JPH0659227B2 - Method for producing D-α-amino acid - Google Patents
Method for producing D-α-amino acidInfo
- Publication number
- JPH0659227B2 JPH0659227B2 JP24899085A JP24899085A JPH0659227B2 JP H0659227 B2 JPH0659227 B2 JP H0659227B2 JP 24899085 A JP24899085 A JP 24899085A JP 24899085 A JP24899085 A JP 24899085A JP H0659227 B2 JPH0659227 B2 JP H0659227B2
- Authority
- JP
- Japan
- Prior art keywords
- amino acid
- substituted
- cells
- formula
- producing
- 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.)
- Expired - Lifetime
Links
- 150000007650 D alpha amino acids Chemical class 0.000 title claims description 20
- 238000004519 manufacturing process Methods 0.000 title claims description 7
- 150000001469 hydantoins Chemical class 0.000 claims description 21
- 229940091173 hydantoin Drugs 0.000 claims description 18
- 244000005700 microbiome Species 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 16
- 235000001014 amino acid Nutrition 0.000 claims description 14
- 150000001413 amino acids Chemical class 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 230000000813 microbial effect Effects 0.000 claims description 11
- 239000002202 Polyethylene glycol Substances 0.000 claims description 10
- 229920001223 polyethylene glycol Polymers 0.000 claims description 10
- 241000235648 Pichia Species 0.000 claims description 8
- 150000005846 sugar alcohols Polymers 0.000 claims description 7
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 6
- 125000000217 alkyl group Chemical group 0.000 claims description 4
- 235000008206 alpha-amino acids Nutrition 0.000 claims description 3
- 125000000547 substituted alkyl group Chemical group 0.000 claims description 2
- 150000001371 alpha-amino acids Chemical class 0.000 claims 1
- 239000000243 solution Substances 0.000 description 19
- 229940024606 amino acid Drugs 0.000 description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 230000003287 optical effect Effects 0.000 description 5
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- NXQJDVBMMRCKQG-UHFFFAOYSA-N 5-phenylimidazolidine-2,4-dione Chemical compound O=C1NC(=O)NC1C1=CC=CC=C1 NXQJDVBMMRCKQG-UHFFFAOYSA-N 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 239000008057 potassium phosphate buffer Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 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 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- 241000320412 Ogataea angusta Species 0.000 description 2
- 150000001370 alpha-amino acid derivatives Chemical class 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- WBYWAXJHAXSJNI-VOTSOKGWSA-M .beta-Phenylacrylic acid Natural products [O-]C(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-M 0.000 description 1
- 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
- 241000186063 Arthrobacter Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- WBYWAXJHAXSJNI-SREVYHEPSA-N Cinnamic acid Chemical compound OC(=O)\C=C/C1=CC=CC=C1 WBYWAXJHAXSJNI-SREVYHEPSA-N 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 241000589565 Flavobacterium Species 0.000 description 1
- 241000588621 Moraxella Species 0.000 description 1
- 241001057811 Paracoccus <mealybug> Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 239000000654 additive Substances 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
- 230000001580 bacterial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 229930016911 cinnamic acid Natural products 0.000 description 1
- 235000013985 cinnamic acid Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 125000005843 halogen group Chemical group 0.000 description 1
- WJRBRSLFGCUECM-UHFFFAOYSA-N hydantoin Chemical group O=C1CNC(=O)N1 WJRBRSLFGCUECM-UHFFFAOYSA-N 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 125000001041 indolyl group Chemical group 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229960005190 phenylalanine Drugs 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- HEBKCHPVOIAQTA-ZXFHETKHSA-N ribitol Chemical compound OC[C@H](O)[C@H](O)[C@H](O)CO HEBKCHPVOIAQTA-ZXFHETKHSA-N 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は、ＤＬ−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
ミノ酸を極めて有利に製造する方法に関するものであ
る。DETAILED DESCRIPTION OF THE INVENTION Industrial Field of the Invention The present invention relates to Hansenul (Hansenul) having the ability to convert DL-5-substituted hydantoins to D-α-amino acids.
a) It relates to a method for producing a D-α-amino acid extremely advantageously by using a microorganism belonging to the genus.
（従来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ＤＬ
−５−置換ヒダントインを化学的に水解して、ＤＬ−α
−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。
また更に、ＤＬ−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Ｎ−カルバモイル−Ｄ−α−
アミノ酸を生成させた後、亜硝酸ソーダ液処理によりＤ
−α−アミノ酸とする方法が知られているが、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。(Prior art and its problems) Corresponding DL as one of methods for producing D-α-amino acid
Chemically hydrolyzing -5-substituted hydantoin to give DL-α
A method is known in which an amino acid is produced and optically resolved to give a D-α-amino acid. However, this method is particularly complicated in the optical resolution step and the yield thereof is not high.
Furthermore, DL-5-substituted hydantoin is allowed to act with a microorganism culture solution, cells, a treated product of the cells, or an enzyme extracted from the cells, and N-carbamoyl-D-α-having optical activity.
After the amino acid is generated, it is treated with a sodium nitrite solution to give D
The method of using -α-amino acid is known, but
This method also requires complicated reaction steps and purification steps.
又、更にＤＬ−５−置換ヒダントインに、ある種の微生
物…例えばシュードモナス（Pseudomonas）、モラキセ
ラ（Moraxella）、パラコッカス（Paracoccus）、アー
スロバクター（Arthrobacter）、アルカリジェネス（Al
caligenes）、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。In addition to DL-5-substituted hydantoin, certain microorganisms such as Pseudomonas, Moraxella, Paracoccus, Arthrobacter, Alkaline Genes (Al
caligenes), Flavobacterium
D- directly by acting on the culture solution, fungus body, or processed product of fungus body of
A method using α-amino acid 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ＤＬ−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。The present inventors have conducted research to find a more efficient method for such a conventional production method, and as a result, used a microorganism of the genus Hansenula to convert DL-5-substituted hydantoin to a D-α-amino acid. I found this to be converted into and provided this first.
多価アルコールやポリエチレングリコールについては、
最近酵素法によってケイ皮酸からＬ−フェニルアラニン
を生成させる反応系に添加することによりその収率が向
上すると云う文献も見られる。For polyhydric alcohol and polyethylene glycol,
Recently, it has been found that the yield is improved by adding it to a reaction system for producing L-phenylalanine from cinnamic acid by an enzymatic method.
しかし、ＤＬ−５−置換ヒダントインからＤ−アミノ酸
への系に添加してどうなるかについては何も知られてい
ない。However, nothing is known about what happens when a DL-5-substituted hydantoin is added to the D-amino acid system.
（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法に対しより効率の
よい方法を見いだすべく研究した結果、ハンセニュラ属
に属する微生物にＤＬ−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、反応溶
液中に多価アルコール又はポリエチレングリコールを添
加することによりＤ−α−アミノ酸の収率が改善される
ことを見いだした。(Means for Solving Problems) The present inventors have conducted research to find a more efficient method than the conventional production method, and as a result, have found that DL-5-substituted hydantoin was added to a microorganism belonging to the genus Hansenula. In the method of contacting and converting into D-α-amino acid, it was found that the yield of D-α-amino acid is improved by adding polyhydric alcohol or polyethylene glycol to the reaction solution.
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。The present invention has been completed as a result of further research based on this finding.
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
又は置換フェニル基を示す。）で表わされるＤＬ−５−
置換ヒダントイン類に、ＤＬ−５−置換ヒダントインを
Ｄ−α−アミノ酸に変換する能力を有するハンセニュラ
（Hansenula）属に属する微生物の培養液、菌体又は菌
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、反応液中に多価アルコール又はポリエチ
レングリコールの存在下で、ＤＬ−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特徴とする一
般式(2) （式中、Ｒは式(1)と同様である。）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, the general formula (1) (In the formula, R represents an alkyl group, a substituted alkyl group, a phenyl group or a substituted phenyl group.) DL-5
The substituted hydantoins are treated with a culture solution, a microbial cell or a treated product of a microorganism belonging to the genus Hansenula having the ability to convert DL-5-substituted hydantoin into D-α-amino acid to give D-α. -In the method of converting to an amino acid, DL-5-substituted hydantoin is converted to a D-α-amino acid in the presence of a polyhydric alcohol or polyethylene glycol in the reaction solution, the general formula (2) (In the formula, R is the same as the formula (1).)
This is a method for producing an 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 シフェリー（Hansenula
ciferrii）、ハンセニュラ ヘンリッシー（Hansenula
henricii）、ハンセニュラ ノフェルメンタス（Hansen
ula nonfermentaus）、ハンセニュラ ポリモルファ（H
ansenula 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。Microorganisms that can be used for the purposes of the present invention include, for example, representatively, the Hansenula ferries.
ciferrii), Hansenula Henry (Hansenula)
henricii), Hansenura Nofermentus (Hansen)
ula nonfermentaus), Hansenula polymorpha (H
ansenula polymorpha), etc., and as long as they can be used for the purpose of the present invention, wild strains existing in nature and microorganisms stored in public microbial conservation institutions are used.
本発明で用いられるＤＬ−５−置換ヒダントイン類と
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。又、本発明で用いられる多価ア
ルコールとは分子内に水酸基を３つ以上含む化合物を意
味し、例えば、グリセロール、キシロース、マンニトー
ル、ソルビトール、アドニトール、グルコースなどであ
る。更にポリエチレングリコールは分子量が４００〜２
００００の物でもよいが、望ましくは分子量４００〜６
０００のものがよい。The DL-5-substituted hydantoins used in the present invention means that the hydrogen atom at the 5-position of hydantoin is an alkyl group, a phenyl group or a substituted derivative thereof, and a substituent attached to the alkyl group or the phenyl group is For example, halogen atom, alkylmercapto group, hydroxyl group,
Examples thereof include an alkoxy group, an amino group, an indolyl group and an alkoxycarbonyl group. The polyhydric alcohol used in the present invention means a compound containing three or more hydroxyl groups in the molecule, and examples thereof include glycerol, xylose, mannitol, sorbitol, adonitol, glucose and the like. Furthermore, polyethylene glycol has a molecular weight of 400 to 2
It may be 0000, but preferably has a molecular weight of 400-6.
000 is good.
本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塩栄養素を
含有させる通常の培地である。培養条件は好気的条件下
にてpH＝３〜９、温度２５〜５０℃の適当な範囲に制御
しつつ行なえばよい。The medium used for culturing the present microorganism is a usual medium containing an assimilable carbon source, a nitrogen source, and inorganic salt nutrients necessary for the growth of the microorganism. The culture conditions may be aerobic conditions while controlling the pH within a suitable range of 3 to 9 and a temperature of 25 to 50 ° C.
ＤＬ−５−置換ヒダントインのＤ−α−アミノ酸への反
応には、前記のようにして培養した微生物の培養液、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。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。For the reaction of DL-5-substituted hydantoin to D-α-amino acid, it can be used in the form of a culture solution, a microbial cell or a treated microbial cell of the microorganism cultured as described above. Although the culture solution of the microorganism may be used as it is, when the components in the culture become an obstacle or when it is desired to use a large amount of cells, the cells separated from the culture solution may be used. The microbial cells reach the purpose of use as they are as viable microbial cells, but they can be used not only in the microbial cells themselves but also in the state of treated microbial cells such as microbial cell grinds or microbial cell extracts. It is also possible to use a product obtained by immobilizing a body or treated product of cells by a known method.
反応基質であるＤＬ−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％の濃度まで用いることが出来る。又、反
応における温度は使用する微生物のＤ−α−アミノ酸へ
の変換する能力を持つ酵素の至適温度が採用されるが、
通常２０〜６０℃の範囲にあり、pHも使用する微生物の
Ｄ−α−アミノ酸への変換する能力を持つ酵素の至適pH
が採用され、通常pH＝５〜９の範囲である。A method in which an aqueous medium is usually used for allowing a culture solution of microorganisms, cells, or a treated product of cells to act on DL-5-substituted hydantoin, which is a reaction substrate, has a reaction substrate concentration of 0.
A concentration of 1 to 10% by weight can be used. The temperature in the reaction is the optimum temperature of the enzyme having the ability to convert the microorganism used to D-α-amino acid.
The optimum pH of an enzyme that is usually in the range of 20 to 60 ° C and has the ability to convert the microorganisms used to D-α-amino acids
Is adopted, and the pH is usually in the range of 5-9.
本発明において、酵素反応溶液への多価アルコールおよ
びポリエチレングリコールの添加量は添加する物質によ
って異なるが、一般的には１〜１００mg/mlであり、例
えば、ポリエチレングリコール（分子量４００）の場合
は５０mg/mlが、又グリセロール、キシロースおよびマ
ンニトールの場合は１〜２mg/mlが望ましい。In the present invention, the amount of polyhydric alcohol and polyethylene glycol added to the enzyme reaction solution varies depending on the substance to be added, but is generally 1 to 100 mg / ml, and for example, in the case of polyethylene glycol (molecular weight 400), 50 mg. / ml is preferably 1-2 mg / ml in the case of glycerol, xylose and mannitol.
前述したようなＤＬ−５−置換ヒダントインを変換して
生成するＤ−α−アミノ酸類の単離は濃縮、中和更にイ
オン交換樹脂処理などの公知の方法を利用することによ
り目的物であるＤ−α−アミノ酸を取得出来る。Isolation of the D-α-amino acids produced by converting the DL-5-substituted hydantoin as described above is carried out by utilizing a known method such as concentration, neutralization and treatment with an ion exchange resin. -Α-amino acid can be obtained.
（発明の作用および効果） 本発明によれば、微生物を用いることによりＤＬ−５−
置換ヒダントインから容易に高収率でＤ−α−アミノ酸
を取得できる。即ち本発明においては、ハンセニュラ属
に属する微生物を用いてＤＬ−５−置換ヒダントインを
Ｄ−α−アミノ酸に変換する反応溶液中に、多価アルコ
ールまたはポリエチレングリコールを添加させることに
より、ＤＬ−５−置換ヒダントインのＤ−α−アミノ酸
への変換率を高めることができ、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。(Operation and Effect of the Invention) According to the present invention, DL-5-
The D-α-amino acid can be easily obtained from the substituted hydantoin in high yield. That is, in the present invention, by adding a polyhydric alcohol or polyethylene glycol to a reaction solution for converting DL-5-substituted hydantoin into D-α-amino acid using a microorganism belonging to the genus Hansenula, DL-5- The conversion rate of substituted hydantoin to D-α-amino acid can be increased, which is an extremely advantageous method for producing D-α-amino acid.
（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 described by the following examples, but the present invention is not limited to these examples.
実施例−１ グルコース２０ｇ／、ＤＬ−５−フェニルヒダントイ
ン５ｇ／、マルツエキス１ｇ／、酵母エキス３ｇ／
、ＫＨ２ＰＯ４ １．５ｇ／、ＭｇＳＯ４−７Ｈ２
Ｏ ０．５ｇ／、ＣａＣｌ２−２Ｈ２Ｏ ０．３３ｇ
／（pH＝６．０）の培地を２５０ml三角フラスコに２
０ml入れ１２０℃、１５分間殺菌した。これに酵母ＹＭ
培地で２８℃、４０時間培養したハンセニュラ ポリモ
ルファ（ＮＲＲＬ Ｙ−２４２３）を−白金耳接種し２
８℃で２４時間培養した。この培養液より遠心分離によ
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。この菌体をＤＬ−５−フェ
ニルヒダントイン１０ｇ／を含む０．１Ｍリン酸カリ
ウムバッファ（pH＝７．５）に３０ｇ／になるように
添加し、更に表−１に示す各種の添加物を入れた溶液５
mlを３２℃、２０時間反応した。反応終了後、反応液４
mlに１２％トリクロロ酢酸溶液１mlを加え全量を５mlと
する。その後、遠心分離（１５０００rpm、１０min.）
にて不溶解物を除去した後、上澄液をアミノ酸アナライ
ザー（日立製）にて生成したアミノ酸を測定した。又、
生成したアミノ酸を分離、精製し旋光度の測定を行なっ
た結果、生成するアミノ酸は全ての場合Ｄ体であること
を確認した。結果は表−１に示す。Example-1 Glucose 20 g /, DL-5-phenylhydantoin 5 g /, malt extract 1 g /, yeast extract 3 g /
, KH 2 PO 4 1.5g /, MgSO 4 -7H 2
O 0.5 g /, CaCl 2 -2H 2 O 0.33 g
/ (PH = 6.0) medium in a 250 ml Erlenmeyer flask 2
0 ml was put and sterilized at 120 ° C. for 15 minutes. Yeast YM
Hansenula polymorpha (NRRL Y-2423) cultured in the medium at 28 ° C. for 40 hours was inoculated with platinum loop 2
The cells were cultured at 8 ° C for 24 hours. The cells were collected from this culture solution by centrifugation and washed once with the same amount of sterilized physiological saline as the culture solution to collect the cells. The cells were added to 0.1 M potassium phosphate buffer (pH = 7.5) containing DL-5-phenylhydantoin at 10 g / so that the concentration became 30 g /, and further various additives shown in Table 1 were added. Solution 5
ml was reacted at 32 ° C. for 20 hours. After the reaction is completed, the reaction liquid 4
1 ml of 12% trichloroacetic acid solution is added to ml to make the total volume 5 ml. After that, centrifugation (15000 rpm, 10 min.)
After the insoluble matter was removed with, the supernatant was measured for amino acids produced with an amino acid analyzer (manufactured by Hitachi). or,
As a result of separating and purifying the produced amino acids and measuring the optical rotation, it was confirmed that the produced amino acids were D-forms in all cases. The results are shown in Table-1.
実施例−２ 実施例−１と同様に調整した菌体を表−２に示す各種Ｄ
Ｌ−５−置換ヒダントイン１０ｇ／およびポリエチレ
ングリコール（分子量４００）５０mg/mlを含む０．１
Ｍリン酸カリウムバッファ（pH＝７．５）に３０ｇ／
になるように添加し、その溶液５mlを３２℃、２０時間
反応した。生成するアミノ酸は前記の方法にて測定し
た。また、これらのアミノ酸を分離・精製し、その旋光
度を測定した結果、生成するアミノ酸は全ての場合Ｄ体
であることを確認した。結果は表−２に示す。 Example-2 Various D's shown in Table 2 are the bacterial cells prepared in the same manner as in Example-1.
0.1 g containing L-5-substituted hydantoin 10 g / and polyethylene glycol (molecular weight 400) 50 mg / ml
30 g / M potassium phosphate buffer (pH = 7.5)
Was added thereto, and 5 ml of the solution was reacted at 32 ° C. for 20 hours. The produced amino acid was measured by the above method. Moreover, as a result of separating and purifying these amino acids and measuring the optical rotation thereof, it was confirmed that all the produced amino acids were D-forms. 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同様に調整して得
られた菌体を、ＤＬ−５−フェニルヒダントイン１０ｇ
／およびポリエチレングリコール（分子量４００）５
０mg/mlを含む０．１Ｍリン酸カリウムバッファ（pH＝
７．５）に３０ｇ／になるように添加し、その溶液５
mlを３２℃、２０時間反応した。生成するアミノ酸は前
記の方法にて測定した。また生成するアミノ酸を分離、
精製し旋光度の測定した結果、生成するアミノ酸は全て
の場合Ｄ体であることを確認した。結果を表−３に示
す。 Example-3 Various cells shown in Table-3 were prepared in the same manner as in Example-1 to obtain 10 g of DL-5-phenylhydantoin.
/ And polyethylene glycol (molecular weight 400) 5
0.1 M potassium phosphate buffer containing 0 mg / ml (pH =
7.5), so that the amount of the solution becomes 30 g /, and the solution 5
ml was reacted at 32 ° C. for 20 hours. The produced amino acid was measured by the above method. In addition, the generated amino acids are separated,
As a result of purification and measurement of optical rotation, it was confirmed that the produced amino acids were D-forms in all cases. The results are shown in Table-3.
Claims (1)
又は置換フェニル基を示す。）で表わされるＤＬ−５−
置換ヒダントイン類に、ＤＬ−５−置換ヒダントインを
Ｄ−α−アミノ酸に変換する能力を有するハンセニュラ
（Hansenula）属に属する微生物の培養液、菌体又は菌
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、反応液中に多価アルコール又はポリエチ
レングリコールの存在下で、ＤＬ−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特徴とする一
般式(2) （式中、Ｒは式(1)と同様である。）で表わされるＤ−
α−アミノ酸の製造方法。1. A general formula (1) (In the formula, R represents an alkyl group, a substituted alkyl group, a phenyl group or a substituted phenyl group.) DL-5
The substituted hydantoins are treated with a culture solution, a microbial cell or a treated product of a microorganism belonging to the genus Hansenula having the ability to convert DL-5-substituted hydantoin into D-α-amino acid to give D-α. -In the method of converting to an amino acid, DL-5-substituted hydantoin is converted to a D-α-amino acid in the presence of a polyhydric alcohol or polyethylene glycol in the reaction solution, the general formula (2) (In the formula, R is the same as the formula (1).)
A method for producing an α-amino acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP24899085A JPH0659227B2 (en) | 1985-11-08 | 1985-11-08 | Method for producing D-α-amino acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP24899085A JPH0659227B2 (en) | 1985-11-08 | 1985-11-08 | Method for producing D-α-amino acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS62111692A JPS62111692A (en) | 1987-05-22 |
| JPH0659227B2 true JPH0659227B2 (en) | 1994-08-10 |
Family
ID=17186381
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP24899085A Expired - Lifetime JPH0659227B2 (en) | 1985-11-08 | 1985-11-08 | Method for producing D-α-amino acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0659227B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9499521B2 (en) | 2014-12-11 | 2016-11-22 | President And Fellows Of Harvard College | Inhibitors of cellular necrosis and related methods |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3702384A1 (en) * | 1987-01-23 | 1988-08-04 | Schering Ag | METHOD FOR PRODUCING L-AMINO ACIDS |
-
1985
- 1985-11-08 JP JP24899085A patent/JPH0659227B2/en not_active Expired - Lifetime
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9499521B2 (en) | 2014-12-11 | 2016-11-22 | President And Fellows Of Harvard College | Inhibitors of cellular necrosis and related methods |
| US9944628B2 (en) | 2014-12-11 | 2018-04-17 | President And Fellows Of Harvard College | Inhibitors of cellular necrosis and related methods |
| US10508102B2 (en) | 2014-12-11 | 2019-12-17 | President And Fellows Of Harvard College | Inhibitors of cellular necrosis and related methods |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS62111692A (en) | 1987-05-22 |
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