fluidity of the solvent. Further, the loss of fluidity makes it impossible to separate tert-leucine amide, resulting in a problem of significantly reducing the purity of optically active tert-leucine. The presence of salt is involved in the gelation of the reaction solution, and when a desalting operation considered to be effective for avoiding gelation is performed, a decrease in yield is inevitable as the number of steps increases. . Furthermore, when a large amount of an organic solvent is added to restore fluidity, tert-leucine amide can be removed, but the recovery rate of the optically active tert-leucine was reduced. The use of a large amount of organic solvent is not preferable from the industrial aspect. Although the gelation is not described in the literature, it has been found that the method described in the literature is difficult to implement industrially as a method for producing optically active tert-leucine.
生体触媒反応液の溶媒を水から有機溶媒へ置換することによりｔｅｒｔ−ロイシンを有機溶媒から析出させる操作を行うと、有機溶媒のゲル化が生じる理由の詳細は明らかではないが、例えば、ｔｅｒｔ−ロイシンの構造異性体であるイソロイシンなどの誘導体では、水素結合の分子間相互作用により低分子でありながらゲルを生成する性質が知られている（英謙二， 高分子論文集， ５２７７３，１９９５年、英謙二， 高分子論文集， ５５，５８５，１９９８年）。ｔｅｒｔ−ロイシンにおいても同様の現象が起こっていると考えられ、ゆえに、有機溶媒のゲル化が生じていると考えられるが、本課題の解決法を示した報告は認められない。 Although the details of the reason why gelation of the organic solvent occurs when the operation of precipitating tert-leucine from the organic solvent by replacing the solvent of the biocatalyst reaction solution from water to an organic solvent is not clear, for example, tert- Derivatives such as isoleucine, which is a structural isomer of leucine, are known to have a low molecular weight due to intermolecular interaction of hydrogen bonds (Kenji Ei, Polymer Journal, 52773, 1995, Eiji Kenji, Polymer Journals, 55, 585, 1998). It is considered that the same phenomenon occurs in tert-leucine, and therefore, it is considered that gelation of the organic solvent occurs, but there is no report showing a solution to this problem.
本発明の目的は、従来技術における上記した課題を解決し、医農薬原料として重要な光学活性Ｌ−またはＤ−ｔｅｒｔ−ロイシンを製造するための操作性に優れた工業的に実施可能な方法を提供することにある。 The object of the present invention is to solve the above-mentioned problems in the prior art and to provide an industrially feasible method excellent in operability for producing optically active L- or D-tert-leucine important as a raw material for medicines and agricultural chemicals. It is to provide.
本発明者らは、光学活性ｔｅｒｔ−ロイシンの工業的に実施可能な製造方法に関して鋭意検討した。 The present inventors diligently investigated an industrially feasible production method of optically active tert-leucine.
一般的な酸、たとえば塩酸、硫酸等の存在下で、アミドを遊離化しないまま反応液の溶媒を水から有機溶媒へ置換することにより光学活性Ｌ−またはＤ−ｔｅｒｔ−ロイシンを有機溶媒に析出させると、未反応のｔｅｒｔ−ロイシンアミドの酸塩が光学活性ｔｅｒｔ−ロイシンと共に析出し、光学活性ｔｅｒｔ−ロイシンの純度を低下させた。そのため、反応液の溶媒を水から有機溶媒へ置換する操作の前に塩基を添加してアミドの遊離化を行う必要がある。ここでアミドの遊離化のために添加する塩基として水酸化ナトリウムなどの無機塩基を使用した場合、生成する塩により、反応液の溶媒を水から有機溶媒へ置換する操作中に反応混合物がゲル化してしまい、工業的操作が困難となるという問題点がある。反応液のゲル化という現象は、塩酸、硫酸をはじめとする鉱酸および蟻酸、プロピオン酸をはじめとする有機酸など多くの酸でｐＨ調整して生体触媒反応を行い、アミドを遊離化して反応液の溶媒を水から有機溶媒へ置換した場合に観察された。 Precipitation of optically active L- or D-tert-leucine in an organic solvent by substituting the solvent of the reaction solution from water to an organic solvent in the presence of a general acid such as hydrochloric acid or sulfuric acid without liberating the amide. As a result, an unreacted tert-leucine amide acid salt was precipitated together with the optically active tert-leucine, thereby reducing the purity of the optically active tert-leucine. Therefore, it is necessary to liberate the amide by adding a base before the operation of replacing the solvent of the reaction solution from water to an organic solvent. Here, when an inorganic base such as sodium hydroxide is used as the base to be added for liberation of the amide, the reaction mixture gels during the operation of replacing the solvent of the reaction solution from water to an organic solvent due to the generated salt. Therefore, there is a problem that industrial operation becomes difficult. The phenomenon of gelation of the reaction solution is the reaction of the biocatalytic reaction by adjusting the pH with various acids such as mineral acids such as hydrochloric acid and sulfuric acid and organic acids such as formic acid and propionic acid, and liberating amides. This was observed when the solvent of the liquid was replaced from water to an organic solvent.
ゲル化に関与すると考えられる成分について広く検討したところ、溶媒置換に供する液に含まれる酸根として酢酸根を含む場合にゲル化を防止できることを見出した。遊離のｔｅｒｔ−ロイシンアミドを原料とし無機酸によるｐＨ調整下で生体触媒反応を行った後に無機塩基により未反応のアミドを遊離化する場合や、ｔｅｒｔ−ロイシンアミドの無機酸塩を原料とし無機塩基によるｐＨ調整下で生体触媒反応を行った後に無機塩基をさらに加えて未反応のアミドを遊離化する場合など、反応系内にｔｅｒｔ−ロイシンアミド酸塩以外の塩が存在する場合には溶媒置換中にゲル化が起こることがあるが、これらの塩のほかに反応液に酢酸根を含ませることによりゲル化を防止することが可能である。このように、ゲル化を回避しながらｔｅｒｔ−ロイシンアミドまたはその塩を含まない高純度の光学活性ｔｅｒｔ−ロイシンを取得できることを見出した。
さらに貧溶媒として相応しい溶媒の種類について検討したところ、酢酸添加下でゲル化を起こさないこと、水と共沸し沸点も比較的低いこと、析出する光学活性ｔｅｒｔ−ロイシンに対する溶解性が低く、しかもｔｅｒｔ−ロイシンアミド酢酸塩の溶解性が高いこと、その他溶媒としての安全性や経済性の面から、炭素数３〜７のアルコール類、特に２−メチル−１−プロパノールが好適に使用できることを見出した。
When the component considered to be involved in gelation has been extensively studied, it has been found that gelation can be prevented when an acetic acid radical is included as an acid radical contained in a solution subjected to solvent substitution. When free tert-leucine amide is used as a raw material and a biocatalytic reaction is carried out under pH adjustment with an inorganic acid, then an unreacted amide is liberated with an inorganic base, or an inorganic acid salt of tert-leucine amide is used as a raw material. If a salt other than tert-leucine amidate is present in the reaction system, such as when an inorganic base is further added to liberate unreacted amide after performing biocatalytic reaction under pH adjustment by In some cases, gelation may occur. In addition to these salts, gelation can be prevented by including an acetate group in the reaction solution. Thus, it was found that high-purity optically active tert-leucine containing no tert-leucine amide or a salt thereof can be obtained while avoiding gelation.
Furthermore, when the kind of solvent suitable as a poor solvent was examined, it did not cause gelation under the addition of acetic acid, it was azeotropic with water and its boiling point was relatively low, the solubility in the precipitated optically active tert-leucine was low, and It has been found that alcohols having 3 to 7 carbon atoms, particularly 2-methyl-1-propanol, can be suitably used from the viewpoint of the high solubility of tert-leucinamide acetate and the safety and economics as other solvents. It was.
このように、溶媒置換前の反応液に酢酸根を存在させ、反応液の溶媒を水からアルコール類へ置換すれば、反応液をゲル化させることなく、かつ光学活性ｔｅｒｔ−ロイシンを析出させｔｅｒｔ−ロイシンアミドまたはその酸塩から分離取得することが可能であることを見出し、本発明を完成するに至った。 In this way, if an acetic acid radical is present in the reaction solution before solvent replacement and the solvent of the reaction solution is replaced from water to alcohols, the optically active tert-leucine can be precipitated without causing the reaction solution to gel. -The present inventors have found that it is possible to separate and acquire from leucine amide or its acid salt, and have completed the present invention.
すなわち、本発明は、光学活性ｔｅｒｔ−ロイシンを得るための（１）から（６）に示す製造方法に関する。
（１）酸根を含有する水溶液中でｔｅｒｔ−ロイシンアミドを立体選択的に加水分解する生体触媒をｔｅｒｔ−ロイシンアミドに作用させた後、反応液に塩基を添加し、続いて反応液の溶媒を水から光学活性ｔｅｒｔ−ロイシンの貧溶媒に置換することにより、生成した光学活性ｔｅｒｔ−ロイシンを析出させ分取する方法において、溶媒置換に供する液中の酸根のうち酢酸根が４５ｍｏｌ％以上であると共に、貧溶媒がアルコール類であることを特徴とする、ｔｅｒｔ−ロイシンアミドからの光学活性ｔｅｒｔ−ロイシンの製造方法。
（２）ｔｅｒｔ−ロイシンアミドを立体選択的に加水分解する生体触媒がキサントバクター属、プロタミノバクター属、ミコバクテリウム属もしくはミコプラナ属に属する微生物、またはこれら微生物から人工的変異手段によって誘導される変異株、細胞融合もしくは遺伝子組換法により誘導される組換株より調製された、酵素または該酵素を有する微生物もしくはその処理物である、（１）に記載の光学活性ｔｅｒｔ−ロイシンの製造方法。
（３）アルコール類が２−プロパノール、２−メチル−１−プロパノール、１−ブタノールおよび２−ブタノールから選ばれる一種以上である、（１）に記載の光学活性ｔｅｒｔ−ロイシンの製造方法。
（４）アルコール類が２−メチル−１−プロパノールである、（１）に記載の光学活性ｔｅｒｔ−ロイシンの製造方法。
（５）光学活性ｔｅｒｔ−ロイシンがＬ−ｔｅｒｔ−ロイシンである、（１）〜（４）の何れか一項に記載の光学活性ｔｅｒｔ−ロイシンの製造方法。
That is, this invention relates to the manufacturing method shown in (1) to (6) for obtaining optically active tert-leucine.
(1) After allowing a biocatalyst that stereoselectively hydrolyzes tert-leucine amide to act on tert-leucine amide in an aqueous solution containing acid radicals, a base is added to the reaction solution, and then the solvent of the reaction solution is changed. In the method of precipitating and separating the optically active tert-leucine produced by substituting the poor solvent for optically active tert-leucine from water, the acetic acid radical is 45 mol% or more of the acid radicals in the solution used for solvent substitution. A method for producing optically active tert-leucine from tert-leucine amide, wherein the poor solvent is an alcohol.
(2) A biocatalyst that stereoselectively hydrolyzes tert-leucinamide is derived from microorganisms belonging to the genus Xantobacter, Protaminobacter, Mycobacterium or Mycoplana, or from these microorganisms by artificial mutation means Of the optically active tert-leucine according to (1), which is an enzyme, a microorganism having the enzyme, or a processed product thereof, which is prepared from a mutant strain, a recombinant strain derived by cell fusion or a gene recombination method Production method.
(3) The method for producing an optically active tert-leucine according to (1), wherein the alcohol is one or more selected from 2-propanol, 2-methyl-1-propanol, 1-butanol and 2-butanol.
(4) The method for producing an optically active tert-leucine according to (1), wherein the alcohol is 2-methyl-1-propanol.
(5) The method for producing optically active tert-leucine according to any one of (1) to (4), wherein the optically active tert-leucine is L-tert-leucine.
本発明の方法によれば、ｔｅｒｔ−ロイシンアミドに立体選択的加水分解活性を有する酵素を作用させ、続いて反応液の溶媒を水から有機溶媒へ置換することにより光学活性Ｌ−またはＤ−ｔｅｒｔ−ロイシンを有機溶媒に析出せしめる際に反応混合物をゲル化させることなくＬ−またはＤ−ｔｅｒｔ−ロイシンを高純度かつ高収率に取得することが可能となる。 According to the method of the present invention, an optically active L- or D-tert is obtained by allowing a tert-leucinamide to act on an enzyme having stereoselective hydrolysis activity, and subsequently substituting the solvent of the reaction solution from water to an organic solvent. -L- or D-tert-leucine can be obtained in high purity and high yield without causing the reaction mixture to gel when leucine is precipitated in an organic solvent.
本発明の実施形態はｔｅｒｔ−ロイシンアミドを立体選択的に加水分解する生体触媒を、酸根を含む液中でｔｅｒｔ−ロイシンアミドに作用させる生体触媒反応を行い、光学活性ｔｅｒｔ−ロイシンならびにその対掌体であるｔｅｒｔ−ロイシンアミドおよびその塩を含む反応液を得、該反応液に塩基を添加することによってｔｅｒｔ−ロイシンアミドを遊離させ、その後、反応液中に酢酸根が存在する状態で、溶媒を水からｔｅｒｔ−ロイシンの貧溶媒に置換することにより、光学活性ｔｅｒｔ−ロイシンを析出させるものである。 In an embodiment of the present invention, a biocatalyst that stereoselectively hydrolyzes tert-leucine amide is allowed to act on tert-leucine amide in a solution containing an acid radical, and the optically active tert-leucine and its enantiomer are subjected to a biocatalytic reaction To obtain a reaction solution containing tert-leucinamide and a salt thereof as a body, and by adding a base to the reaction solution, tert-leucineamide is liberated, and in the presence of acetate radicals in the reaction solution, Is substituted from water with a poor solvent of tert-leucine to precipitate optically active tert-leucine.
［生体触媒反応］
まず、生体触媒反応により、ｔｅｒｔ−ロイシンアミドの立体選択的加水分解を行う。すなわち、酸根を含む水溶液中で、ｔｅｒｔ−ロイシンアミドを立体選択的に加水分解する生体触媒をｔｅｒｔ−ロイシンアミドに作用させて、光学活性ｔｅｒｔ−ロイシンとその対掌体であるｔｅｒｔ−ロイシンアミドを含む反応液を得る。酸根としては、塩酸根、硫酸根、リン酸根などの無機酸根、蟻酸根、酢酸根、プロピオン酸根などの有機酸根が例示される。
[Biocatalytic reaction]
First, stereoselective hydrolysis of tert-leucinamide is performed by a biocatalytic reaction. That is, a biocatalyst that stereoselectively hydrolyzes tert-leucine amide in an aqueous solution containing an acid radical is allowed to act on tert-leucine amide, so that optically active tert-leucine and its counter tert-leucine amide are converted. A reaction solution containing is obtained. Examples of acid radicals include inorganic acid radicals such as hydrochloric acid radicals, sulfate radicals, and phosphate radicals, and organic acid radicals such as formic acid radicals, acetate radicals, and propionate radicals.
生体触媒反応に使用される生体触媒は、Ｌ−またはＤ−ｔｅｒｔ−ロイシンに対応するＬ−またはＤ−ｔｅｒｔ−ロイシンアミドを立体選択的に加水分解する生体触媒であればよく、このような生体触媒を有する微生物として例えば、キサントバクター属、プロタミノバクター属、ミコバクテリウム属またはミコプラナ属等に属する微生物、具体的にはキサントバクター フラバス（Ｘａｎｔｈｏｂａｃｔｅｒ ｆｌａｖｕｓ）ＮＣＩＢ １００７１Ｔ、プロタミノバクター アルボフラバス（Ｐｒｏｔａｍｉｎｏｂａｃｔｅｒ ａｌｂｏｆｌａｖｕｓ）ＡＴＣＣ８４５８、ミコバクテリウム メタノリカ（Ｍｙｃｏｂａｃｔｅｒｉｕｍ ｍｅｔｈａｎｏｌｉｃａ）ＢＴ−８４（ＦＥＲＭ Ｐ８８２３）、ミコバクテリウム メタノリカ（Ｍｙｃｏｂａｃｔｅｒｉｕｍ ｍｅｔｈａｎｏｌｉｃａ）Ｐ−２３（ＦＥＲＭ Ｐ８８２５）、ミコプラナ ラモサ（Ｍｙｃｏｐｌａｎａ ｒａｍｏｓａ）ＮＣＩＢ９４４０Ｔ、ミコプラナ ディモルファ（Ｍｙｃｏｐｌａｎａ ｄｉｍｏｒｐｈａ）ＡＴＣＣ４２７９Ｔ、バリオボラックス パラドクサス（Ｖａｒｉｏｖｏｒａｘ ｐａｒａｄｏｘｕｓ）ＤＳＭ１４４６８が挙げられるが、これらに限定されるものではない。また、これら微生物から人工的変異手段によって誘導される変異株、あるいは細胞融合もしくは遺伝子組換法等の遺伝学的手法により誘導される組換株、例えばｐＭＣＡ１／ＪＭ１０９（ＦＥＲＭ ＡＰ−２００５６）等の何れの菌株であっても上記能力を有するものであれば本発明に使用できる。また、これら微生物は、菌体または菌体処理物、例えば菌体濃縮液、乾燥菌体、菌体破砕物、菌体抽出物もしくは精製酵素、またはこれらの担体固定物等の形態で使用することができる。 The biocatalyst used for the biocatalytic reaction may be a biocatalyst that stereoselectively hydrolyzes L- or D-tert-leucine amide corresponding to L- or D-tert-leucine. Examples of microorganisms having a catalyst include microorganisms belonging to the genus Xantobacter, Protaminobacter, Mycobacterium or Mycoplana, specifically Xantobacter flavus NCIB 10071T, Protaminobacter alboflavus (Protaminobacter alboflavus) ATCC 8458, Mycobacterium methanolica BT-84 (FERM P8823), Mycobacterium methanolica (Myco) acterium methanolica) P-23 (FERM P8825), Mikopurana Lamosa (Mycoplana ramosa) NCIB9440T, Mikopurana Dimorufa (Mycoplana dimorpha) ATCC4279T, Barrio borax Paradokusasu (Variovorax paradoxus) DSM14468 including without being limited thereto. Further, mutant strains derived from these microorganisms by artificial mutation means, or recombinant strains derived by genetic techniques such as cell fusion or gene recombination methods, such as pMCA1 / JM109 (FERM AP-20056), etc. Any strain having the above-mentioned ability can be used in the present invention. In addition, these microorganisms should be used in the form of microbial cells or processed microbial products, for example, microbial cell concentrate, dried microbial cells, microbial cell lysates, microbial cell extracts or purified enzymes, or fixed carriers thereof. Can do.
生体触媒反応に適したｔｅｒｔ−ロイシンアミド初期濃度は０．０１ｗｔ％〜飽和濃度、好ましくは１〜２０ｗｔ％、より好ましくは５〜２０ｗｔ％である。低濃度域では反応液体積当りの生産性が非常に低くなり、高濃度域では酵素の失活を招きやすい。なお、ｔｅｒｔ−ロイシンアミドの塩を原料として使用することも可能である。 The initial concentration of tert-leucinamide suitable for the biocatalytic reaction is 0.01 wt% to a saturated concentration, preferably 1 to 20 wt%, more preferably 5 to 20 wt%. Productivity per volume of the reaction solution is very low in the low concentration range, and the enzyme is easily deactivated in the high concentration range. It is also possible to use a tert-leucinamide salt as a raw material.
生体触媒の使用量はその比活性または活性量によって決定され、例えばｐＭＣＡ１／ＪＭ１０９（ＦＥＲＭ ＡＰ−２００５６）の場合にはｔｅｒｔ−ロイシンアミドに対して乾燥重量として重量比０．０００５〜３とするのが好ましく、０．０００５〜０．０５がより好ましい。 The amount of biocatalyst used is determined by the specific activity or the amount of activity. For example, in the case of pMCA1 / JM109 (FERM AP-20056), the dry weight with respect to tert-leucine amide is 0.0005-3. Is preferable, and 0.0005 to 0.05 is more preferable.
生体触媒反応に適した反応温度は生体触媒により異なり一概に規定することはできないが、一般的には１０〜７０℃の範囲が好ましい。 The reaction temperature suitable for the biocatalytic reaction differs depending on the biocatalyst and cannot be defined unconditionally, but generally it is preferably in the range of 10 to 70 ° C.
生体触媒反応に適したｐＨは使用する生体触媒により異なり一概に規定することはできないが、例えばｐＭＣＡ１／ＪＭ１０９（ＦＥＲＭ ＡＰ−２００５６）の場合にはｐＨ６〜１０が好適であり、ｐＨ６〜９がより好適である。遊離ｔｅｒｔ−ロイシンアミドを原料とする場合、原料水溶液のｐＨは１０．５程度となるため、生体触媒反応を至適ｐＨ条件で実施するためには酸を添加してｐＨを調整する必要がある。添加する酸の量は、使用する酵素により異なり一概に規定することはできないが、例えばｐＭＣＡ１／ＪＭ１０９（ＦＥＲＭ ＡＰ−２００５６）の場合には、ｐＨ調整に効果を有する範囲としてｔｅｒｔ−ロイシンアミドに対して０．００５〜１ｍｏｌ倍量、好ましくは０．００５〜０．５ｍｏｌ倍量である。また、ｔｅｒｔ−ロイシンアミドの酸塩を原料として使用し、その水溶液のｐＨよりも生体触媒の至適ｐＨが高い場合には、塩基を添加して至適ｐＨ付近で生体触媒反応を行うことが好ましい。原料としてｔｅｒｔ−ロイシンアミドを使用して酸を添加した場合においてもｔｅｒｔ−ロイシンアミドの酸塩を使用した場合においても、ｐＨが調整された水溶液は、添加した酸またはｔｅｒｔ−ロイシンアミドの酸塩に由来する酸根を含有する。 The pH suitable for the biocatalytic reaction differs depending on the biocatalyst used, and cannot be defined unconditionally. For example, in the case of pMCA1 / JM109 (FERM AP-20056), pH 6-10 is preferable, and pH 6-9 is more Is preferred. When free tert-leucinamide is used as a raw material, the pH of the aqueous raw material solution is about 10.5. Therefore, in order to carry out the biocatalytic reaction under optimum pH conditions, it is necessary to adjust the pH by adding an acid. . The amount of acid to be added differs depending on the enzyme to be used and cannot be specified in general. For example, in the case of pMCA1 / JM109 (FERM AP-20056), the range effective for pH adjustment is tert-leucinamide. 0.005 to 1 mol times, preferably 0.005 to 0.5 mol times. In addition, when an acid salt of tert-leucinamide is used as a raw material and the optimum pH of the biocatalyst is higher than the pH of the aqueous solution, a biocatalytic reaction can be performed near the optimum pH by adding a base. preferable. Whether the acid is added using tert-leucinamide as a raw material or the acid salt of tert-leucinamide is used, the aqueous solution whose pH is adjusted is the acid added or acid salt of tert-leucinamide. Contains acid radicals derived from
生体触媒を用いた反応系にさらに、Ｆｅ２＋、Ｍｎ２＋、Ｚｎ２＋、Ｎｉ２＋、Ｃｏ２＋などの各種金属イオンを反応溶液中に１〜５０ｐｐｍ添加することにより、立体選択的加水分解速度を向上させることができる。この際、添加する金属イオンは反応液内において非常に低濃度で添加するため、ゲル化の要因としては無視し得る。ｔｅｒｔ−ロイシンアミド水溶液への添加順序は特に限定されないが、より高い反応速度を得るために酸、金属イオン、生体触媒の順であることが好ましい。 Further, by adding 1 to 50 ppm of various metal ions such as Fe 2+ , Mn 2+ , Zn 2+ , Ni 2+ and Co 2+ to the reaction system using a biocatalyst, the stereoselective hydrolysis rate is improved. Can be made. At this time, the metal ions to be added are added at a very low concentration in the reaction solution, and thus can be ignored as a cause of gelation. The order of addition to the aqueous tert-leucinamide solution is not particularly limited, but is preferably in the order of acid, metal ion, and biocatalyst in order to obtain a higher reaction rate.
生体触媒反応の反応様式は、回分でも連続でも良い。反応装置は、撹拌機を備えた槽でも、固定化触媒を充填した塔でも、それらの組み合わせでも良い。 The reaction mode of the biocatalytic reaction may be batch or continuous. The reactor may be a tank equipped with a stirrer, a column packed with an immobilized catalyst, or a combination thereof.
生体触媒反応は、Ｌ−またはＤ−ｔｅｒｔ−ロイシンアミドの９５％以上がＬ−またはＤ−ｔｅｒｔ−ロイシンに変換されるまで、好ましくはＬ−またはＤ−ｔｅｒｔ−ロイシンアミドが検出されなくなるまで行うと、収率上のみならず品質上も好適である。 The biocatalytic reaction is performed until 95% or more of the L- or D-tert-leucine amide is converted to L- or D-tert-leucine, preferably until no L- or D-tert-leucine amide is detected. And it is suitable not only in yield but also in quality.
生体触媒反応終了後、反応液中に存在する生体触媒は、例えば遠心分離、濾過膜あるいは吸着分離などの通常の分離手段により生体触媒を除くことが望ましい。さらに限外濾過し、または活性炭等の吸着剤を用いて微生物由来の有機物の大部分を除去するとより好適である。また、生体触媒反応に使用した生体触媒は、反応に使用した後も、遠心分離もしくはろ過操作などにより回収し、生体触媒反応の容器に戻すことにより再利用することができる。 After completion of the biocatalyst reaction, it is desirable that the biocatalyst present in the reaction solution is removed from the biocatalyst by ordinary separation means such as centrifugation, filtration membrane, or adsorption separation. Further, it is more preferable to perform ultrafiltration or remove most of organic substances derived from microorganisms using an adsorbent such as activated carbon. In addition, the biocatalyst used for the biocatalyst reaction can be reused by collecting it by centrifugal separation or filtration operation and returning it to the biocatalyst reaction container.
［アミドの遊離化］
生体触媒反応終了後または生体触媒除去後の反応液はｔｅｒｔ−ロイシンとｔｅｒｔ−ロイシンアミドおよびその塩を含有している。塩酸や硫酸等の無機酸とｔｅｒｔ−ロイシンアミドの塩はアルコールへの溶解挙動がｔｅｒｔ−ロイシンと類似しているが、後工程で光学活性ｔｅｒｔ−ロイシンとその対掌体であるｔｅｒｔ−ロイシンアミドを分離する目的から、塩基を添加してｔｅｒｔ−ロイシンアミドと塩を形成している酸と添加した塩基との塩を形成させることによりアミドの遊離化を行い、ｔｅｒｔ−ロイシンとｔｅｒｔ−ロイシンアミドの溶解性を大幅に異なる状態とする。塩基としては金属水酸化物、金属アルコラートなど通常用いられる強塩基および、アルカリ金属酢酸塩などの酢酸塩を用いることができる。ただし、光学活性ｔｅｒｔ−ロイシンアミド酢酸塩は、アルコールへの溶解度が高いことから、塩基添加による遊離化を行うことなくｔｅｒｔ−ロイシンアミドとｔｅｒｔ−ロイシンを分離できる。従って、塩基添加は溶媒置換に供する液に含まれる酢酸根以外の酸根を対象として行えばよく、塩基の添加量は、好ましくは溶媒置換に供する液に含まれる酢酸根以外の酸根に対して０．９５〜１．０５当量、さらに好ましくは１当量である。これより不足の場合には、得られる光学活性ｔｅｒｔ−ロイシン中にその対掌体であるｔｅｒｔ−ロイシンアミドが含有されるために好ましくなく、また過剰の場合にはｔｅｒｔ−ロイシンアミド塩として溶解除去されるべき酢酸分までもが酢酸塩として光学活性ｔｅｒｔ−ロイシン中に残留するために不要な塩分増加を招き好ましくない。
[Liberation of amide]
The reaction solution after completion of the biocatalytic reaction or after removal of the biocatalyst contains tert-leucine, tert-leucine amide and a salt thereof. Salts of inorganic acids such as hydrochloric acid and sulfuric acid and tert-leucine amide are similar to tert-leucine in the dissolution behavior in alcohols. However, optically active tert-leucine and its enantiomer tert-leucine amide are used in the subsequent steps. For the purpose of separating tert-leucine and tert-leucine amide, the amide is liberated by forming a salt between the acid forming the salt with tert-leucine amide and the added base, tert-leucine and tert-leucine amide The solubilities of the are significantly different. As the base, a commonly used strong base such as a metal hydroxide or metal alcoholate and an acetate such as an alkali metal acetate can be used. However, since optically active tert-leucine amide acetate has high solubility in alcohol, tert-leucine amide and tert-leucine can be separated without liberation by addition of a base. Therefore, the base addition may be performed on an acid radical other than the acetate radical contained in the liquid used for solvent substitution, and the amount of the base added is preferably 0 with respect to the acid radical other than the acetate radical contained in the liquid used for solvent substitution. .95 to 1.05 equivalents, more preferably 1 equivalent. When the amount is shorter than this, the enantiomer tert-leucine amide is contained in the obtained optically active tert-leucine, which is not preferable. When the amount is excessive, it is dissolved and removed as a tert-leucine amide salt. Even the acetic acid content to be formed remains in the optically active tert-leucine as the acetic acid salt, which causes an undesirable increase in salinity.
［酢酸根の添加］
溶媒置換の際にゲル化を引き起こさないという観点から、溶媒置換に供する液に含まれる酸根のうち４５ｍｏｌ％以上を酢酸根とすることが必要であり、含まれる酸根のうち５０ｍｏｌ％以上を酢酸根とすることがより好ましい。溶媒置換に供する液に酢酸根を含ませるには、酢酸、酢酸塩等を溶媒置換の前までに反応液に添加すればよい。また、アミドの遊離化と酢酸根の添加の両方の目的を兼ね、アルカリ金属酢酸塩などの酢酸塩を添加することも有効である。
[Addition of acetate radical]
From the viewpoint of not causing gelation at the time of solvent substitution, it is necessary to use 45 mol% or more of the acid radicals contained in the solution to be subjected to solvent substitution as acetate radicals, and 50 mol% or more of the acid radicals contained are acetate radicals. More preferably. In order to include acetic acid radicals in the solution used for solvent replacement, acetic acid, acetate, etc. may be added to the reaction solution before solvent replacement. It is also effective to add acetates such as alkali metal acetates for the purpose of both liberation of amides and addition of acetate radicals.
［溶媒置換］
続いて、溶媒置換を行う。反応液の溶媒を水からｔｅｒｔ−ロイシンの貧溶媒へと置換し、未反応のｔｅｒｔ−ロイシンアミドは溶解させ、光学活性ｔｅｒｔ−ロイシンを析出させたのちに回収する方法により、光学活性ｔｅｒｔ−ロイシンを容易に取得することができる。溶媒置換の方法は、減圧または常圧で濃縮した後にｔｅｒｔ−ロイシンの貧溶媒（以下単に貧溶媒とする）を加える方法、貧溶媒を加えて水と共沸させる方法など、通常の溶媒置換方法を用いることができる。
[Solvent substitution]
Subsequently, solvent replacement is performed. The solvent of the reaction solution is replaced with a poor solvent of tert-leucine from water, the unreacted tert-leucine amide is dissolved, and the optically active tert-leucine is precipitated and then recovered. Can be easily obtained. Solvent replacement methods include normal solvent replacement methods such as a method of adding a tert-leucine poor solvent (hereinafter simply referred to as a poor solvent) after concentration at reduced pressure or normal pressure, a method of adding a poor solvent and azeotroping with water, etc. Can be used.
貧溶媒として用いる有機溶媒は、ｔｅｒｔ−ロイシンアミドの溶解度が高く、光学活性ｔｅｒｔ−ロイシンの溶解度が低い溶媒としてアルコール類が好適である。より好適には、２−プロパノール、２−メチル−１−プロパノール、１−ブタノールおよび２−ブタノール等の炭素数３〜７のアルコール類が好適に使用される。さらには水と共沸し、水への溶解性が低い性質を有する有機溶媒は、溶媒の置換操作が容易になることから、より好適であり、特に２−メチル−１−プロパノールが有効である。この場合、生体触媒を除去した反応液、または該反応液を濃縮した後に有機溶媒を添加し、常圧または減圧下にて共沸させながら水を留去させ、貧溶媒へ置換していく方法を用いることができる。貧溶媒中の水分量は少ないほど光学活性ｔｅｒｔ−ロイシンの収率が高くなる。貧溶媒中の水分量は、好ましくは１０％未満、より好ましくは１％未満である。さらに、貧溶媒への置換を行い、光学活性ｔｅｒｔ−ロイシンを析出させたのちに回収する操作の温度は、特に限定されず、常用の温度域である。 The organic solvent used as the poor solvent is preferably an alcohol as a solvent having high solubility of tert-leucine amide and low solubility of optically active tert-leucine. More preferably, alcohols having 3 to 7 carbon atoms such as 2-propanol, 2-methyl-1-propanol, 1-butanol and 2-butanol are preferably used. Furthermore, an organic solvent having a property of being azeotroped with water and having low solubility in water is more preferable since the operation of replacing the solvent becomes easy, and 2-methyl-1-propanol is particularly effective. . In this case, the reaction liquid from which the biocatalyst has been removed, or a method of adding an organic solvent after concentrating the reaction liquid, distilling off water while azeotroping under normal pressure or reduced pressure, and replacing with a poor solvent Can be used. The smaller the amount of water in the poor solvent, the higher the yield of optically active tert-leucine. The amount of water in the poor solvent is preferably less than 10%, more preferably less than 1%. Furthermore, the temperature of the operation for recovering after substituting with a poor solvent and precipitating the optically active tert-leucine is not particularly limited, and is a usual temperature range.
［光学活性ｔｅｒｔ−ロイシンの回収］
析出した光学活性ｔｅｒｔ−ロイシンは、遠心分離や濾過などの通常の固液分離手段により容易に回収することができる。回収された光学活性ｔｅｒｔ−ロイシンは残留するｔｅｒｔ−ロイシンアミドを抽出する溶媒で洗浄した後、通風乾燥や真空乾燥などにより乾燥させ、ｔｅｒｔ−ロイシンアミドの混入のないものとして得られる。
[Recovery of optically active tert-leucine]
The precipitated optically active tert-leucine can be easily recovered by ordinary solid-liquid separation means such as centrifugation or filtration. The recovered optically active tert-leucine is washed with a solvent for extracting the remaining tert-leucine amide and then dried by ventilation drying, vacuum drying, or the like, and is obtained as a mixture free of tert-leucine amide.
次に、本発明を実施例および比較例をもってより具体的に説明する。ただし、本発明はこれらの例にのみ制限されるものではない。なお、ｔｅｒｔ−ロイシンアミドおよびｔｅｒｔ−ロイシンの定量には以下に示す高速液体クロマトグラフィー（ＨＰＬＣ）分析条件を用いた。
〔ＨＰＬＣ分析条件１〕
カラム：Ｌｉｃｈｒｏｓｏｒｂ ＲＰ−１８（４．６φ×２５０ｍｍ）
溶離液：過塩素酸５０ｍＭ水溶液
流速：０．５ｍｌ／ｍｉｎ
検出：ＲＩ
〔ＨＰＬＣ分析条件２〕
カラム：スミキラルＯＡ−５０００（４．６φ×５０ｍｍ）
溶離液：硫酸銅１ｍＭ水溶液
流速：０．６ｍｌ／ｍｉｎ
検出：ＵＶ ２５４ｎｍ
Next, the present invention will be described more specifically with reference to examples and comparative examples. However, the present invention is not limited only to these examples. The following high performance liquid chromatography (HPLC) analysis conditions were used for quantification of tert-leucine amide and tert-leucine.
[HPLC analysis condition 1]
Column: Licrosorb RP-18 (4.6φ × 250 mm)
Eluent: Perchloric acid 50 mM aqueous solution Flow rate: 0.5 ml / min
Detection: RI
[HPLC analysis condition 2]
Column: Sumichiral OA-5000 (4.6φ × 50 mm)
Eluent: Copper sulfate 1 mM aqueous solution Flow rate: 0.6 ml / min
Detection: UV 254nm
実施例１
［生体触媒の培養］
生体触媒としては、Ｌ−ｔｅｒｔ−ロイシンアミド立体選択的加水分解酵素を有する形質転換株ｐＭＣＡ１／ＪＭ１０９（ＦＥＲＭ ＡＰ−２００５６）の菌体を用いた。該株を下記の培地にて回分培養を行い、さらに遠心分離濃縮によって培養濃縮液を得た。
培地組成（ｐＨ７．０）
グリセリン ６０ｇ
ポリペプトン ４８ｇ
チアミン塩酸塩 ４．８ｍｇ
ＫＨ２ＰＯ４ ４．８ｇ
ＭｇＳＯ４・７Ｈ２Ｏ １．８ｇ
ＭｎＣｌ２・４Ｈ２Ｏ １２ｍｇ
ＦｅＳＯ４・７Ｈ２Ｏ ７２ｍｇ
Ｈ２Ｏ １２００ｍＬ
［光学活性ｔｅｒｔ−ロイシンの製造］
１００ｍＬのフラスコに６．５１ｇ（０．０５００ｍｏｌ）のＤＬ−ｔｅｒｔ−ロイシンアミドと水５８．１ｇを加えて溶解した後、３５％塩酸０．５２ｇ（０．００５０ｍｏｌ）を添加して溶液ｐＨを１０．５から８．６に調整した。さらに、塩化マンガン四水和物２．３ｍｇを添加し、生体触媒としてＬ−ｔｅｒｔ−ロイシンアミド立体選択的加水分解酵素を有する形質転換株ｐＭＣＡ１／ＪＭ１０９（ＦＥＲＭ ＡＰ−２００５６）を上記の培養濃縮液０．７５ｇ（乾燥菌体の重量として０．０６５ｇを含有）を接種し、４０℃において２０時間撹拌により立体選択的加水分解を行った。
反応の進行は高速液体クロマトグラフィー（ＨＰＬＣ）条件１で確認した。生体触媒添加後２０時間で原料ＤＬ−ｔｅｒｔ−ロイシンアミド中のＬ−ｔｅｒｔ−ロイシンアミドが検出限界以下となるまでＬ−ｔｅｒｔ−ロイシンに変換された。
反応後、反応液に活性炭および濾過助剤を加えて濾過することにより菌体を除去した濾液を取得した。濾液に酢酸０．３０ｇ（０．００５０ｍｏｌ、溶媒置換に供する液中の酸根における酢酸根（以下単に酢酸根）は５０モル％）、水酸化ナトリウム０．２０ｇ（０．００５０ｍｏｌ）を加えた後、濾液に２−メチル−１−プロパノール８０ｍＬを加え、２０ｋＰａにて含水濃度が１％に達するまで共沸脱水を行い、溶媒置換操作を行った。共沸脱水は沸点６０℃で進行し、共沸脱水終了時には沸点は７６℃であった。溶媒置換により析出した光学活性Ｌ−ｔｅｒｔ−ロイシンを吸引濾過により濾取し、７０℃に加温した２−メチル−１−プロパノール２２ｍＬで洗浄し、さらに２０℃のアセトン５５ｍＬで洗浄した後、４０℃で真空乾燥を行い、白色粉末を３．８５ｇ取得した。このＬ−ｔｅｒｔ−ロイシンの化学純度をＨＰＬＣ条件１で分析したところ、光学活性Ｌ−ｔｅｒｔ−ロイシンの化学純度は９１．５％であり、Ｌ−ｔｅｒｔ−ロイシン以外に塩化ナトリウムを含んでいた。ｔｅｒｔ−ロイシンアミドおよびその酸塩は検出されなかった。光学純度をＨＰＬＣ条件２で分析したところ、光学純度は９９％以上であった。また、原料ＤＬ−ｔｅｒｔ−ロイシンアミド中のＬ−ｔｅｒｔ−ロイシンアミドに対する収率は９６．０ｍｏｌ％であった。
Example 1
[Biocatalyst culture]
As a biocatalyst, cells of a transformed strain pMCA1 / JM109 (FERM AP-20056) having L-tert-leucinamide stereoselective hydrolase were used. The strain was subjected to batch culture in the following medium, and a culture concentrate was obtained by centrifugal concentration.
Medium composition (pH 7.0)
Glycerin 60g
Polypeptone 48g
Thiamine hydrochloride 4.8mg
KH2PO4 4.8g
MgSO4 · 7H2O 1.8g
MnCl2 · 4H2O 12mg
FeSO4 · 7H2O 72mg
H2O 1200mL
[Production of optically active tert-leucine]
In a 100 mL flask, 6.51 g (0.0500 mol) of DL-tert-leucine amide and 58.1 g of water were added and dissolved, and then 0.52 g (0.0050 mol) of 35% hydrochloric acid was added to adjust the solution pH to 10. Adjusted from .5 to 8.6. Further, 2.3 mg of manganese chloride tetrahydrate was added, and the transformant pMCA1 / JM109 (FERM AP-20056) having L-tert-leucinamide stereoselective hydrolase as a biocatalyst was added to the above culture concentrate. 0.75 g (containing 0.065 g as the weight of the dried cells) was inoculated and subjected to stereoselective hydrolysis by stirring at 40 ° C. for 20 hours.
The progress of the reaction was confirmed by high performance liquid chromatography (HPLC) condition 1. 20 hours after the addition of the biocatalyst, L-tert-leucine amide in the raw material DL-tert-leucine amide was converted to L-tert-leucine until it was below the detection limit.
After the reaction, activated carbon and a filter aid were added to the reaction solution and filtered to obtain a filtrate from which the cells were removed. After adding 0.30 g of acetic acid (0.0050 mol, acetic acid radical (hereinafter simply referred to as acetic acid radical) in the liquid to be used for solvent substitution) to the filtrate, 0.20 g (0.0050 mol) of sodium hydroxide was added, To the filtrate, 80 mL of 2-methyl-1-propanol was added, and azeotropic dehydration was performed at 20 kPa until the water concentration reached 1%, and a solvent replacement operation was performed. Azeotropic dehydration proceeded at a boiling point of 60 ° C., and the boiling point was 76 ° C. at the end of azeotropic dehydration. The optically active L-tert-leucine precipitated by solvent substitution was collected by suction filtration, washed with 22 mL of 2-methyl-1-propanol heated to 70 ° C., and further washed with 55 mL of acetone at 20 ° C. Vacuum drying was performed at 0 ° C. to obtain 3.85 g of a white powder. When the chemical purity of this L-tert-leucine was analyzed under HPLC condition 1, the chemical purity of the optically active L-tert-leucine was 91.5%, and sodium chloride was included in addition to L-tert-leucine. Tert-leucine amide and its acid salt were not detected. When the optical purity was analyzed under HPLC condition 2, the optical purity was 99% or more. Moreover, the yield with respect to L-tert-leucinamide in raw material DL-tert-leucineamide was 96.0 mol%.
実施例２
実施例１と同様に立体選択的加水分解反応を行い、菌体を除去した後、添加する酢酸量を０．６０ｇ（０．０１００ｍｏｌ、酢酸根６７モル％）、水酸化ナトリウム量を０．２０ｇ（０．００５０ｍｏｌ）とした以外は実施例１と同様の操作を行った。白色粉末３．４３ｇを取得した。このＬ−ｔｅｒｔ−ロイシンの化学純度をＨＰＬＣ条件１で分析したところ、光学活性Ｌ−ｔｅｒｔ−ロイシンの化学純度は９１．５％であり、Ｌ−ｔｅｒｔ−ロイシン以外に塩化ナトリウムを含んでいた。ｔｅｒｔ−ロイシンアミドおよびその酸塩は検出されなかった。光学純度をＨＰＬＣ条件２で分析したところ、光学純度は９９％以上であった。また、原料ＤＬ−ｔｅｒｔ−ロイシンアミド中のＬ−ｔｅｒｔ−ロイシンアミドに対する収率は９５．６ｍｏｌ％であった。
Example 2
A stereoselective hydrolysis reaction was carried out in the same manner as in Example 1 to remove the cells, and then the amount of acetic acid to be added was 0.60 g (0.0100 mol, acetate root 67 mol%), and the amount of sodium hydroxide was 0.20 g. The same operation as in Example 1 was performed except that (0.0050 mol) was used. 3.43 g of white powder was obtained. When the chemical purity of this L-tert-leucine was analyzed under HPLC condition 1, the chemical purity of the optically active L-tert-leucine was 91.5%, and sodium chloride was included in addition to L-tert-leucine. Tert-leucine amide and its acid salt were not detected. When the optical purity was analyzed under HPLC condition 2, the optical purity was 99% or more. Moreover, the yield with respect to L-tert-leucinamide in raw material DL-tert-leucineamide was 95.6 mol%.
実施例３
菌体除去後に酢酸および水酸化ナトリウムに代えて酢酸ナトリウム０．４１ｇ（０．００５０ｍｏｌ、酢酸根５０モル％）を添加してから溶媒置換を行った以外は実施例１と同様の操作を行った。溶媒置換に際し、ゲル化は起こらず、白色粉末３．４１ｇを取得した。ＨＰＬＣ条件１で分析したところ、この粉末中のＬ−ｔｅｒｔ−ロイシン濃度は９１．４％であり、Ｌ−ｔｅｒｔ−ロイシンのほかに塩化ナトリウムが含まれていた。ｔｅｒｔ−ロイシンアミドおよびその酸塩は検出されなかった。ＨＰＬＣ条件２で分析したところ、Ｌ−ｔｅｒｔ−ロイシンの光学純度は９９％以上であった。また、原料ＤＬ−ｔｅｒｔ−ロイシンアミド中のＬ−ｔｅｒｔ−ロイシンアミドに対する収率は９５．１ｍｏｌ％であった。
Example 3
After removing the cells, the same operation as in Example 1 was performed except that the solvent was replaced after adding 0.41 g of sodium acetate (0.0050 mol, 50 mol% of acetate root) instead of acetic acid and sodium hydroxide. . Upon solvent substitution, gelation did not occur and 3.41 g of white powder was obtained. When analyzed under HPLC condition 1, the concentration of L-tert-leucine in this powder was 91.4%, and sodium chloride was contained in addition to L-tert-leucine. Tert-leucine amide and its acid salt were not detected. When analyzed under HPLC condition 2, the optical purity of L-tert-leucine was 99% or more. Moreover, the yield with respect to L-tert-leucinamide in raw material DL-tert-leucineamide was 95.1 mol%.
実施例４
ＤＬ−ｔｅｒｔ−ロイシンアミドに代えてＤＬ−ｔｅｒｔ−ロイシンアミド酢酸塩８．６１ｇ（０．０５００ｍｏｌ、酢酸根１００％）を用い、３５％塩酸に代えて水酸化ナトリウム１．６０ｇ（０．０４００ｍｏｌ）を加えてｐＨを８．４に調整し、菌体除去後、酢酸を添加せずに水酸化ナトリウム０．４０ｇ（０．０１００ｍｏｌ）を添加してから溶媒置換を行った以外は実施例１と同様の操作を行い、白色粉末７．２３ｇを取得した。ＨＰＬＣ条件１で分析したところ、この粉末中のＬ−ｔｅｒｔ−ロイシン濃度は４３．３％であり、Ｌ−ｔｅｒｔ−ロイシンのほかに酢酸ナトリウムが含まれていた。ｔｅｒｔ−ロイシンアミドおよびその酸塩は検出されなかった。ＨＰＬＣ条件２で分析したところ、Ｌ−ｔｅｒｔ−ロイシンの光学純度は９９％以上であった。また、原料ＤＬ−ｔｅｒｔ−ロイシンアミド中のＬ−ｔｅｒｔ−ロイシンアミドに対する収率は９５．６ｍｏｌ％であった。
Example 4
Instead of DL-tert-leucinamide, 8.61 g (0.0500 mol, acetate radical 100%) of DL-tert-leucineamide acetate was used, and 1.60 g (0.0400 mol) of sodium hydroxide was substituted for 35% hydrochloric acid. PH was adjusted to 8.4, and after removing the cells, Example 1 was repeated except that 0.40 g (0.0100 mol) of sodium hydroxide was added without adding acetic acid and then solvent replacement was performed. The same operation was performed to obtain 7.23 g of white powder. When analyzed under HPLC condition 1, the concentration of L-tert-leucine in this powder was 43.3%, and sodium acetate was contained in addition to L-tert-leucine. Tert-leucine amide and its acid salt were not detected. When analyzed under HPLC condition 2, the optical purity of L-tert-leucine was 99% or more. Moreover, the yield with respect to L-tert-leucinamide in raw material DL-tert-leucineamide was 95.6 mol%.
比較例１
実施例１と同様に立体選択的加水分解反応を行い、菌体を除去した後、酢酸を添加しない以外は実施例１と同様の操作を行った。溶媒を水から２−メチル−１−プロパノールに置換する途中、反応混合物全体がゲル化し、流動性が失われたため、Ｌ−ｔｅｒｔ−ロイシンを回収することができなかった。
Comparative Example 1
A stereoselective hydrolysis reaction was carried out in the same manner as in Example 1, and after removing the cells, the same operation as in Example 1 was carried out except that acetic acid was not added. During the replacement of the solvent from water with 2-methyl-1-propanol, the entire reaction mixture gelled and lost its fluidity, so L-tert-leucine could not be recovered.
比較例２
実施例１と同様に立体選択的加水分解反応を行い、菌体を除去した後、添加する酢酸量を０．０６ｇ（０．００１０ｍｏｌ、酢酸根１７モル％）とした以外は実施例１と同様の操作を行った。溶媒を水から２−メチル−１−プロパノールに置換する途中、反応混合物全体がゲル化し、流動性が失われたため、Ｌ−ｔｅｒｔ−ロイシンを回収することができなかった。
Comparative Example 2
Similar to Example 1, after performing a stereoselective hydrolysis reaction and removing the cells, the amount of acetic acid to be added was changed to 0.06 g (0.0010 mol, acetate root 17 mol%). Was performed. During the replacement of the solvent from water with 2-methyl-1-propanol, the entire reaction mixture gelled and lost its fluidity, so L-tert-leucine could not be recovered.
比較例３
実施例１と同様に立体選択的加水分解反応を行い、菌体を除去した後、添加する酢酸量を０．２０ｇ（０．００３３ｍｏｌ、酢酸根４０モル％）とした以外は実施例１と同様の操作を行った。溶媒を水から２−メチル−１−プロパノールに置換する途中、反応混合物全体がゲル化し、流動性が失われたため、Ｌ−ｔｅｒｔ−ロイシンを回収することができなかった。
Comparative Example 3
A stereoselective hydrolysis reaction was carried out in the same manner as in Example 1, and after removing the cells, the amount of acetic acid to be added was changed to 0.20 g (0.0033 mol, acetate root 40 mol%). Was performed. During the replacement of the solvent from water with 2-methyl-1-propanol, the entire reaction mixture gelled and lost its fluidity, so L-tert-leucine could not be recovered.
比較例４〜１０
実施例１と同様に立体選択的加水分解反応を行い、菌体を除去した後、酢酸に換えて表１の化合物（０．００５０ｍｏｌ）を添加した以外は実施例１と同様の操作を行った。いずれも溶媒を水から２−メチル−１−プロパノールに置換する途中、反応混合物全体がゲル化し、流動性が失われたため、Ｌ−ｔｅｒｔ−ロイシンを回収することができなかった。
A stereoselective hydrolysis reaction was carried out in the same manner as in Example 1, and after removing the cells, the same operation as in Example 1 was carried out except that the compound shown in Table 1 (0.0050 mol) was added instead of acetic acid. . In either case, L-tert-leucine could not be recovered because the entire reaction mixture gelled and the fluidity was lost during the replacement of the solvent from water with 2-methyl-1-propanol.
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| CN109563026A (en) * | 2016-07-28 | 2019-04-02 | 昭和电工株式会社 | The manufacturing method of a-amino acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6387998A (en) * | 1986-09-30 | 1988-04-19 | Mitsubishi Gas Chem Co Inc | Production of d-alpha-amino acid |
| JP2001328970A (en) * | 2000-05-18 | 2001-11-27 | Mitsubishi Rayon Co Ltd | Method for producing optically active alpha-amino acid and optically active alpha-amino acid amide |
| JP2002253293A (en) * | 2001-03-02 | 2002-09-10 | Mitsubishi Gas Chem Co Inc | METHOD FOR PRODUCING OPTICALLY ACTIVE L-tert-LEUCINE |
| JP2003225094A (en) * | 2001-12-06 | 2003-08-12 | Degussa Ag | D-AMIDASE DERIVED FROM VARIOVORAX, GENE ENCODING THE SAME, PLASMID, VECTOR AND MICROORGANISM INCLUDING SUCH NUCLEIC ACID, HYBRIDIZING NUCLEIC ACID, PRIMER FOR PRODUCING NUCLEIC ACID, METHOD FOR PRODUCING IMPROVED rec-AMIDASE, CODE FOR rec-AMIDASE AND NUCLEIC ACID, USE OF D-AMIDASE AND NUCLEIC ACID, AND WHOLE CELL CATALYST |
-
2009
- 2009-08-06 JP JP2009183537A patent/JP2011036135A/en not_active Ceased
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6387998A (en) * | 1986-09-30 | 1988-04-19 | Mitsubishi Gas Chem Co Inc | Production of d-alpha-amino acid |
| JP2001328970A (en) * | 2000-05-18 | 2001-11-27 | Mitsubishi Rayon Co Ltd | Method for producing optically active alpha-amino acid and optically active alpha-amino acid amide |
| JP2002253293A (en) * | 2001-03-02 | 2002-09-10 | Mitsubishi Gas Chem Co Inc | METHOD FOR PRODUCING OPTICALLY ACTIVE L-tert-LEUCINE |
| JP2003225094A (en) * | 2001-12-06 | 2003-08-12 | Degussa Ag | D-AMIDASE DERIVED FROM VARIOVORAX, GENE ENCODING THE SAME, PLASMID, VECTOR AND MICROORGANISM INCLUDING SUCH NUCLEIC ACID, HYBRIDIZING NUCLEIC ACID, PRIMER FOR PRODUCING NUCLEIC ACID, METHOD FOR PRODUCING IMPROVED rec-AMIDASE, CODE FOR rec-AMIDASE AND NUCLEIC ACID, USE OF D-AMIDASE AND NUCLEIC ACID, AND WHOLE CELL CATALYST |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109563026A (en) * | 2016-07-28 | 2019-04-02 | 昭和电工株式会社 | The manufacturing method of a-amino acid |
| CN109563026B (en) * | 2016-07-28 | 2021-12-28 | 昭和电工株式会社 | Process for producing alpha-amino acid |
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