JPS63207399A - Production of d-alpha-amino acid - Google Patents
Production of d-alpha-amino acidInfo
- Publication number
- JPS63207399A JPS63207399A JP3912887A JP3912887A JPS63207399A JP S63207399 A JPS63207399 A JP S63207399A JP 3912887 A JP3912887 A JP 3912887A JP 3912887 A JP3912887 A JP 3912887A JP S63207399 A JPS63207399 A JP S63207399A
- Authority
- JP
- Japan
- Prior art keywords
- substituted
- reaction
- amino acid
- continuously
- bacterial cells
- 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
- 150000007650 D alpha amino acids Chemical class 0.000 title claims description 24
- 238000004519 manufacturing process Methods 0.000 title claims description 9
- 238000000034 method Methods 0.000 claims abstract description 26
- 244000005700 microbiome Species 0.000 claims abstract description 24
- 150000001469 hydantoins Chemical class 0.000 claims abstract description 18
- 238000006243 chemical reaction Methods 0.000 claims abstract description 15
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims abstract description 5
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 4
- 230000001580 bacterial effect Effects 0.000 claims description 33
- 229940091173 hydantoin Drugs 0.000 claims description 15
- 239000000126 substance Substances 0.000 claims 2
- 125000000547 substituted alkyl group Chemical group 0.000 claims 1
- 238000000108 ultra-filtration Methods 0.000 abstract description 16
- 241000235648 Pichia Species 0.000 abstract description 10
- 239000000758 substrate Substances 0.000 abstract description 10
- 102000004190 Enzymes Human genes 0.000 abstract description 8
- 108090000790 Enzymes Proteins 0.000 abstract description 8
- 239000000047 product Substances 0.000 abstract description 8
- 238000003860 storage Methods 0.000 abstract description 7
- 230000000813 microbial effect Effects 0.000 abstract description 6
- 239000007864 aqueous solution Substances 0.000 abstract description 5
- 239000007795 chemical reaction product Substances 0.000 abstract description 5
- 239000007788 liquid Substances 0.000 abstract description 5
- PBNUQCWZHRMSMS-UHFFFAOYSA-N 5-propan-2-ylimidazolidine-2,4-dione Chemical compound CC(C)C1NC(=O)NC1=O PBNUQCWZHRMSMS-UHFFFAOYSA-N 0.000 abstract description 3
- 241000320412 Ogataea angusta Species 0.000 abstract description 3
- 239000012295 chemical reaction liquid Substances 0.000 abstract description 3
- 239000000706 filtrate Substances 0.000 abstract description 3
- 150000001875 compounds Chemical class 0.000 abstract 2
- 239000011541 reaction mixture Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 19
- 230000003287 optical effect Effects 0.000 description 10
- 239000012528 membrane Substances 0.000 description 9
- 235000001014 amino acid Nutrition 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 239000012510 hollow fiber Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 150000001371 alpha-amino acids Chemical class 0.000 description 2
- 235000008206 alpha-amino acids Nutrition 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- NXQJDVBMMRCKQG-UHFFFAOYSA-N 5-phenylimidazolidine-2,4-dione Chemical compound O=C1NC(=O)NC1C1=CC=CC=C1 NXQJDVBMMRCKQG-UHFFFAOYSA-N 0.000 description 1
- 241000588986 Alcaligenes Species 0.000 description 1
- 241000186063 Arthrobacter Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- KZSNJWFQEVHDMF-SCSAIBSYSA-N D-valine Chemical compound CC(C)[C@@H](N)C(O)=O KZSNJWFQEVHDMF-SCSAIBSYSA-N 0.000 description 1
- 229930182831 D-valine Natural products 0.000 description 1
- 241000589565 Flavobacterium Species 0.000 description 1
- 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 1
- 241001057811 Paracoccus <mealybug> Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 125000003545 alkoxy group Chemical group 0.000 description 1
- 125000004453 alkoxycarbonyl group Chemical group 0.000 description 1
- 125000004414 alkyl thio group Chemical group 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 125000005843 halogen group Chemical group 0.000 description 1
- WJRBRSLFGCUECM-UHFFFAOYSA-N hydantoin Chemical compound O=C1CNC(=O)N1 WJRBRSLFGCUECM-UHFFFAOYSA-N 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 125000001041 indolyl group Chemical group 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 235000019710 soybean protein Nutrition 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000007218 ym medium Substances 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５−置換ヒダントイン類をＤ−α−アミノ酸に
変換する能力を有するハンセニュラ（Ｈａｎｓｅｎｕｌ
ａ）属に属する微生物を用いることにより゛Ｄ−α−ア
ミノ酸を極めて高収率かつ連続的に製造する方法に関す
るものである。Detailed Description of the Invention (Industrial Application Field) The present invention is directed to the use of Hansenul, which has the ability to convert 5-substituted hydantoins into D-α-amino acids.
The present invention relates to a method for continuously producing D-α-amino acids at extremely high yields by using microorganisms belonging to the genus a).
（従果の技術とその問題点）
Ｄ−α−アミノ酸の製造方法の一つとして対応する５−
置換ヒダントインを化学的に氷解して、ＤＬ−アミノ酸
を製造し、これを光学分割してＤ−α−アミノ酸とする
方法が知られている。しかし、この方法は特に光学分割
の工程が煩雑であり、その収率も高くない。また、５−
置換ヒダントインに微生物の培養液、菌体、菌体処理物
または菌体から抽出した酵素を作用させて光学活性を有
するＤ−Ｎ−カルバミル−α−アミノ酸を生成させた後
、亜硝酸ソーダ液によりＤ−α−アミノ酸とする方法が
しられているが、しかし、この方法も反応工程および精
製工程が煩雑である。また、更に５−置換ヒダントイン
にある種の微生物−ｍ−例えばシュードモナス（Ｐｓｅ
ｕｄｏｍｏｎａｓ） 、モラキセラ（Ｍｏｒａｘｅｌｌ
ａ） 、バラコツカス（ＰａｒａｃｏｃｃｕＳ）、アー
スロバフタ−（八ｒｔｈｒｏｂａｃｔｅｒ）、アルカリ
ジェネス（Ａｌｃａｌｉｇｅｎｅｓ）、フラボバクテリ
ウム（Ｆｌａｖｏｂａｃｔｅｒｉｕｍ）−−−−の培養
液、菌体、菌体処理物を作用させて直接にＤ−α−アミ
ノ酸とする方法も知られているが収率は高くない。(Technology of subsidiary fruit and its problems) 5- corresponding as one of the methods for producing D-α-amino acid
A known method is to chemically thaw a substituted hydantoin to produce a DL-amino acid, which is then optically resolved into a D-α-amino acid. However, this method requires particularly complicated optical resolution steps, and the yield is not high. Also, 5-
Substituted hydantoin is reacted with microorganism culture solution, bacterial cells, treated bacterial cells, or enzymes extracted from bacterial cells to produce optically active D-N-carbamyl-α-amino acids, and then treated with a sodium nitrite solution. A method of producing a D-α-amino acid is known, but this method also involves complicated reaction steps and purification steps. Additionally, 5-substituted hydantoins may be added to certain microorganisms such as Pseudomonas (Pse).
udomonas), Moraxell
a) D- directly by acting with a culture solution, bacterial cells, or treated bacterial cells of Paracoccus S, Arthrobacter, Alcaligenes, Flavobacterium. A method for producing α-amino acids is also known, but the yield is not high.
本発明者らは、この様な従来の製造方法に対し、より効
率のよい方法を見い出すべく研究した結果、ハンセニュ
ラ（Ｈａｎｓｅｎｕｌａ）属の微生物を用いて５−置換
ヒダントインをＤ−α−アミノ酸に変換させることを見
い出し、これを先に提供した。工業的に実施する場合に
は、酵素源である菌体等の使用量をできるだけ減少させ
るとか、反応速度の増大、酵素の安定性向上、あるいは
酵素または菌体を連続的に使用し、より経済性を高める
事が望ましい。その−例として、ホローファイバーを菌
体の分離に利用して反復回分培養によるエタノール生産
（Ｊ、 Ｆｅｒｍｅｎ ｔ、 Ｔｅｃｈｏｌ、 、 ６
２．４１ 、１９８４）や限外ろ過膜を利用したプロテ
アーゼによる大豆タンパク質の連続加水分解（Ｂｉｏｔ
ｅｃｈｎｏｌ 、 Ｂｉｏｅｎｇ、 ＋ ２３＋ ２２
５７＋１９８１）等の報告があるが、ハンセニュラ属を
用いてＤ−α−アミノ酸を製造する上で、限外ろ過膜を
利用して連続的に製造する方法は報告されていない。As a result of our research to find a more efficient method for conventional production methods, we converted 5-substituted hydantoin into D-α-amino acid using a microorganism of the genus Hansenula. We found a way to do this and provided this first. When implementing it industrially, it is more economical to reduce the amount of bacteria used as an enzyme source, increase the reaction rate, improve enzyme stability, or use enzymes or bacteria continuously. It is desirable to increase gender. As an example, hollow fibers are used to isolate bacterial cells to produce ethanol by repeated batch culture (J. Ferment, Techol., 6).
2.41, 1984) and continuous hydrolysis of soybean protein by protease using an ultrafiltration membrane (Biot
echnol, Bioeng, +23+22
57+1981), but there has been no report on a method for continuously producing D-α-amino acids using an ultrafiltration membrane using Hansenula.
（問題点を解決するための手段）
本発明者らは、この様な従来の製造方法に対してより効
率の良い方法を見いだすべく研究した結果、ハンセニュ
ラ属に属する微生物が５−置換ヒダントインを接触させ
Ｄ−α−アミノ酸に変換させる方法において、限外ろ過
した菌体または菌体処理物を循環させながら連続的に作
用させることにより、Ｄ−α−アミノ酸を連続的かつ高
収率で得られることを見いだした。この発明はこの知見
に基づいて更に研究した結果、完成されるに至ったもの
である。(Means for Solving the Problems) As a result of research to find a more efficient method than the conventional production method, the present inventors found that microorganisms belonging to the genus Hansenula come into contact with 5-substituted hydantoin. In the method of converting D-α-amino acids into D-α-amino acids, D-α-amino acids can be obtained continuously and in high yield by continuously reacting with ultrafiltered bacterial cells or bacterial cell-treated products while circulating them. I found out. This invention was completed as a result of further research based on this knowledge.
すなわち本発明は一般式（１）
（式中、Ｒはアルキル基、置換アルキル基、フェニル基
、または、置換フェニル基を示す、）で表わされる５−
置換ヒダントイン類にハンセニュラ（Ｈａｎｓｅｎｕｌ
ａ）属に属する微生物の培養液、菌体または菌体処理物
を作用させてＤ、−α−アミノ酸に変換させる方法にお
いて、限外ろ過した菌体または菌体処理物を循環させな
がら連続的にＤ−α−アミノ酸に変換させる事を特徴と
する一般式％式％
（式中、Ｒは（１）に同じ）で表わされるＤ−α−アミ
ノ酸の製造方法である。That is, the present invention provides a 5-
Hansenul is a substituted hydantoin.
a) In a method of converting into D, -α-amino acid by reacting a culture solution, cells or processed material of microorganisms belonging to the genus, continuously circulating the ultrafiltered microorganisms or processed material of microorganisms. This is a method for producing a D-α-amino acid represented by the general formula % (wherein R is the same as in (1)), which is characterized by converting the D-α-amino acid into a D-α-amino acid.
本発明の目的のために使用されうる微生物は、例えば代
表例としては、ハンセニュラ・シフェリ−（）ｌａｎｓ
ｅｎｕｌａ ｃｉｆｅｒｒｉｉ）、ハンセニュラーヘン
リッシー（Ｈａｎｓｅｎｕｌａ ｈｅｎｒｉｃｉｉ）、
ハンセニュラーノ 。Microorganisms that can be used for the purposes of the present invention include, for example, Hanssenula siferii () lans.
enula ciferrii), Hansenula henricii,
Hansenurano.
フェルメンタス（Ｈａｎｓｅｎｕｌａ ｎｏｆｅｒｍｅ
ｎｔａｕｓ）Ｓハンセニュラ・ポリモルフｙ （Ｈａｎ
ｓｅｎｕｌａ ｐｏｌｙｍｏｒｐｈａ）などが挙げられ
、これらは本発明の目的に使用されうるかぎり自然界に
存在する野生株および公的な微生物保存機関に保存され
ている微生物が用いられる。Fermentas (Hansenula noferme)
ntaus) S Hansenula polymorph y (Han
senula polymorpha), and as long as they can be used for the purpose of the present invention, wild strains existing in nature and microorganisms preserved in public microorganism preservation institutions are used.
これら微生物の培養に用いられる培地は通常資化しうる
炭素源、窒素源および微生物の生育に必要な無機栄養素
を含有させる通常の培地である。The medium used for culturing these microorganisms is a normal medium containing an assimilable carbon source, a nitrogen source, and inorganic nutrients necessary for the growth of the microorganism.
培養条件は好気的条件下にてｐＨ・３〜９、温度１５〜
４０℃の適当の範囲に制御しつつ行えばよい。Culture conditions are aerobic, pH 3-9, temperature 15-9.
It may be carried out while controlling the temperature within an appropriate range of 40°C.
本発明で用いられる５−置換ヒダントインとは、ヒダン
トインの５位の水素原子がアルキル基、フェニル基また
はそれらの置換誘導体でありアルキル基またはフェニル
基に付随する置換基としては、例えばハロゲン原子、ア
ルキルメルカプト基、ヒドロキシル基、アルコキシ基、
アミノ基、インドリル基、アルコキシカルボニル基など
がある。The 5-substituted hydantoin used in the present invention means that the hydrogen atom at position 5 of the hydantoin is an alkyl group, a phenyl group, or a substituted derivative thereof, and examples of the substituents attached to the alkyl group or phenyl group include a halogen atom, an alkyl Mercapto group, hydroxyl group, alkoxy group,
These include amino groups, indolyl groups, and alkoxycarbonyl groups.
５−置換ヒダントインをＤ−α−アミノ酸に変換せしめ
る方法は前記の微生物の培養液、菌体または菌体処理物
の形態で使用できる。微生物の培養液をそのまま使用し
てもよいが培養液中の成分が障害になる場合や菌体量を
多く使用したい場合には、培養液から分離した菌体を用
いればよい。The method for converting a 5-substituted hydantoin into a D-α-amino acid can be used in the form of a culture solution, a bacterial cell, or a processed product of the microorganism. The culture solution of the microorganism may be used as it is, but if components in the culture solution become a hindrance or if a large amount of microbial cells is desired to be used, microbial cells separated from the culture solution may be used.
菌体は生菌体のままで使用目的を達するが、菌体そのも
のではなく菌体磨砕物や菌体抽出物のような菌体処理物
の状態でも用いることが可能であり、更に上記の菌体ま
たは菌体処理物を公知の方法で固定化したものも使用す
ることができる。Although the purpose of use is achieved as a viable bacterial cell, it is also possible to use a processed bacterial cell product such as a bacterial cell grind product or a bacterial cell extract instead of the bacterial cell itself. It is also possible to use cells or treated cells that have been immobilized by known methods.
反応基質である５−置換ヒダントインに微生物の培養液
、菌体または菌体処理物を作用させるには通常、水性媒
体中で行う方法が用いられ、反応基質の濃度は０．１−
１０重量％の濃度で用いられる。The reaction substrate, 5-substituted hydantoin, is usually reacted with a microorganism culture solution, cells, or a processed product of the microorganism in an aqueous medium, and the concentration of the reaction substrate is 0.1-
It is used at a concentration of 10% by weight.
γ
また酵素反応における温度およびｐＨは使用する微生物
の５−置換ヒダントインをＤ−α−アミノ酸に変換する
能力を持つ酵素の至適温度および至適ｐＨが採用される
が通常、温度は１５〜６０℃、ｐＨは５〜９の範囲であ
る。γ In addition, the temperature and pH in the enzyme reaction are the optimum temperature and pH of the microorganism used for the enzyme that has the ability to convert 5-substituted hydantoin into D-α-amino acid, but the temperature is usually 15 to 60. ℃ and pH range from 5 to 9.
本発明で、限外ろ過するための方法として、例えば、ベ
リコンカセット（ＭＩＬＬＩＰＯＲＥ ？ＬＩＮＩＴＡ
Ｍ Ｔ台ＳＹＳＴＥＭ 、日本ミリボア、リミテッド社
製）および中空糸膜（Ａｍｌｃｏｎ社）による方法等が
ある。限外ろ過するための透過膜は、基質および反応生
成物は透過させるが、菌体または菌体処理により得られ
る酵素は透過させない孔径の微孔を有する事が必要であ
り、菌体を用いる場合は、平均孔径０゜０５〜０．３μ
ｍの微孔を有する微孔膜や限外ろ過膜が用いられ、菌体
処理物により得られる酵素を用いる場合は、分画分子量
が１０００〜１００万程度である膜が用いられる。次に
、この様な発明方法を適用した実施態様のひとつを第１
図を用いて説明する。In the present invention, as a method for ultrafiltration, for example, a Vericon cassette (MILLIPORE?LINITA) is used.
There are methods using a hollow fiber membrane (manufactured by Amlcon Co., Ltd.) and a hollow fiber membrane (manufactured by Amlcon Co., Ltd.). The permeable membrane for ultrafiltration must have micropores with a pore size that allows the substrate and reaction products to pass through, but not the bacterial cells or enzymes obtained by treating the bacterial cells. has an average pore diameter of 0°05~0.3μ
A microporous membrane or an ultrafiltration membrane having micropores of m is used, and when an enzyme obtained from a bacterial cell treatment product is used, a membrane having a molecular weight cut-off of about 1,000 to 1,000,000 is used. Next, one of the embodiments to which such an invention method is applied will be described in the first example.
This will be explained using figures.
基質溶液貯槽（２）からポンプ（５）により基質溶液を
１０−５００ｍ１／ｈで反応槽（１）に送液し、反応液
および菌体をポンプ（６）により限外ろ過装置（３）に
送液し限外ろ過により菌体と反応生成物および未反応の
基質と分離し菌体は反応槽（２）に戻し、反応生成物お
よび基質を含む、ろ液を１０−５００ｍ１／ｈで反応液
貯槽（４）に送液する。また、ろ液を再び反応槽に戻す
事も可能である。この様にして得られたＤ−α−アミノ
酸を酵素反応溶液中から分離するにはイオン交換樹脂な
どを用いる通常の方法が採用できる。The substrate solution is sent from the substrate solution storage tank (2) to the reaction tank (1) at a rate of 10-500 m1/h using the pump (5), and the reaction solution and bacterial cells are transferred to the ultrafiltration device (3) using the pump (6). The liquid is sent, and the bacterial cells, reaction products, and unreacted substrates are separated by ultrafiltration, and the bacterial cells are returned to the reaction tank (2), and the filtrate containing the reaction products and substrates is reacted at a rate of 10 to 500 m1/h. The liquid is sent to the liquid storage tank (4). It is also possible to return the filtrate to the reaction tank. To separate the D-α-amino acid thus obtained from the enzyme reaction solution, a conventional method using an ion exchange resin or the like can be employed.
生成したＤ−α−アミノ酸の定量はニンヒドリンを用い
る比色法および液体クロマトグラフィで測定する方法を
用いた。光学異性体は結晶の比旋光度の測定および光学
分割カラム（キラルパックＷＨ−ダイセル社製）を用い
る液体クロマトグラフィによってＤ一体を確認した。The produced D-α-amino acid was quantified using a colorimetric method using ninhydrin and a method using liquid chromatography. The optical isomer was confirmed to be D-integrated by measuring the specific optical rotation of the crystal and by liquid chromatography using an optical resolution column (Chiralpak WH-manufactured by Daicel).
（発明の作用および効果）
本発明によれば、ハンセニュラ属の微生物を用いること
により５−置換ヒダントインから高収率かつ連続的にＤ
−α−アミノ酸を取得することができる。即ち、本発明
においてはハンセニュラ（Ｈａｎｓｅｎｕｌａ）属に属
する微生物の培養液、菌体または菌体処理物を用いて５
−置換ヒダントインをＤ−α−アミノ酸に変換するに際
し、限外ろ過した菌体または菌体処理物を循環させなが
ら連続的に作用させることにより、５−置換ヒダントイ
ンのＤ−α−アミノ酸への変換率が高められると同時に
連続的に製造でき、Ｄ−α−アミノ酸の製造方法として
は極めて有利な方法である。(Operations and Effects of the Invention) According to the present invention, by using a microorganism belonging to the genus Hansenula, 5-substituted hydantoin can be continuously produced in high yield.
-α-amino acids can be obtained. That is, in the present invention, a culture solution, bacterial cells, or treated bacterial cells of a microorganism belonging to the genus Hansenula are used to
- When converting substituted hydantoin to D-α-amino acid, 5-substituted hydantoin is converted to D-α-amino acid by continuously acting while circulating ultrafiltered bacterial cells or bacterial cell-treated products. This method is extremely advantageous as a method for producing D-α-amino acids, as the production rate can be increased and the production can be carried out continuously.
（実施例）
以下の例により本発明を具体的に説明するが、本発明は
これらの例のみに限定されるものではない。(Examples) The present invention will be specifically explained using the following examples, but the present invention is not limited to these examples.
実施例−１
グルコース２０ｇ／ｌ、ＤＬ−５−フェニルヒダントイ
ン５ｇ／ｌ 、マルツエキスＩｇ／ｌ 、 Ｈａエキス
３ｇ／ｌ 、 ＫＨ２ＰＯ４，１，５ｇ／Ｉ ＳＭｇＳ
Ｏ４，，７８ｚＯ０，５ｇ／ｌ。Example-1 Glucose 20g/l, DL-5-phenylhydantoin 5g/l, Malt extract Ig/l, Ha extract 3g/l, KH2PO4,1.5g/I SMgS
O4,,78zO0,5g/l.
ＣａＣ１ｚ、ＨｚＯ０，３３ｇ／ｌ （ｐＨ＝６）の培
地を２５０ｍ ｌ三角フラスコに２０ｍ１入れ、１２０
℃、１５分間殺菌した。Pour 20ml of a medium containing CaC1z, HzO0, 33g/l (pH=6) into a 250ml Erlenmeyer flask,
Sterilized at ℃ for 15 minutes.
これに酵母ＹＭ培地で２８℃、２４時間培養したハンセ
ニュラ ポリモルファ（ＮＲＲＬ Ｙ−２４２３）を１
白金耳接種し、２８℃、２４時間培養した。この培養液
を遠心分離により菌体を採取し、培養液と同量の殺菌し
た生理食塩水にて１回洗浄し菌体を集めた。To this, 1 portion of Hansenula polymorpha (NRRL Y-2423) cultured in yeast YM medium at 28°C for 24 hours was added.
A platinum loop was inoculated and cultured at 28°C for 24 hours. Bacterial cells were collected by centrifugation of this culture solution, and washed once with sterilized physiological saline in the same amount as the culture solution to collect the microbial cells.
第１図に連続限外ろ過膜型反応装置を示した。反応槽（
１）に菌体をＤＬ−５−イソプロピルヒダントイン１０
ｇ／ｌ水溶液（ｐＨ−７，５）−終末３００ｍ１−・−
に１０ｇ／ｌになる様に添加し２８℃で７０時間反応し
た。Figure 1 shows a continuous ultrafiltration membrane type reactor. Reaction tank (
1) Inject the bacterial cells with DL-5-isopropylhydantoin 10
g/l aqueous solution (pH-7,5) - final 300ml - -
was added to the solution at a concentration of 10 g/l and reacted at 28°C for 70 hours.
基質貯槽（２）からＤＬ−５−イソプロビルヒダントイ
ンＬｏｇ／Ｉ水溶液をポンプ（５）により４０ｍ１／ｈ
で反応槽（１）に送液し、反応液および菌体をポンプ（
６）により４０ｍ１／ｈで限外ろ過装置（３）（ペリコ
ンカセット阿ＴＬＬＩＰＯＲＥ ＦＩＩＮＩＴＡＭ−Ｔ
河ＳＹＳＴＥＭ、 日本ミリボア。DL-5-isoprobylhydantoin Log/I aqueous solution is pumped from the substrate storage tank (2) at 40 ml/h using the pump (5).
to the reaction tank (1), and pump the reaction solution and bacterial cells (
6) at a rate of 40 m1/h using an ultrafiltration device (3)
Kawa SYSTEM, Japan Millibore.
リミテッド社製）に送液し限外ろ過により菌体と反応生
成物および未反応の基質と分離し菌体は反応槽（１）に
戻した。生成したアミノ酸を前述の方法で測定した。ま
た生成したアミノ酸を光学分割カラムによる光学活性測
定によりＤ一体であった。Ltd.), the bacterial cells were separated from the reaction product and unreacted substrate by ultrafiltration, and the bacterial cells were returned to the reaction tank (1). The amino acids produced were measured using the method described above. Furthermore, the produced amino acid was determined to be D-integrated by optical activity measurement using an optical resolution column.
結果を表−１に示した。The results are shown in Table-1.
表−１
反応時間（ｈ） Ｄ−バリン（ｍｇ／ｍｔ）実施例
−２
実施例−１と同様な方法にて調整した菌体を表−２に示
す各種ＤＬ−５−置換ヒダントインＬｏｇ／ｌ水溶液（
ｐＨ７，５） −・−終末３００ｍ１−に１０ｇ／ｌに
な様に添加して実施例−１で示した限外ろ過装置を用い
て２８℃で７０間反応した。生成したアミノ酸を前述の
方法で測定した。また生成したアミノ酸を光学分割カラ
ムによる光学活性測定によりＤ一体であった。結果を表
−２に示した。Table 1 Reaction time (h) D-valine (mg/mt) Example 2 Various DL-5-substituted hydantoin Log/l shown in Table 2 using bacterial cells prepared in the same manner as in Example 1 Aqueous solution (
pH 7.5) --- Final 300 ml was added at 10 g/l and reacted at 28 DEG C. for 70 hours using the ultrafiltration apparatus shown in Example-1. The amino acids produced were measured using the method described above. Furthermore, the produced amino acid was determined to be D-integrated by optical activity measurement using an optical resolution column. The results are shown in Table-2.
実施例−３
表−３に示す各種微生物を実施例−１と同様に調整して
得られた菌体を、ＤＬ−５−イソプロピルヒダントイン
１０ｇ／ｌ水溶液（ｐＨ７，５） （終末３００ｍ１
）に１０ｇ／ｌになる様に添加して実施例−１で示し
た限外ろ過装置を用いて２８℃で７０時間反応した。生
成したアミノ酸を前述の方法で測定した。Example 3 The various microorganisms shown in Table 3 were prepared in the same manner as in Example 1, and the resulting bacterial cells were added to a 10 g/l aqueous solution of DL-5-isopropylhydantoin (pH 7.5) (final 300 ml).
) at a concentration of 10 g/l and reacted at 28° C. for 70 hours using the ultrafiltration apparatus shown in Example-1. The amino acids produced were measured using the method described above.
また生成したアミノ酸を光学分割カラムによる光学活性
測定によりＤ一体であった。結果を表−３明細吉の二゛
１８°（内８に変更なし）表−２
表−３Furthermore, the produced amino acid was determined to be D-integrated by optical activity measurement using an optical resolution column. The results are shown in Table-3.
第１図は本発明を実施するために用いる連続限外ろ過膜
型反応装置の概略図であり、示した符号は、それぞれ次
を意味する。
（１）：反応槽
（２）二基質溶液貯槽
（３）：限外ろ過装置
（４）：反応液貯槽
（５）：ポンプ
（６）：ポンプ
（７）二基質送液ライン
（８）：菌体循環ライン
（９）：反応液送液ライン
特許出願人 三井東圧化学株式会社
手続補正書（方式）
昭和６２年０５月０７日
特許庁長官 黒 １）明 雄 殿
１、事件の表示
昭和６２年特許願第０３９１２８号
２、発明の名称
り一α−アミノ酸の製造方法。
３、補正をする者
事件との関係 特許出願人
住所 東京都千代田区霞が関三丁目２番５号４、補正命
令の日付（発送日）
昭和６２年０４月２８日
５、補正の対象
明細書第１３、発明の詳細な説明の欄）６、補正の内容FIG. 1 is a schematic diagram of a continuous ultrafiltration membrane type reactor used to carry out the present invention, and the indicated symbols have the following meanings. (1): Reaction tank (2) Bisubstrate solution storage tank (3): Ultrafiltration device (4): Reaction liquid storage tank (5): Pump (6): Pump (7) Bisubstrate liquid feeding line (8): Bacterial body circulation line (9): Reaction liquid feeding line Patent applicant Mitsui Toatsu Chemical Co., Ltd. Procedural amendment (method) May 7, 1988 Commissioner of the Patent Office Black 1) Akio Tono 1, Incident indication Showa 1962 Patent Application No. 039128 2, Title of the invention: Process for producing α-amino acids. 3. Relationship with the case of the person making the amendment Patent applicant address: 3-2-5-4 Kasumigaseki, Chiyoda-ku, Tokyo Date of amendment order (shipment date) April 28, 1985 5. Specification subject to amendment No. 13. Detailed description of the invention column) 6. Contents of amendment
Claims (1)
、または、置換フェニル基を示す。）で表わされる５−
置換ヒダントイン類にハンセニュラ（Ｈａｎｓｅｎｕｌ
ａ）属に属する微生物の培養液、菌体または菌体処理物
を作用させてＤ−α−アミノ酸に変換させる方法におい
て、限外ろ過した菌体または菌体処理物を循環させなが
ら連続的にＤ−α−アミノ酸に変換させる事を特徴とす
る一般式（２） ▲数式、化学式、表等があります▼ （式中、Ｒは（１）に同じ） で表わされるＤ−α−アミノ酸の製造方法。[Claims] General formula (1) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R represents an alkyl group, a substituted alkyl group, a phenyl group, or a substituted phenyl group.) 5 −
Hansenul is a substituted hydantoin.
a) In a method of converting into D-α-amino acid by reacting a culture solution, cells, or a processed product of microorganisms belonging to the genus, continuously while circulating the ultrafiltered bacterial cells or processed product of microorganisms. Production of D-α-amino acids represented by general formula (2) ▲Mathematical formulas, chemical formulas, tables, etc.▼ (in the formula, R is the same as in (1)), which is characterized by conversion to D-α-amino acids Method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3912887A JPS63207399A (en) | 1987-02-24 | 1987-02-24 | Production of d-alpha-amino acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3912887A JPS63207399A (en) | 1987-02-24 | 1987-02-24 | Production of d-alpha-amino acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS63207399A true JPS63207399A (en) | 1988-08-26 |
Family
ID=12544464
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3912887A Pending JPS63207399A (en) | 1987-02-24 | 1987-02-24 | Production of d-alpha-amino acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63207399A (en) |
-
1987
- 1987-02-24 JP JP3912887A patent/JPS63207399A/en active Pending
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