JPS6391097A - Separation of d-phenylalanine - Google Patents
Separation of d-phenylalanineInfo
- Publication number
- JPS6391097A JPS6391097A JP23715986A JP23715986A JPS6391097A JP S6391097 A JPS6391097 A JP S6391097A JP 23715986 A JP23715986 A JP 23715986A JP 23715986 A JP23715986 A JP 23715986A JP S6391097 A JPS6391097 A JP S6391097A
- Authority
- JP
- Japan
- Prior art keywords
- phenylalanine
- acetyl
- acylase
- reaction
- crystals
- 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
- 238000000926 separation method Methods 0.000 title claims abstract description 11
- COLNVLDHVKWLRT-MRVPVSSYSA-N D-phenylalanine Chemical compound OC(=O)[C@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-MRVPVSSYSA-N 0.000 title abstract description 18
- 229930182832 D-phenylalanine Natural products 0.000 title abstract description 18
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims abstract description 73
- 229960005190 phenylalanine Drugs 0.000 claims abstract description 41
- 108700023418 Amidases Proteins 0.000 claims abstract description 33
- 102000005922 amidase Human genes 0.000 claims abstract description 33
- CBQJSKKFNMDLON-SNVBAGLBSA-N N-acetyl-D-phenylalanine Chemical compound CC(=O)N[C@@H](C(O)=O)CC1=CC=CC=C1 CBQJSKKFNMDLON-SNVBAGLBSA-N 0.000 claims abstract description 27
- 230000003287 optical effect Effects 0.000 claims abstract description 22
- 239000013078 crystal Substances 0.000 claims abstract description 21
- 239000007788 liquid Substances 0.000 claims abstract description 16
- 239000000706 filtrate Substances 0.000 claims abstract description 11
- CBQJSKKFNMDLON-UHFFFAOYSA-N N-acetylphenylalanine Chemical compound CC(=O)NC(C(O)=O)CC1=CC=CC=C1 CBQJSKKFNMDLON-UHFFFAOYSA-N 0.000 claims abstract description 9
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims abstract description 8
- CBQJSKKFNMDLON-JTQLQIEISA-M N-acetyl-L-phenylalaninate Chemical compound CC(=O)N[C@H](C([O-])=O)CC1=CC=CC=C1 CBQJSKKFNMDLON-JTQLQIEISA-M 0.000 claims abstract 3
- 230000003301 hydrolyzing effect Effects 0.000 claims abstract 3
- 238000006243 chemical reaction Methods 0.000 claims description 35
- 238000000034 method Methods 0.000 claims description 20
- 239000012141 concentrate Substances 0.000 claims 1
- 239000000243 solution Substances 0.000 abstract description 19
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 abstract description 18
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 9
- 239000007864 aqueous solution Substances 0.000 abstract description 8
- 239000002994 raw material Substances 0.000 abstract description 4
- 230000000202 analgesic effect Effects 0.000 abstract description 3
- 239000007787 solid Substances 0.000 abstract description 2
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 abstract 1
- 239000012670 alkaline solution Substances 0.000 abstract 1
- 238000004090 dissolution Methods 0.000 abstract 1
- 238000001914 filtration Methods 0.000 abstract 1
- 239000000413 hydrolysate Substances 0.000 abstract 1
- 150000002500 ions Chemical class 0.000 abstract 1
- 230000007935 neutral effect Effects 0.000 abstract 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- CBQJSKKFNMDLON-JTQLQIEISA-N N-acetyl-L-phenylalanine Chemical compound CC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 CBQJSKKFNMDLON-JTQLQIEISA-N 0.000 description 10
- 230000007062 hydrolysis Effects 0.000 description 6
- 235000011121 sodium hydroxide Nutrition 0.000 description 5
- 238000002425 crystallisation Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000003828 vacuum filtration Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 230000008025 crystallization Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- VJUNTPRQTFDQMF-UHFFFAOYSA-N 1-benzylimidazolidine-2,4-dione Chemical compound O=C1NC(=O)CN1CC1=CC=CC=C1 VJUNTPRQTFDQMF-UHFFFAOYSA-N 0.000 description 2
- -1 L-phenylalaninyl Chemical group 0.000 description 2
- OKJIRPAQVSHGFK-UHFFFAOYSA-N N-acetylglycine Chemical compound CC(=O)NCC(O)=O OKJIRPAQVSHGFK-UHFFFAOYSA-N 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical compound O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- XWKAVQKJQBISOL-ZETCQYMHSA-N (2s)-2-anilinopropanoic acid Chemical compound OC(=O)[C@H](C)NC1=CC=CC=C1 XWKAVQKJQBISOL-ZETCQYMHSA-N 0.000 description 1
- QDGAVODICPCDMU-UHFFFAOYSA-N 2-amino-3-[3-[bis(2-chloroethyl)amino]phenyl]propanoic acid Chemical compound OC(=O)C(N)CC1=CC=CC(N(CCCl)CCCl)=C1 QDGAVODICPCDMU-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 229910001429 cobalt ion Inorganic materials 0.000 description 1
- XLJKHNWPARRRJB-UHFFFAOYSA-N cobalt(2+) Chemical compound [Co+2] XLJKHNWPARRRJB-UHFFFAOYSA-N 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 108091022884 dihydropyrimidinase Proteins 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000004973 liquid crystal related substance Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Natural products CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 description 1
- 238000011197 physicochemical method Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
Landscapes
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
二工訟旦Ｕ二皿
本発明は、光学的に純度の高いＤ−フェニルアラニンの
分離方法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for separating D-phenylalanine with high optical purity.
Ｄ−フェニルアラニンは鎮痛作用を有し、鎮痛剤のほか
、抗生物質の合成原料などとしても最近注目を集めてい
る医薬、医薬原料として有用なアミノ酸である。D-phenylalanine has an analgesic effect and is an amino acid useful as a medicine and a raw material for pharmaceuticals, which has recently attracted attention not only as an analgesic but also as a synthetic raw material for antibiotics.
従来の技′ｒ及び発明が一゛しようとしている。・ｉｒ
占Ｄ−フェニルアラニンの製造及び分離方法に関しては
、ベンジルヒダントインをある種の微生物が生産する酵
素ヒダントイナーゼを作用させて酵素的にＤ−フェニル
アラニンを得る方法や、（発酵と工業 Ｖｏｌ １３
８ ＡＩＯＰ９３７外 ）また、優先晶出法、ジャスト
レオマー法などの物理化学的な手法による光学分割法に
よる方法が多数知られている。Conventional techniques and inventions are coming together.・IR
Regarding the production and separation method of D-phenylalanine, there is a method of enzymatically obtaining D-phenylalanine by treating benzylhydantoin with the enzyme hydantoinase produced by a certain microorganism, and a method of enzymatically obtaining D-phenylalanine (Fermentation and Industry Vol. 13)
8 AIOP937) In addition, many methods are known that use optical resolution methods using physicochemical methods such as preferential crystallization method and just-reomer method.
前者においては原料ベンジルヒダントインの工・ 業
的な入手に難があり、後者は完全な光学分割はできず、
かなりの量の異性体（Ｌ一体）が混入される。For the former, it is difficult to obtain the raw material benzylhydantoin, and for the latter, complete optical resolution is not possible.
A significant amount of the isomer (L mono) is mixed in.
Ｄ−フェニルアラニンは用途によっては光学異性体の混
入は厳して制限されており、含有量によってはその商品
価値を低下させる。したがってＤ−フェニルアラニンの
製造においては、極力Ｌ−フェニルアラニンの含有を減
少させる必要があるが、化学的方法によるＤ−フェニル
アラニンの製造においては、得られたＤ一体及びＬ一体
のラセミ体から高純度のＤ一体のみを得る分離に大変困
難をきたす。Depending on the use of D-phenylalanine, inclusion of optical isomers is strictly restricted, and depending on the content, its commercial value may be lowered. Therefore, in the production of D-phenylalanine, it is necessary to reduce the content of L-phenylalanine as much as possible, but in the production of D-phenylalanine by chemical methods, high purity D It is very difficult to separate to obtain only one piece.
例えばＮ−アセチルグリシンとベンズアルデヒドの縮合
などの化学的方法で得られたラセミ体反応液から、通常
のアミノ酸の光学分割法として知られているＮ−アセチ
ル−Ｌ−フェニルアラニンのみ加水分解させるアシラー
ゼ酵素を用いたアシラーゼ反応後、反応液を固液分離に
付した場合、加水分解により生成したＬ−フェニルアラ
ニンの溶解変分がＮ−アセチル−Ｄ−フェニルアラニン
の結晶に付着される。For example, from a racemic reaction solution obtained by a chemical method such as the condensation of N-acetylglycine and benzaldehyde, we use an acylase enzyme that hydrolyzes only N-acetyl-L-phenylalanine, which is known as a conventional optical resolution method for amino acids. When the reaction solution is subjected to solid-liquid separation after the reaction with the acylase used, the dissolved portion of L-phenylalanine produced by hydrolysis is attached to the crystals of N-acetyl-D-phenylalanine.
またアシラーゼ酵素による選択的加水分解反応において
は、効率のよい分離法を考慰するのは勿論だが、Ｌ−フ
ェニルアラニンの生成物濃度が高くなる？二１反応を「
１害する。したがってＮ−アセチル−ＤＬ−フェニルア
ラニンのアシラーゼ反応による光学分割においては１回
のアシラーゼ反応のみでは、限度があり、たとえ酵素を
多用してもＮ−アセチル−Ｌ−フェニルアラニンを充分
に反応せしめることはできず、得られるＮ−アセチル−
Ｄ−フェニルアラニン中にかなりの未反応し一体が混入
され、最終的に加水分解して得られるＤ−フェニルアラ
ニンの高光学純度を得るには問題があることもわかった
。Furthermore, in selective hydrolysis reactions using acylase enzymes, it is natural to consider efficient separation methods, but does this result in a high concentration of L-phenylalanine products? 21 reaction is ``
1 harm. Therefore, in the optical resolution of N-acetyl-DL-phenylalanine by acylase reaction, there is a limit to just one acylase reaction, and even if a large number of enzymes are used, N-acetyl-L-phenylalanine cannot be sufficiently reacted. The resulting N-acetyl-
It was also found that a considerable amount of unreacted solids were mixed into D-phenylalanine, which caused a problem in obtaining high optical purity of D-phenylalanine obtained by final hydrolysis.
問題点を解決するための手段
本発明者らはこのような問題点を踏まえ、化学的方法に
より得られたＮ−アセチル−ＤＬ−フェニルアラニンの
ラセミ体反応液からＮ−アセチル−Ｄ−フェニルアラニ
ンのみを効率よく分離し、高光学的純度を有するＤ−フ
ェニルアラニンを得る方法を鋭意検討し、本発明方法に
達したものである。Means for Solving the Problems In view of the above problems, the present inventors extracted only N-acetyl-D-phenylalanine from a racemic reaction solution of N-acetyl-DL-phenylalanine obtained by a chemical method. The method of the present invention was developed after intensive research into a method for efficiently separating D-phenylalanine with high optical purity.
すなわち、本発明方法は、１）Ｎ−アセチル−ＤＬ−フ
ェニルアラニンをアシラーゼ酵素を用いて光学分割を行
い、得られたアシラーゼ反応液を、２）濃縮後冷却して
出来るだけのＬ−フェニルアラニニルアラニンのみを完
全に溶解してろ液として出来るだけＬ−フェニルアラニ
ンを分離するかのいずれかの方法で、Ｌ−フェニルアラ
ニンを分離後、３）主にＮ−アセチル−Ｄ−フェニルア
ラニン及び未反応のＮ−アセチル−Ｌ−フェニルアラニ
ンよりなるろ液または結晶を、再度アシラーゼ反応に付
して、光学分割を行い、４）得られた反応終了液のＰ
ｌ−Ｉを再度１以下にして、生成残存するＬ−フェニル
アラニンを溶解、固液分離して、５）Ｎ−アセチルーＤ
−フェニルアラニンを加水分解に付することよりなる、
光学的高純度のＤ−フェニルアラニンを得る方法である
。That is, the method of the present invention involves 1) optically resolving N-acetyl-DL-phenylalanine using an acylase enzyme, and 2) concentrating and cooling the resulting acylase reaction solution to obtain as much L-phenylalaninyl as possible. After separating L-phenylalanine by either completely dissolving only alanine and separating as much L-phenylalanine as possible as a filtrate, 3) mainly N-acetyl-D-phenylalanine and unreacted N- The filtrate or crystals consisting of acetyl-L-phenylalanine is subjected to the acylase reaction again, optical resolution is performed, and 4) the P of the resulting reaction-completed liquid is
5) N-acetyl-D
- consisting of subjecting phenylalanine to hydrolysis,
This is a method for obtaining optically highly pure D-phenylalanine.
本発明は以下の様にして実施する。The present invention is carried out as follows.
Ｎ−アセチル−ＤＬ−フェニルアラニンのアシラーゼ酵
素によるアシラーゼ加水分解反応は、Ｎ−アセチルーＤ
Ｌ−フェニルアラニンの５〜４０％水溶液を苛性ソーダ
で中性付近もしくは弱アルカリ付近まで中和した水性媒
体中で、温度３０〜６０°Ｃ１反応時間１０〜７０ Ｈ
ｒで実施するのが好ましく、またアシラーゼの安定化の
ために通常行なわれている様にＣｏＣｄ２・６Ｉ−ｈｏ
を添加し系内のコバルトイオン濃度が１０−６Ｍ〜１０
−２Ｍの濃度となるようにして実施するのが望ましい。The acylase hydrolysis reaction of N-acetyl-DL-phenylalanine by the acylase enzyme produces N-acetyl-D
A 5-40% aqueous solution of L-phenylalanine was neutralized with caustic soda to near neutrality or weakly alkaline in an aqueous medium at a temperature of 30-60°C and a reaction time of 10-70 H.
CoCd2.6I-ho is preferably carried out, and CoCd2.6I-ho
is added to increase the cobalt ion concentration in the system from 10-6M to 10
It is desirable to carry out the experiment so that the concentration is -2M.
上記の様な方法で得られたアシラーゼ反応終了液中には
、加水分解により生成したＬ−フェニルアラニンの溶解
度が２％前後であるため、溶解度分取外のＬ−フェニル
アラニンの結晶が析出しており、未反応のＮ−アセチル
−Ｌ−フェニルアラニンとＮ−アセチル−Ｄ−フェニル
アラニン及び溶解変分のＬ−フェニルアラニンは溶液と
なっている。この反応水溶液からなるべく多くのＬ−フ
ェニルアラニンを系外へ除去するために、アシラーゼ反
応マスを濃縮した後、冷却を行ない固液分離によりＬ−
フェニルアラニンを系外へ除去する。Since the solubility of L-phenylalanine produced by hydrolysis is around 2% in the acylase reaction completed liquid obtained by the above method, crystals of L-phenylalanine that were not separated by solubility are precipitated. , unreacted N-acetyl-L-phenylalanine and N-acetyl-D-phenylalanine, and dissolved L-phenylalanine are in a solution. In order to remove as much L-phenylalanine as possible from this reaction aqueous solution out of the system, the acylase reaction mass is concentrated, cooled, and L-phenylalanine is subjected to solid-liquid separation.
Remove phenylalanine from the system.
その際、好ましくはＬ−フェニルアラニンの濃度が２０
％程度となるまで濃縮を行ない、０〜１０°Ｃまで冷却
してＬ−フェニルアラニンの結晶を固液分離するのがよ
い。At that time, preferably the concentration of L-phenylalanine is 20
% and then cooled to 0 to 10°C to separate solid-liquid crystals of L-phenylalanine.
また本発明においては、上記の濃縮、冷却による方法以
外に、Ｌ−フェニルアラニンを分離する方法としてアシ
ラーゼ反応終了後、直ちにＰＨを塩酸などで１以下にし
て、生成しているＬ−フェニルアラニンを完全に溶解さ
せ、ろ液として分離することもできる。その場合はＮ−
アセチル−Ｄ−フェニルアラニン及び未反応のＮ−アセ
チル−し−フェニルアラニンは結晶として析出するので
これを単離する。その際、水でよく洗浄して付着してい
るＬ−フェニルアラニンを十分除去するのがよい。In addition to the concentration and cooling methods described above, in the present invention, as a method for separating L-phenylalanine, immediately after the acylase reaction is completed, the pH is lowered to 1 or less with hydrochloric acid, etc., to completely remove the L-phenylalanine produced. It can also be dissolved and separated as a filtrate. In that case, N-
Acetyl-D-phenylalanine and unreacted N-acetyl-phenylalanine precipitate as crystals, which are isolated. At that time, it is preferable to wash thoroughly with water to sufficiently remove attached L-phenylalanine.
このようにしてＮ−フェニルアラニンを固液分離して得
られた、Ｎ−アセチル−Ｄ−フェニルアラニン及び未反
応のＮ−アセチル−Ｌ−フェニルアラニン及び若干のＬ
−フェニルアラニンを含むろ液または結晶は、Ｌ−フェ
ニルアラニン濃度が０．５％以内となる様に水で希釈さ
れ、この水溶液中に新たにアシラーゼ酵素を添加して再
度反応を行なわせる。N-acetyl-D-phenylalanine, unreacted N-acetyl-L-phenylalanine and some L-phenylalanine obtained by solid-liquid separation of N-phenylalanine in this way
- The filtrate or crystals containing phenylalanine is diluted with water so that the L-phenylalanine concentration is within 0.5%, and acylase enzyme is newly added to this aqueous solution and the reaction is performed again.
これにより、アシラーゼ酵素反応阻害もなく、未反応の
Ｎ−アセチル−Ｌ−フェニルアラニンは再度加水分解さ
せることができて極力減らすことができる。As a result, there is no inhibition of the acylase enzyme reaction, and unreacted N-acetyl-L-phenylalanine can be hydrolyzed again and can be reduced as much as possible.
二回目のアシラーゼ反応が終了したら、溶液のＰＨを塩
酸などで１以下とすることにより、生成しているＬ−フ
ェニルアラニンを溶解させ、未反応物のほとんど含まれ
ないＮ−アセチル−Ｄ−フェニルアラニンを析出させ固
液分離により単離する。その際好ましくはアシラーゼ反
応終了液は濃縮などによりＮ−アセチル−Ｄ−フェニル
アラニンの濃度を高くしておく方がよい。After the second acylase reaction is completed, the pH of the solution is lowered to 1 or less using hydrochloric acid, etc. to dissolve the L-phenylalanine that has been produced, and N-acetyl-D-phenylalanine, which contains almost no unreacted substances, is dissolved. It is precipitated and isolated by solid-liquid separation. In this case, it is preferable to increase the concentration of N-acetyl-D-phenylalanine in the acylase reaction-completed solution by concentrating it or the like.
かくして光学純度の高い、すなわちＤ一体音量９９％以
上のＮ−アセチル−Ｄ−フェニルアラニンを得ることが
できる。In this way, N-acetyl-D-phenylalanine with high optical purity, that is, a D-integral volume of 99% or more, can be obtained.
本発明のアシラーゼ反応において使用するアシラーゼ酵
素は、選択的にＮ−アセチルアミノ酸のＬ一体のみを加
水分解するものであればいかなる菌体より取得されたも
のでもよいが、アシラーゼ反応は通常中性付近もしくは
弱アルカリ付近で行なわれており、従って本発明におい
ても至適Ｐ Ｉ−Ｉが６〜９付近のアシラーゼを使用す
るのが好まし知釧苗−ストレプトミセス屈などの公知の
放線菌などから得られたものが使用できる。The acylase enzyme used in the acylase reaction of the present invention may be obtained from any bacterial cell as long as it selectively hydrolyzes only the L-unit of N-acetylamino acids; Acylases are also used in the vicinity of weak alkalinity, and therefore, in the present invention, it is preferable to use an acylase with an optimum P I-I of around 6 to 9. You can use what you get.
本発明方法により得られたＮ−アセチル−Ｄ−フェニル
アラニンの加水分解は、常法に従い実施できる。即ち塩
酸使用量がＮ−アセチル−Ｄ−フェニルアラニンの１．
２５倍モル程度、Ｎ−アセチル−Ｄ−フェニルアラニン
の濃度が１０〜３０％の塩酸水溶液中で、加熱還流を数
時間行ない冷却、中和して析出した結晶を固液分離する
ことにより高収量で光学純度の高い（光学純度９９％以
上）のＤ−フェニルアラニンを得ることができる。Hydrolysis of N-acetyl-D-phenylalanine obtained by the method of the present invention can be carried out according to a conventional method. That is, the amount of hydrochloric acid used is 1.5% of N-acetyl-D-phenylalanine.
In a hydrochloric acid aqueous solution containing about 25 times the mole of N-acetyl-D-phenylalanine and a concentration of 10 to 30%, it is heated under reflux for several hours, cooled and neutralized, and the precipitated crystals are solid-liquid separated, resulting in a high yield. D-phenylalanine with high optical purity (optical purity of 99% or more) can be obtained.
また副生したＬ−フェニルアラニンは、単離し−ＤＬ−
フェニルアラニンとして再使用することもできる。In addition, the by-produced L-phenylalanine was isolated and -DL-
It can also be reused as phenylalanine.
以下実施例を示す。Examples are shown below.
実施例Ｉ
Ｎ−アセチル−ＤＬ−フェニルアラニン１００ｇをイオ
ン交換水及び２０％苛性ソーダ水溶液にて溶解し、ｐＨ
７，５に合わせる。ＣｏＣｌ２・６Ｈ２０を反応系内の
Ｃｏｚ＋濃度が５Ｘ１０−４Ｍとなる様に添加した。反
応液の全重量が５５０ｇとなる様にして、大野製薬■製
アシラーゼ酵素１．Ｂ（１８゜０００Ｕ／ｇ）を添加し
て４０°Ｇ／４０Ｈｒで反応を行った。反応開始後１０
Ｉ（ｒ付近より結晶が析出し始めた。Example I 100 g of N-acetyl-DL-phenylalanine was dissolved in ion-exchanged water and 20% caustic soda aqueous solution, and the pH
Adjust to 7.5. CoCl2.6H20 was added so that the Coz+ concentration in the reaction system was 5X10-4M. Acylase enzyme 1. manufactured by Ohno Pharmaceutical ■ was added so that the total weight of the reaction solution was 550 g. B (18°000 U/g) was added and the reaction was carried out at 40°G/40Hr. 10 after starting the reaction
Crystals began to precipitate near I(r).
反応終了後、反応液を減圧下（約１００＋ｘ／）Ｉｇ）
で濃縮し、水留去を行ない濃縮液１ｓｏｇｃＬ−フェニ
ルアラニン２０％濃縮液）を得た。After the reaction is completed, the reaction solution is reduced under reduced pressure (approximately 100+x/)Ig)
and water was distilled off to obtain a concentrated solution (1sogcL-phenylalanine 20% concentrated solution).
反応終了マスのＨＬＣ分析の結果、Ｎ−アセチル−Ｌ−
フェニルアラニンよりＬ−フェニルアラニンへの転換率
は８８％であった。As a result of HLC analysis of the reaction completed mass, N-acetyl-L-
The conversion rate of phenylalanine to L-phenylalanine was 88%.
次に濃縮液を冷却して、１０°（：、／２Ｈｒ晶出後、
ヌッチェにより真空ろ過を行ない、Ｌ−フェニルアラニ
ンの粗結晶を約４０ｇ（Ｄｒｙ換算３３．０．！？）ど
ろ液１４１ｇを得た。ろ液中のＬ−フェニルアラニン濃
度は１．５％であった。Next, the concentrated liquid was cooled to 10° (:, /2Hr after crystallization,
Vacuum filtration was performed using a Nutsche filter to obtain approximately 40 g of crude crystals of L-phenylalanine (33.0.!? in dry terms) and 141 g of filtrate. The L-phenylalanine concentration in the filtrate was 1.5%.
得られたろ液を水で希釈して全体を４５０ｇとし、さら
に２０％苛性ソーダ水溶液にてＰＨを７．５に合わせた
。ついで大野製薬■製アシラーゼ酵素０．７ ｇを添加
して４０℃／ ４０１−ｂで２回目の反応を行なった。The obtained filtrate was diluted with water to give a total weight of 450 g, and the pH was adjusted to 7.5 with a 20% aqueous sodium hydroxide solution. Then, 0.7 g of acylase enzyme (manufactured by Ohno Pharmaceutical Co., Ltd.) was added, and a second reaction was carried out at 40°C/401-b.
２回目アシラーゼ反応終了後の反応液中のＬ−フェニル
アラニン濃度は０．４７％より１．１１％まで上昇し、
Ｎ−アセチル−Ｌ−フェニルアラニンの大部分がＬ−フ
ェニルアラニンへ転換していることを示す。After the second acylase reaction, the L-phenylalanine concentration in the reaction solution increased from 0.47% to 1.11%,
This shows that most of N-acetyl-L-phenylalanine has been converted to L-phenylalanine.
得られた反応終了液を約３００９まで減圧下に濃縮し、
室温にて濃塩酸を加えてＰＨ１とした。引続き１０°Ｇ
／２１（ｒ品出を行ない、ヌッチェで真空ろ過後水洗、
乾燥を行ない、Ｎ−アセチル−Ｄ−フェニルアラニンの
結晶４２．８ｇを得た。The obtained reaction-completed liquid was concentrated under reduced pressure to about 300%,
The pH was adjusted to 1 by adding concentrated hydrochloric acid at room temperature. Continued at 10°G
/21 (r) After vacuum filtration with Nutsche and washing with water,
After drying, 42.8 g of N-acetyl-D-phenylalanine crystals were obtained.
この結晶は純度９９．６％、旋光度〔α沼＝−４０，２
°（Ｃ＝ ２ 、 ｃｌ−１３０ｘ−ｘ ）−ｃあり、
光学的にほぼ純品のＮ−アセチル−Ｄ−フェニルアラニ
ンであることが確認された。This crystal has a purity of 99.6% and an optical rotation [α=-40.2
°(C=2, cl-130x-x) - with c,
It was confirmed that it was optically almost pure N-acetyl-D-phenylalanine.
上記により得られたＮ−アセチル−Ｄ−フェニルアラニ
ン２５ｇを、濃塩酸及び水に溶解して、全容量が１００
ｇ、塩酸濃度を７％として、加熱還流を８ Ｈｒ行ない
、冷却後３２％苛性ソーダ水溶液にテＰ Ｈを５．０と
し、１０ ’Ｃ／ ２ Ｈｒ晶析を行なった。25 g of N-acetyl-D-phenylalanine obtained above was dissolved in concentrated hydrochloric acid and water until the total volume was 100 g.
g, hydrochloric acid concentration was set to 7%, heating and refluxing was carried out for 8 hours, and after cooling, crystallization was carried out in a 32% caustic soda aqueous solution at pH 5.0 for 10'C/2 hours.
ヌッチェによる真空ろ過し、水にて洗浄後、結晶を乾燥
して白色のＤ−フェニルアラニン精結晶１５．２ｇを得
た。After vacuum filtration using a Nutsche filter and washing with water, the crystals were dried to obtain 15.2 g of white D-phenylalanine crystals.
本品は純度１００．１％、〔α〕譬−＋３４６°（Ｃ＝
２、水）であり、光学異性体分離用カラム（ダイセル社
製キラルパック）で分析した結果、Ｌ一体は０．３％の
混入であった。This product has a purity of 100.1%, [α] +346° (C =
2, water), and as a result of analysis using a column for optical isomer separation (Chiral Pack, manufactured by Daicel), it was found that 0.3% of L was present.
比較例
実施例１と同様に行なった。ただしアシラーゼ使用量は
倍量の２．８ｇとし、２回目のアシラーゼ反応はカット
した。得られたＮ−アセチル−Ｄ−フェニルアラニンの
旋光度〔α躍−−３６．２° （Ｃ＝２、Ｇ−１３０Ｉ
４ ）と低く、さらにこれより得られたＤ−フェニルア
ラニンの〔α）宕＝ ＋２９．４°と低かった。Comparative Example The same procedure as in Example 1 was carried out. However, the amount of acylase used was doubled to 2.8 g, and the second acylase reaction was cut. The optical rotation of the obtained N-acetyl-D-phenylalanine [α jump - 36.2° (C=2, G-130I
4), and furthermore, the D-phenylalanine obtained therefrom was as low as [α) = +29.4°.
この場合り一体の８％が混入していた。In this case, 8% of grains were mixed in.
実施例２
実施例１と同様にして一回目の光学分割反応を行ない、
濃縮液を得た。濃縮液に室温にて濃塩酸８６ｇを加えて
ＰＨを１．０とし、５°Ｃに冷却、２Ｈｒ晶出を行なっ
た。析出している未反応のＮ−アセチル−Ｌ−フェニル
アラニンを含むＮ−アセチル−Ｄ−フェニルアラニンの
結晶をろ別し、乾燥後４６ｇを得た。Example 2 The first optical resolution reaction was carried out in the same manner as in Example 1,
A concentrated solution was obtained. 86 g of concentrated hydrochloric acid was added to the concentrated solution at room temperature to adjust the pH to 1.0, and the mixture was cooled to 5°C and crystallized for 2 hours. The precipitated N-acetyl-D-phenylalanine crystals containing unreacted N-acetyl-L-phenylalanine were filtered off and dried to obtain 46 g.
この結晶は、純度９８．７％、旋光度〔α〕乞−−３２
．４°（Ｃ−２、Ｃｌ−１３０１１）。光学純度は約９
０％であった。This crystal has a purity of 98.7% and an optical rotation [α] of -32
．． 4° (C-2, Cl-13011). Optical purity is approximately 9
It was 0%.
得られた結晶４０ｇを用いて水及び２０％苛性ソーダ水
溶液にてＰ １１を７．５、全容量を２２０ｇとなる様
にし、大野製薬■製アシラーゼ酵素０．７タを添加して
４０°Ｇ／４０Ｈｒで２回目の反応を行なった。反応液
中には新たにＬ−フェニルアラニンが重量で２．９ｇ生
成しており、Ｎ−アセチル−Ｌ−フェニルアラニンの大
部分はＬ−フェニルアラニンへ転換していることを示す
。Using 40 g of the obtained crystals, adjust the P 11 to 7.5 and the total volume to 220 g with water and a 20% caustic soda aqueous solution, add 0.7 g of acylase enzyme manufactured by Ohno Pharmaceutical ■, and heat at 40°G/ A second reaction was carried out for 40 hours. 2.9 g of L-phenylalanine was newly produced in the reaction solution, indicating that most of the N-acetyl-L-phenylalanine was converted to L-phenylalanine.
上記により得られた反応終了液を室温にて濃塩酸を加え
ＰＨ１，０とし、さらに１０ ’Ｃ／ ２１１ ｒ晶出
後ヌツチェで真空ろ過、水洗、乾燥を行ない、Ｎ−アセ
チルーＤ−フェニルアラニンの結晶３２７を得た。The reaction-completed solution obtained above was adjusted to pH 1.0 by adding concentrated hydrochloric acid at room temperature, and after crystallization at 10'C/211r, vacuum filtration was performed using a Nutsche filter, washing with water, and drying were performed to obtain crystals of N-acetyl-D-phenylalanine. I got 327.
コノ結晶ハ純度９９．８％。（α〕”、’ ＝−４０，
３°（Ｃ＝２、ＣＨ３０Ｈ）であり、はぼ純品のＮ−ア
セチル−Ｄ−フェニルアラニンであることが確認された
。Konocrystal purity is 99.8%. (α〕”,' =-40,
3° (C=2, CH30H), and it was confirmed that this was pure N-acetyl-D-phenylalanine.
上記により得られたＮ−アセチル−Ｄ−フェニルアラニ
ン２５７を用いて実施例１と同様に加水分解を行ない、
Ｄ−フェニルアラニン精結晶１４．８夕を得た。本品は
純度９９．８％、〔α九＝ ＋３４．８゜（Ｃ＝２、水
）であり光学異性体分離用カラム（ダイセル社製、キラ
ルパック）で分析した結果、Ｌ一体は０．２％の混入で
あった。Hydrolysis was carried out in the same manner as in Example 1 using N-acetyl-D-phenylalanine 257 obtained above,
14.8 minutes of D-phenylalanine crystals were obtained. This product has a purity of 99.8% [α9 = +34.8° (C = 2, water)], and as a result of analysis using a column for optical isomer separation (Chiral Pack, manufactured by Daicel), the L content was 0. The contamination was 2%.
Claims (2)
シラーゼ酵素を用いて光学分割を行い、得られたアシラ
ーゼ反応液を ２）濃縮、冷却して反応液中のＬ−フェニルアラニンを
結晶として固液分離した後、 ３）主にＮ−アセチル−Ｄ−フェニルアラニン及び未反
応のＮ−アセチル−Ｌ−フェニルアラニンよりなるろ液
を再度光学分割を行い、 ４）得られた反応終了液のＰＨを１以下にして生成残存
するＬ−フェニルアラニンを溶解させ、 ５）固液分離したＮ−アセチル−Ｄ−フェニルアラニン
を加水分解に付すことよりなる、光学的に純度の高いＤ
−フェニルアラニンの分離方法(1) 1) Perform optical resolution of N-acetyl-DL-phenylalanine using an acylase enzyme, and 2) Concentrate and cool the resulting acylase reaction solution to crystallize L-phenylalanine in the reaction solution and separate it into solid-liquid. After that, 3) optically resolve the filtrate, which mainly consists of N-acetyl-D-phenylalanine and unreacted N-acetyl-L-phenylalanine, and 4) reduce the pH of the resulting reaction-completed liquid to 1 or less. and 5) hydrolyzing the solid-liquid separated N-acetyl-D-phenylalanine.
-Method for separating phenylalanine
シラーゼ酵素を用いて光学分割を行い、得られたアシラ
ーゼ反応液を ２）ＰＨ１以下にして、反応液中の生成Ｌ−フェニルア
ラニンをろ液として固液分離した後、 ３）主にＮ−アセチル−Ｄ−フェニルアラニン及び未反
応のＮ−アセチル−Ｌ−フェニルアラニンよりなる結晶
を再度光学分割を行い、 ４）得られた反応終了液のＰＨを１以下にして生成残存
するＬ−フェニルアラニンを溶解させ、 ５）固液分離したＮ−アセチル−Ｄ−フェニルアラニン
を加水分解に付すことよりなる、光学的に純度の高いＤ
−フェニルアラニンの分離方法(2) 1) Perform optical resolution of N-acetyl-DL-phenylalanine using an acylase enzyme, and 2) lower the pH of the resulting acylase reaction solution to 1 or less to solidify the L-phenylalanine produced in the reaction solution as a filtrate. After liquid separation, 3) optically resolve the crystals mainly consisting of N-acetyl-D-phenylalanine and unreacted N-acetyl-L-phenylalanine, and 4) lower the pH of the resulting reaction-completed liquid to 1 or less. and 5) hydrolyzing the solid-liquid separated N-acetyl-D-phenylalanine to obtain optically pure D.
-Method for separating phenylalanine
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23715986A JPH0634751B2 (en) | 1986-10-07 | 1986-10-07 | Method for separating D-phenylalanine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23715986A JPH0634751B2 (en) | 1986-10-07 | 1986-10-07 | Method for separating D-phenylalanine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6391097A true JPS6391097A (en) | 1988-04-21 |
| JPH0634751B2 JPH0634751B2 (en) | 1994-05-11 |
Family
ID=17011269
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP23715986A Expired - Lifetime JPH0634751B2 (en) | 1986-10-07 | 1986-10-07 | Method for separating D-phenylalanine |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0634751B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4912042A (en) * | 1989-08-17 | 1990-03-27 | Eastman Kodak Company | Preparation of D-malic acid or derivative |
-
1986
- 1986-10-07 JP JP23715986A patent/JPH0634751B2/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4912042A (en) * | 1989-08-17 | 1990-03-27 | Eastman Kodak Company | Preparation of D-malic acid or derivative |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0634751B2 (en) | 1994-05-11 |
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