JPH02174684A - Production of n-carbamoyl-d-phenylglycine - Google Patents
Production of n-carbamoyl-d-phenylglycineInfo
- Publication number
- JPH02174684A JPH02174684A JP32631188A JP32631188A JPH02174684A JP H02174684 A JPH02174684 A JP H02174684A JP 32631188 A JP32631188 A JP 32631188A JP 32631188 A JP32631188 A JP 32631188A JP H02174684 A JPH02174684 A JP H02174684A
- Authority
- JP
- Japan
- Prior art keywords
- reaction
- cdpg
- microorganisms
- phenylglycine
- carbamoyl
- 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
- GIOUOHDKHHZWIQ-SSDOTTSWSA-N N-carbamoyl-D-phenylglycine Chemical compound NC(=O)N[C@@H](C(O)=O)C1=CC=CC=C1 GIOUOHDKHHZWIQ-SSDOTTSWSA-N 0.000 title claims abstract 7
- 238000004519 manufacturing process Methods 0.000 title claims description 7
- 238000006243 chemical reaction Methods 0.000 claims abstract description 41
- 244000005700 microbiome Species 0.000 claims abstract description 28
- 238000000034 method Methods 0.000 claims abstract description 20
- NXQJDVBMMRCKQG-UHFFFAOYSA-N 5-phenylimidazolidine-2,4-dione Chemical compound O=C1NC(=O)NC1C1=CC=CC=C1 NXQJDVBMMRCKQG-UHFFFAOYSA-N 0.000 claims abstract description 5
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 2
- 238000007039 two-step reaction Methods 0.000 claims 3
- 239000007788 liquid Substances 0.000 abstract description 8
- 241000894006 Bacteria Species 0.000 abstract description 3
- 241000589516 Pseudomonas Species 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- 239000000725 suspension Substances 0.000 abstract description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 abstract 3
- 230000000717 retained effect Effects 0.000 abstract 2
- 239000003513 alkali Substances 0.000 abstract 1
- 239000012295 chemical reaction liquid Substances 0.000 abstract 1
- 239000007787 solid Substances 0.000 abstract 1
- 230000001580 bacterial effect Effects 0.000 description 10
- 239000000243 solution Substances 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 239000002994 raw material Substances 0.000 description 7
- 239000007864 aqueous solution Substances 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- AZRZBCASYOBNKQ-UHFFFAOYSA-N 6-chloro-3,5-diaminopyrazine-3-carboxamide Chemical compound CN(C)C(N)=NC(=O)C1=NC(Cl)=C(N)N=C1N AZRZBCASYOBNKQ-UHFFFAOYSA-N 0.000 description 2
- ZGUNAGUHMKGQNY-SSDOTTSWSA-N D-alpha-phenylglycine Chemical compound OC(=O)[C@H](N)C1=CC=CC=C1 ZGUNAGUHMKGQNY-SSDOTTSWSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229940091173 hydantoin Drugs 0.000 description 2
- 150000001469 hydantoins Chemical class 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- NXQJDVBMMRCKQG-ZETCQYMHSA-N (5s)-5-phenylimidazolidine-2,4-dione Chemical compound O=C1NC(=O)N[C@H]1C1=CC=CC=C1 NXQJDVBMMRCKQG-ZETCQYMHSA-N 0.000 description 1
- BGNGWHSBYQYVRX-UHFFFAOYSA-N 4-(dimethylamino)benzaldehyde Chemical compound CN(C)C1=CC=C(C=O)C=C1 BGNGWHSBYQYVRX-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000193755 Bacillus cereus Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000589565 Flavobacterium Species 0.000 description 1
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 1
- 241001057811 Paracoccus <mealybug> Species 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- WJRBRSLFGCUECM-UHFFFAOYSA-N hydantoin Chemical compound O=C1CNC(=O)N1 WJRBRSLFGCUECM-UHFFFAOYSA-N 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、一般式（り
（式中、Ｒはフェニル基を示す、）で表される５−フェ
ニルヒダントイン（以下、５−Ｐｅｎと略記する。）を
一般式（Ｕ）
Ｒ−ＣＨ−ＣＯＯＨ
（式中、Ｒは一般式（１）と同じ。）で表されるＮ Ｈ
ＣＯＮ Ｈｚ
（■）
（式中、Ｒは一般式（１）と同じ。）で表されるＳ−カ
ルバモイル−Ｄ−フェニルグリシン（以下、ＣＤＰＧと
略記する。）に変換せしめる方法で、抗生物質等の原料
として工業的に重要な物質であるＤ−フェニルグリシン
の原料であるＣＤＰＧを極めて存利に製造する方法に関
するものである。Detailed Description of the Invention [Field of Industrial Application] The present invention provides 5-phenylhydantoin (hereinafter abbreviated as 5-Pen) represented by the general formula (R (in the formula, R represents a phenyl group)). ) is represented by the general formula (U) R-CH-COOH (wherein, R is the same as the general formula (1)).
CON Hz (■) (In the formula, R is the same as general formula (1).) This is a method of converting it into S-carbamoyl-D-phenylglycine (hereinafter abbreviated as CDPG). The present invention relates to a method for extremely efficiently producing CDPG, which is a raw material for D-phenylglycine, which is an industrially important raw material.
〔従来の技術及び発明が解決しようとする課題〕医薬の
中間原料としての０−フェニルグリシンは、化学合成に
よって得られるＯＬ体を光学分割して製造する方法およ
び本発明のように微生物を利用して５−置換ヒダントイ
ン類をＤ−’（−カルバミル−αアミノ酸類に変換させ
る方法としては特蘭昭５３−９１１８９号が知られてい
る。[Prior art and problems to be solved by the invention] 0-phenylglycine as an intermediate raw material for pharmaceuticals can be produced by optically resolving the OL form obtained by chemical synthesis, and by using microorganisms as in the present invention. As a method for converting 5-substituted hydantoins into D-'(-carbamyl-α amino acids), Tokuran No. 53-91189 is known.
このＯＬ分割方法においては、Ｌ体の回収、さらに、ラ
セミ化等の分ｖＩＦ４作について煩雑である。In this OL splitting method, recovery of the L-isomer and production of vIF4 such as racemization are complicated.
一方、上述の方法については、ローアミノ酸の前駆体で
あるＮ−カルバモイル−Ｄ−アミノ酸の収率が高くなく
、また、残存の５−Ｐｅ１（の回収が必要となり、さら
に、その回収操作は煩雑である。On the other hand, with the above method, the yield of N-carbamoyl-D-amino acid, which is a precursor of raw amino acids, is not high, and it is necessary to recover residual 5-Pe1, and furthermore, the recovery operation is complicated. It is.
本発明が解決しようとする課題は、従来のＤ−Ｎカルバ
ミル−α−アミノ酸の製造方法よりも、さらに工業的に
有利、かつ、安価な良い製造方法を提供することにある
。The problem to be solved by the present invention is to provide a production method that is more industrially advantageous and cheaper than the conventional production method of DN carbamyl-α-amino acid.
Ｃ間ｕ点を解決するための手段］
本発明者らは、このような従来のＣＩＩＰＧの製造より
効率の良い方法を見出すべ（鋭意研究した結果、５−Ｐ
ｅＨに微生物を作用させてＣＤＰＧに変換させる方法に
おいて、２段階に分けて微生物と反応させる方法を見出
し、本発明を完成させるに至った。[Means for solving the C-to-U point] The present inventors have found a method that is more efficient than the conventional CIIPG manufacturing method (as a result of intensive research, 5-P
In a method of reacting eH with microorganisms to convert it into CDPG, we have discovered a method of reacting with microorganisms in two stages, and have completed the present invention.
すなわち、本発明は５−ＰｅＨに微生物を作用させてＣ
ＤＰＧに変換させる方法において、２段階に分けて、そ
れぞ−れ微生物との反応ｐｌ＋を、１段階目は６，０〜
８．５．２段階目は７．０〜９．０で、さらに、その微
生物を、または微生物を固定化した状態で反応させるこ
とを特徴とするＣＤＰＧの製造方法に関するものである
。That is, the present invention allows microorganisms to act on 5-PeH to reduce C.
In the method of converting to DPG, the reaction pl+ with microorganisms is divided into two steps, and the first step is 6,0~
8.5. The second stage is 7.0 to 9.0 and relates to a method for producing CDPG, which is further characterized by reacting the microorganism or with the microorganism immobilized.
本発明において使用される微生物は、５−ＰｅＨをＣＤ
ＰＣに変換させる酵素を存するものであれば、どの微生
物でもよい０例えば、シュードモナス（Ｐｓｅｕｄｏｍ
ｏｎａｓ） 、バチルス（Ｂａｃｉｌｌｕｓ）、モラキ
七う（１ｏｒａｘｅｌｌａ） 、バラコツカス（Ｐａｒ
ａｃｏｃｃｕｓ）、アースロバクク−（Ａｒｔｈｒｏｂ
ａｃｔＱｒ）、アルカリジェネス（Ａｌｃａｌｉｚｅｎ
ｅｓ） 、フラボバクテリウム（Ｆ１３ｖｃｂａｃｔｅ
ｒｉｕ＊）等である。The microorganism used in the present invention converts 5-PeH into CD
Any microorganism may be used as long as it has an enzyme that converts it into PC. For example, Pseudomonas
onas), Bacillus, 1 oraxella, Par
acoccus), Arthrobacillus
actQr), Alcalize
es), Flavobacterium (F13vcbacte
riu*) etc.
微生物の培養に用いられる培地は、通常、責化しうる炭
素源、窒素源及び微生物の生育に必要な無機栄養素を含
存させる、通常の培地である。The medium used for culturing microorganisms is a conventional medium that usually contains responsible carbon sources, nitrogen sources, and inorganic nutrients necessary for the growth of microorganisms.
培養条件は、好気的条件下にて、ｐＨ・４〜９、温度２
５〜４５℃の適当な範囲に制御しつつ行えば望ましい。The culture conditions are aerobic, pH 4-9, temperature 2.
It is preferable to carry out the process while controlling the temperature within an appropriate range of 5 to 45°C.
Ｄ−５−置換ヒダントインだに微生物を作用せしめる方
法は、微生物の菌体または菌体の処理物を水溶液中で接
触せしめる方法である。A method for causing microorganisms to act on D-5-substituted hydantoin is a method in which microorganism cells or a treated product of microorganism cells are brought into contact with each other in an aqueous solution.
微生物を用いて、５−ＰｅＨをＣＤＰＧへの変換におい
て、原料である５−ＰｅＨの水に対する溶解度は２〜３
ｇ／ｌ！と小さいばかりでなく、溶解性も良くないため
、原料である５−ＰｅＨをｇ層状態で反応させることに
なる。When converting 5-PeH to CDPG using microorganisms, the solubility of the raw material 5-PeH in water is 2 to 3.
g/l! Not only is it small, but its solubility is also poor, so the raw material 5-PeH is reacted in the g-layer state.
しかし、生成物であるＣＤＰＧの存在下でｉ：ｌ：５−
ＰｅＨの溶解度も大きくなる。また、微生物と○反応：
こおいて、反応液中の原料である５−Ｐｅ！’！の溶、
騨亥と５−ＰｅＨからＣＤＰＧｌ・の反応速度を考慮す
れ：烈ｐｌ（・９．５で最高になるが、あまりｐＨが高
くなると菌体自身の溶菌という新たな問題が生してくる
。また、生成したｃｏｐｃ１度が高まるにしたがい、）
害菌、さらに５−ＰｅＨがらＣＤＰＣへの反応阻害がＬ
こめちれるため、微生物との反応については、ｐＨ５Ｃ
ＣＩＰＧ’ＪＩ、さらに生成したＣＤＰＣをＤ−フェニ
ルグリシンへの転換を考慮して、残存５−ＰｅＨ濃度を
できるだ１す少なくする必要がある。However, in the presence of the product CDPG i:l:5-
The solubility of PeH also increases. Also, ○ reaction with microorganisms:
Here, 5-Pe! which is a raw material in the reaction solution! '! melting,
Consider the reaction rate of CDPGl from Denghai and 5-PeH: The highest value is 9.5, but if the pH becomes too high, a new problem arises: lysis of the bacterial cells themselves. , as the generated copc1 degree increases)
Harmful bacteria, as well as 5-PeH, inhibit the reaction to CDPC.
For reactions with microorganisms, pH 5C
Considering the conversion of CIPG'JI and the generated CDPC into D-phenylglycine, it is necessary to reduce the residual 5-PeH concentration as much as possible.
本発明の反応は、５−ＰｅＨからＣＤＰＧへの微生物に
よる変換反応を効率よく行い、さらに、溶液中の残存５
−ＰｅＨをできるかぎり少なく、好ましくは残存５−Ｐ
ｅ１ｔ濃度／生成ＣＤＰＧが０．０５以下にするために
、２段階に分５すで微生物と反応させるのである。The reaction of the present invention efficiently performs the conversion reaction of 5-PeH to CDPG by microorganisms, and furthermore,
-PeH as low as possible, preferably remaining 5-P
In order to make the e1t concentration/generated CDPG less than 0.05, the reaction with microorganisms is carried out in two stages.
まず、１段階目の反応は、主にＣＤＰＧを生成させるこ
とを目的とし、２段階目の反応は、未反応の５−ＰｅＨ
を徹底的にＣ［ｌＰＧに変換させることにある。First, the first step reaction is mainly aimed at producing CDPG, and the second step reaction is to generate unreacted 5-PeH.
The goal is to thoroughly convert C[lPG.
すなわち、１段階目では、原料である５ −Ｐｅｔ（が
困体のままである懸濁液で、５−ＰｅＨの濃度は、通常
は、０．５〜１０重量％である。そのと濁液に菌体また
は菌体を固定化したものを作用させる。That is, in the first stage, a suspension in which the raw material 5-Pet (5-PeH) remains in a state is produced, and the concentration of 5-PeH is usually 0.5 to 10% by weight. is treated with bacterial cells or immobilized bacterial cells.
反応中のｐＨは、生成Ｃ［ｌＰＧを中和するためにｐｉ
スクントを用いてアルカリ水溶液を滴下して反応液のｐ
Ｈを保持する。 ｐＨの範囲は、６．０〜８．５であり
、特に好ましくは、７．０〜８．５である。The pH during the reaction was adjusted to
Using a scunto, drop an alkaline aqueous solution to reduce the p of the reaction solution.
Hold H. The pH range is 6.0 to 8.5, particularly preferably 7.0 to 8.5.
アルカリ水溶液には、水に化ナトリウム、水酸化カリウ
ムおよびアンモニア水等がある。Examples of aqueous alkaline solutions include sodium chloride, potassium hydroxide, and aqueous ammonia.
反応温度は、使用する微生物のＣＤＰＧへの変換する能
力を持つ酵素の至１ＩｙＩ温度が採用されるが、通常、
２０〜６０’Ｃの範囲にあり、特に好ましくは２０〜５
０℃である０反応液中の生成したＣＤＰＧが菌体の活性
阻害濃度付近に達したら反応を止める。The reaction temperature is the lowest temperature of the microorganism used for the enzyme capable of converting it into CDPG, but usually
It is in the range of 20 to 60'C, particularly preferably 20 to 5'C.
The reaction is stopped when the CDPG produced in the 0 reaction solution at 0°C reaches a concentration close to the inhibition of bacterial cell activity.
ＣＤＰＧにより阻害を受ける濃度は、微生物により異な
るが、通常は、１〜５重量％である。このようにして得
られた反応液を、遠心分Ｕあるいはセラ７り膜等の限界
濾過膜により反応清澄液と菌体および残存５・ＰｅＨ結
晶とに固液公社させる。この時の反応清澄液には、生成
したＣＤＰＧと飽和熔解分の５−Ｐｅ屓２〜５ｇ／ｆ＞
が含すれろ。The concentration at which CDPG is inhibited varies depending on the microorganism, but is usually 1 to 5% by weight. The reaction solution thus obtained is separated into a clear reaction solution, bacterial cells, and residual 5.PeH crystals using a centrifugal filter or an ultrafiltration membrane such as a cellar membrane. At this time, the reaction clear liquid contained 2 to 5 g/f of the produced CDPG and 5-Pe of the saturated melt.
Include it.
２段階目の反応では、さろに、この反応清！金液に新た
に面体または菌体を固定化したものを作用させる。反応
中のｐＨは、生成したＣＤＰＧを中和するためｐＨスタ
７トを用いてアルカリ水−Ｖ＠液を滴下し１、反応液の
ｐｉを保持する。 ｐＨの範囲は７．０〜９．０であり
、特に、好ましくは、８．０〜９゜０である。In the second stage of the reaction, this reaction is clear! A new face piece or one with immobilized bacterial cells is applied to the gold liquid. The pH during the reaction is adjusted by dropping alkaline water-V@ solution using a pH starter to neutralize the generated CDPG, and maintain the pi of the reaction solution. The pH range is 7.0-9.0, particularly preferably 8.0-9.0.
反応温度は、１段Ｐ）冒の反応温度と同じ温度を採用し
、残存５−Ｐｅ）ＩＪ度／生成ＣＤＰＧが０．０５以下
になった時に反応を止める。生成したＣＤり［、の単離
：よ、濃縮し＋１）１を等電点（ｐｆ＋・２）にするこ
とにより、目的物であるＣＤＰＧ壱近出させ取得できろ
。The reaction temperature is the same as that of the first stage P), and the reaction is stopped when the ratio of residual 5-Pe) IJ degree/generated CDPG becomes 0.05 or less. Isolation of the generated CD: By concentrating and bringing 1 to its isoelectric point (pf+2), the target product, CDPG, can be extracted and obtained.
生成したＣ０ＰＧの定ｇ！は、エールリッヒ試薬を用い
る比色法および液体クロマトグラフィーで測定する方法
を用いた。Constant g of generated C0PG! used a colorimetric method using Ehrlich's reagent and a measurement method using liquid chromatography.
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７りＷＨ、ダイセル社製）を用いる液体
クロマトグラフィーによ１て０体を確認した。Zero optical isomers were confirmed by measuring the specific rotation of the crystal and liquid chromatography using an optical resolution column (Chiralpa 7 WH, manufactured by Daicel Corporation).
以下の例により本発明を具体的に説明するが、本発明；
よ、これらの例のみに限定されるものではない。The present invention will be specifically explained with reference to the following examples.
However, it is not limited to these examples.
実施例１
グリセロール６ｇ／ｌ、ヒダントインＸｇ／ｆ、肉エキ
ス２０ｇ／ｊ！、 ＫＨｚＰＯｎ ２ｇ／Ｌ Ｍｇ５
Ｏａ −７Ｈｚ０１ ｇ／ ｊ！ ５ＦｅＳＯａ
・７ ＨｘＯ２抛ｇ／ｆ！ＳＭｎ５Ｏ６２０＋ｇ／ｌ
％ＣｕＳ０．２０ ｍｇ＃！ （ｐＨ−５，５）の培地
を２５０ｍ ｌ三角フラスコに２０ｍ ｉ入れ、１２０
″Ｃ１１５分間５３国した。Example 1 Glycerol 6g/l, hydantoin Xg/f, meat extract 20g/j! , KHzPOn 2g/L Mg5
Oa -7Hz01 g/j! 5FeSOa
・7 HxO2 g/f! SMn5O620+g/l
%CuS0.20 mg#! Pour 20ml of medium (pH-5.5) into a 250ml Erlenmeyer flask,
``C 115 minutes and 53 countries.
これにブイヨン培地で、２８℃、２４Ｐｆ間培養したシ
ュードモナス・ストリアタ（Ｐｓｅｕｄｏａｏｎａｓ
５ｔｒｉａｔａ；ＩＦｏ １２９９６）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、培養液と同量の殺蘭した生理食塩
水にて１回洗浄し、菌体を集めた。This was then cultured in broth medium at 28°C for 24Pf.
5triata; IFo 12996) was inoculated into one platinum loop and cultured at 28°C for 24 hours. Bacterial cells were collected by centrifugation of this culture solution, washed once with the same amount of orchid-killed physiological saline as the culture solution, and collected.
１段階目の反応は、この菌体を０Ｌ−５−フェニルヒダ
ントイン３０ｇ／ １水溶液（ｐＨ・８．５）・−・終
末１０００層ｌ・・・にＩｇ／ｊ！になるように添加し
た。この）容ン夜２Ｈを２Ｎ−＼ａＯＨを用いて８．５
に調製し、液温を２８゛ｃにした。さらに、窒素を封入
し、撹拌下に反応させた０反応中のｐｇ＋まｐ＋スタフ
・トを用いて８．５に保持した０反応液中のＣＤｐＧｃ
さ変が約２０ｇ／ １に達したとき、反２液を１０ＱＯ
Ｑｒｐｍ、１０分間、迷心分社し、上澄液と菌体および
残存５−ＰｅＨ結晶とに固液分離させた。In the first step, the bacterial cells were transferred to 0 L-5-phenylhydantoin 30 g/1 aqueous solution (pH 8.5) --- Ig/j! It was added so that 8.5 using 2N-\aOH
The liquid temperature was adjusted to 28°C. Furthermore, CDpGc in the 0 reaction solution, which was kept at 8.5 using pg+map+staft in the 0 reaction which was sealed with nitrogen and reacted under stirring.
When the change reaches about 20g/1, add 10QO of anti-2 liquid.
Qrpm for 10 minutes to perform solid-liquid separation into supernatant, bacterial cells, and remaining 5-PeH crystals.
２段階目の反応は、得られた上澄液１０００ｉ １に菌
体を１１！／ｆになるように添加し、ｐ）ｌスタットを
用いて２ＮＪａＯＨでｐ）Ｉ ９．０に調製し、液温を
２８゛ｃにした。さらに、窒素を封入し、撹拌下に２８
°Ｃで反応させた。結果を第１表に示した。In the second step of the reaction, 11 microorganisms were added to 1000 i of the obtained supernatant. p)I was adjusted to 9.0 with 2N JaOH using a p)l stat, and the liquid temperature was adjusted to 28°C. Furthermore, nitrogen was filled in and the mixture was heated for 28 hours while stirring.
The reaction was carried out at °C. The results are shown in Table 1.
（以下、余白）
第１表
実施例２
実施例１と同様の方法いて調製した国体を０．００４
ＭＩＰｒ ｒａバッフｙ−（ｐＨ７，０） −・・終末
１００！ｌ ｆ！−に４ｇ／ｌになるように添加し、ア
クリルアミドモノマー７．５ｇ、 Ｎ、Ｎ”−メチレ
ンビスアクリルアミド４００ｍｇ 、２．５重量％Ｋｔ
Ｓ２０ｍ水溶液５閣！および５重１％Ｎ、Ｎ、Ｎ’、Ｎ
’−テトラメチルエチレンジアミン水溶液５−１を加え
、窒素封入し、水冷下、３０分間重合させ、ブレンダー
にて破砕後、０．０１ＭＪ５酸バッファーにて洗浄し固
定化ゲル１２０ｇを得た。(Hereinafter, blank space) Table 1 Example 2 Kokutai prepared in the same manner as Example 1 was 0.004
MIPr ra buffer y-(pH 7,0) ---End 100! lf! 7.5 g of acrylamide monomer, 400 mg of N,N''-methylenebisacrylamide, 2.5 wt% Kt.
Five S20m aqueous solutions! and 5 weight 1% N, N, N', N
'-Tetramethylethylenediamine aqueous solution 5-1 was added, sealed with nitrogen, polymerized for 30 minutes under water cooling, crushed in a blender, and washed with 0.01 MJ5 acid buffer to obtain 120 g of immobilized gel.
１段階目の反応は、この固定化ゲル１００ｇを実施例１
と同様な方法にて得られた上澄液１０００ａ Ｉ！に添
加し、ｐＨスタンドを用いて２Ｎ−ＮａｏＨ水溶液を滴
下しｐＨ９，０にｔＪＲ製し、窒素封入ご、攪拌下に２
８°Ｃで反応させた。結果を第２表に示した。In the first step reaction, 100 g of this immobilized gel was added to Example 1.
Supernatant liquid 1000a obtained in the same manner as I! 2N-NaoH aqueous solution was added dropwise using a pH stand to adjust the pH to 9.0.
The reaction was carried out at 8°C. The results are shown in Table 2.
（以下、余白）
第２表
第３表
実施例３
実施例１と間を兼の方法にて調製した培地にブイヨン培
地で、２８°Ｃ１２４時間培養したバチルス・セレウス
（Ｂａｃｉｌｌｕｓ ｃｅｒｅｕｓ；ＩＦＯ３１３１）
を１白金耳接種し、２８℃、２４時間培養した。(Hereinafter, blank spaces) Table 2 Table 3 Example 3 Bacillus cereus (IFO3131) was cultured at 28°C for 124 hours in a broth medium prepared in the same manner as in Example 1.
One platinum loop of the following was inoculated and cultured at 28°C for 24 hours.
菌体の調製方法および反応方法は、実施例１と同様の方
法で行った。ただし、１段階目の反応ｐＨを７．０とし
、２段階目の反応ｐ）Ｉを８．０とした。結果を第３表
に示した。The bacterial cell preparation method and reaction method were the same as in Example 1. However, the reaction pH in the first step was 7.0, and the reaction p)I in the second step was 8.0. The results are shown in Table 3.
〔発明の効果：
本発明：よ、反応に使用した微生物の活性低下による反
応ｍ害を抑制し、高収率で、しかも、工業的に有利に、
かつ、安価にＣＤＰＧ ＠製造することができる有用な
方法である。[Effects of the invention: The present invention: suppresses reaction damage caused by decreased activity of microorganisms used in the reaction, achieves high yield, and is industrially advantageous;
Moreover, it is a useful method that can produce CDPG@ at low cost.
Claims (3)
ニルヒダントインに微生物を作用させて、Ｎ−カルバモ
イル−Ｄ−フェニルグリシンに変換させる方法において
、２段階で反応させることを特徴とする一般式（II） ▲数式、化学式、表等があります▼（II） （式中、Ｒは一般式（ I ）と同じ。）で表されるＮ−
カルバモイル−Ｄ−フェニルグリシンの製造方法。(1) General formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) (In the formula, R represents a phenyl group.) By allowing microorganisms to act on 5-phenylhydantoin, N- In the method of converting to carbamoyl-D-phenylglycine, general formula (II) is characterized by a two-step reaction ▲There are numerical formulas, chemical formulas, tables, etc.▼(II) (In the formula, R is the general formula (I ) is the same as ).
Method for producing carbamoyl-D-phenylglycine.
Ｈを６．０〜８．５で、２段階目のｐＨを７．０〜９．
０で反応させる請求項１記載の方法。(2) In a two-step reaction method, p in the first step
H at 6.0-8.5, and the second stage pH at 7.0-9.
2. The method according to claim 1, wherein the reaction is carried out at 0.0.
微生物を固定化した状態で反応させる請求項１または請
求項２記載の方法。(3) The method according to claim 1 or 2, in which the microorganism or microorganisms are reacted in an immobilized state in the two-step reaction method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63326311A JP2866660B2 (en) | 1988-12-26 | 1988-12-26 | Method for producing N-carbamoyl-D-phenylglycine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63326311A JP2866660B2 (en) | 1988-12-26 | 1988-12-26 | Method for producing N-carbamoyl-D-phenylglycine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02174684A true JPH02174684A (en) | 1990-07-06 |
| JP2866660B2 JP2866660B2 (en) | 1999-03-08 |
Family
ID=18186345
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63326311A Expired - Fee Related JP2866660B2 (en) | 1988-12-26 | 1988-12-26 | Method for producing N-carbamoyl-D-phenylglycine |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2866660B2 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5455788A (en) * | 1977-10-05 | 1979-05-04 | Kanegafuchi Chem Ind Co Ltd | Preparation of d-n-carbamoylphenylglycine |
| JPS5586A (en) * | 1979-03-16 | 1980-01-05 | Kanegafuchi Chem Ind Co Ltd | Preparation of d-n-carbamyl(p-hydroxyphenyl)glycine |
-
1988
- 1988-12-26 JP JP63326311A patent/JP2866660B2/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5455788A (en) * | 1977-10-05 | 1979-05-04 | Kanegafuchi Chem Ind Co Ltd | Preparation of d-n-carbamoylphenylglycine |
| JPS5586A (en) * | 1979-03-16 | 1980-01-05 | Kanegafuchi Chem Ind Co Ltd | Preparation of d-n-carbamyl(p-hydroxyphenyl)glycine |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2866660B2 (en) | 1999-03-08 |
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