JPS62163698A - Production of gamma-l-glutamyl-l-alpha-amino-n-butyrylglycine - Google Patents
Production of gamma-l-glutamyl-l-alpha-amino-n-butyrylglycineInfo
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- JPS62163698A JPS62163698A JP61003682A JP368286A JPS62163698A JP S62163698 A JPS62163698 A JP S62163698A JP 61003682 A JP61003682 A JP 61003682A JP 368286 A JP368286 A JP 368286A JP S62163698 A JPS62163698 A JP S62163698A
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- acid
- amino
- butyrylglycine
- glutamyl
- bacterial cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明はｒ−Ｌ−グルタミル−Ｌ−α−アミノ−ｎ−ブ
チリルグリシン（以下オ７タルミン酸と記す。）の製造
方法に関するものである。さらに詳しくはγ−グルタミ
ルシステインシンセターゼ（以下ＧＳＨ−１と記す。）
及びグルタチオンシンセターゼ（以下ＧＳＨ−Ｉｆと記
す。）活性を有する大腸菌を用いてＬ−グルタミン酸、
Ｌ−α−アミノ酪酸、及びグリシンよりオフタルミン酸
を効率良く酵素的に製造する方法に関する。なお、オフ
タルミン酸は眼の水晶体の成分で眼薬の原料として使用
できることが示唆されている。Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a method for producing r-L-glutamyl-L-α-amino-n-butyrylglycine (hereinafter referred to as otalminic acid). be. More specifically, γ-glutamylcysteine synthetase (hereinafter referred to as GSH-1)
and L-glutamic acid, using Escherichia coli having glutathione synthetase (hereinafter referred to as GSH-If) activity.
The present invention relates to a method for efficiently enzymatically producing ophthalmic acid from L-α-aminobutyric acid and glycine. It has been suggested that ophthalmic acid is a component of the crystalline lens of the eye and can be used as a raw material for eye medicines.
（従来の技術）
オフタルミン酸の製造に関する従来の技術としては、ｒ
−グルタミル−α−アミノ−ｎ−ブチリックアシド（以
下ｒ −ＧＡＢと記す。）とグリシン、アデノシン三リ
ン酸（以下ＡＴＰと記す。）及び酵素活性化剤としての
塩化マグネシウムを含有する水溶液をＧＳＨ−ＩＩと接
触させる事ｔ−特徴とする製造法が知られている。（例
えば、特開昭５８−１３４９９７ ）（発明が解決しよ
うとする問題点〕
しかしながら上記の方法は
（１） 原料のｒ −ＧＡＢが高価であること″（２
） 高価なＡＴＰ ｉ多量に使用すること（３）
酵素の単離精製等の操作を必要とし、又単位菌体あたり
のオ７タルミン酸収量が低い等の問題点を有していた。(Prior art) As a conventional technology for producing ophthalmic acid, r
GSH -Production methods characterized by contacting with II are known. (For example, JP-A-58-134997) (Problems to be solved by the invention) However, the above method has the following problems: (1) The raw material r-GAB is expensive'' (2)
) Using large amounts of expensive ATP i (3)
This method requires operations such as isolation and purification of the enzyme, and also has problems such as a low yield of otalminic acid per unit cell.
（問題点を解決するための手段）
本発明者等は上記問題点の解決のため、種々検討の結果
、Ｌ−グルタミン酸、Ｌ−α−アミノ酪酸及びグリシン
を同時にＧＳＨＩ 、 ＧＳＨＩＩと接触させ、反応さ
せることによシ１回操作で簡便にしかも効率よくオフタ
ルミン酸を製造できることを見出し本発明を完成した。(Means for Solving the Problems) In order to solve the above problems, the present inventors, as a result of various studies, brought L-glutamic acid, L-α-aminobutyric acid, and glycine into contact with GSHI and GSHII at the same time, resulting in a reaction. The present invention has been completed based on the discovery that ophthalmic acid can be produced simply and efficiently in a single operation.
すなわち本発明は、アセチルリン酸の存在下、Ｌ−グル
タミン酸、Ｌ−α−アミノ酪酸、グリシン及びアデノシ
ン三リン酸を含む水溶液にγ−グルタミルシスティンシ
ンセターゼ、グルタチオンシンセターゼ、及びアセテー
トキナーゼ活性を有する大腸菌又はその処理物を接触さ
せメ宝−Ｌ−グルタミル−Ｌ−α−アミノ−ｎ−プチリ
ルグリシンの製造方法である。以下本発明の方法につい
て詳細に説明する。That is, the present invention has γ-glutamylcysteine synthetase, glutathione synthetase, and acetate kinase activities in an aqueous solution containing L-glutamic acid, L-α-aminobutyric acid, glycine, and adenosine triphosphate in the presence of acetyl phosphate. This is a method for producing Meto-L-glutamyl-L-α-amino-n-butyrylglycine by contacting E. coli or a treated product thereof. The method of the present invention will be explained in detail below.
本発明に用いられる大腸菌はＧＳＨＩ活性、ＧＳＨＩＩ
活性を有するもの、及びアセチルリン酸（以下ＡＣＰと
記す）とアデノシン三リン酸（以下ＡＤＰと記す）から
ＡＴＰを生成し得るアセテートキナーゼ活性を有する大
腸菌であればいずれも使用できる。The E. coli used in the present invention has GSHI activity, GSHII
Any E. coli can be used as long as it has an acetate kinase activity that can generate ATP from acetyl phosphate (hereinafter referred to as ACP) and adenosine triphosphate (hereinafter referred to as ADP).
これらの酵素活性を全て具備するもの、いずれかの酵素
活性を持つものを組合せたものが使用できる。さらにこ
れらの大腸菌を変異処理して得られる変異株や、遺伝子
組換えによシ各酵素活性を増強した菌株も用いることが
できる。その具体例としては例えば特開昭５９−１９２
０８８号記載の、遺伝子組換手法によｊ５ＧｓＨＩなら
びにＧＳＨＩＩの活性を共に増強した大腸菌をあげるこ
とができる。Those having all of these enzyme activities or a combination of those having any of these enzyme activities can be used. Furthermore, mutant strains obtained by subjecting these Escherichia coli to mutation treatment and strains in which the activities of various enzymes are enhanced through genetic recombination can also be used. As a specific example, for example, Japanese Patent Application Laid-Open No. 59-192
Examples include Escherichia coli in which both j5GsHI and GSHII activities are enhanced by genetic recombination techniques, as described in No. 088.
大腸菌の培養には特定の方法を用いることは必要とせず
通常の方法で培養すればよい。培養によシ得られた菌体
、その処理物はオ７タルミン酸の生成反応に用いられる
。又、菌体の無細胞抽出液を反応に使用することができ
る。菌体そのものを使用してもよいが、そのときは基質
、とくにＡＴＰの透過性を良くするためにトルエンのよ
うな有機溶媒あるいは界面活性剤による処理が有効であ
る。It is not necessary to use a specific method for culturing E. coli, and it is sufficient to use a conventional method. The bacterial cells obtained by culturing and the processed product thereof are used in the production reaction of 7-talumic acid. Furthermore, a cell-free extract of bacterial cells can be used in the reaction. The bacterial cells themselves may be used, but in that case, treatment with an organic solvent such as toluene or a surfactant is effective in order to improve permeability to the substrate, especially ATP.
菌体はその懸濁液を反応に使用することができる。A suspension of the bacterial cells can be used for the reaction.
菌体又は酵素の固定化物例えば高分子の担体に包括固定
化したものを用い、繰返し、又は連続的に反応を行う方
法が効率的である。固定化菌体ないし酵素は例えば菌体
ないし酵素をポリアクリルアミドゲル、アルギン酸カル
シツム、カラギーナン等の高分子の担体に包括固定化し
、ヘキサメチレンジアミンとグルタルアルデヒドによっ
て硬化処理全行うことによって得られる。菌体の場合は
、トルエンのような有機溶媒で更に処理すると高活性で
耐久性に富む固定化菌体が得られる。この固定化菌体は
ガラスカラム内に充填し、固定化菌体カラムとして、オ
フタルミン酸の連続生産にも用いられる。An efficient method is to carry out the reaction repeatedly or continuously using immobilized bacterial cells or enzymes, such as those entrappingly immobilized on a polymeric carrier. Immobilized microbial cells or enzymes can be obtained, for example, by entrapping and immobilizing microbial cells or enzymes on a polymeric carrier such as polyacrylamide gel, calcium alginate, carrageenan, etc., and then completely curing with hexamethylene diamine and glutaraldehyde. In the case of bacterial cells, highly active and durable immobilized bacterial cells can be obtained by further treatment with an organic solvent such as toluene. The immobilized cells are packed into a glass column and used as an immobilized cell column for continuous production of ophthalmic acid.
オ７タルミン酸生成反応は菌体あるいは菌体処理物を５
〜２００ｍＭのＬ−グルタミン酸、５〜５０−のＬ−α
−アミノ酪酸、５〜５０ｍＭのグリシン、１〜５ｍＭの
マグネシウムイオン、０．５〜１０ｍＭのＡＴＰ、及び
５〜８０ｍＭのＡＣＰｔ−含む反応液で２０〜４０℃、
反応ｐＨ６〜９で数時間接触させることによって行える
。反応液の好ましい実施態様の例はＬ−グルタ之ン酸約
８０ｍＭ、Ｌ−α−アミノ酪酸約２０ｍＭ、グリシン約
２０ｍＭ、マグネシウムイオン約２．５ｍＭ、 ＡＴＰ
約１ ｍＭ 、 ＡＣＰ約６０蛎である。反応温度は３
５〜４０℃声は中性付近がよシ好ましい。The 7-thalumic acid production reaction is carried out by
~200mM L-glutamic acid, 5-50-L-α
- Aminobutyric acid, 5-50mM glycine, 1-5mM magnesium ion, 0.5-10mM ATP, and 5-80mM ACPt- in a reaction solution containing 20-40°C,
This can be carried out by contacting for several hours at a reaction pH of 6 to 9. A preferred embodiment of the reaction solution includes approximately 80 mM L-glutanoic acid, approximately 20 mM L-α-aminobutyric acid, approximately 20 mM glycine, approximately 2.5 mM magnesium ion, and ATP.
Approximately 1 mM, ACP approximately 60 mg. The reaction temperature is 3
It is preferable that the voice be around neutral between 5 and 40 degrees Celsius.
なおＬ−α−アミノ酪酸のかわシにり、Ｌ−α−アミノ
酪酸を使用しても良いがその場合の添加濃度は１０〜１
００ｍＭ （好適には４０ ｍＭ）が適当である。又原
料は追補添加しても良く、特にＡＣＰの追補添加は良い
結果が得られる。ＡＴＰ再生系に必要な酢＃Ｉ中ナーゼ
については大腸菌自体、強い酢酸キナーゼ活性を有して
いるので別途添加する必要はない。In addition, L-α-aminobutyric acid may be used, but in that case, the concentration of addition is 10 to 1.
00mM (preferably 40mM) is suitable. Further, additional raw materials may be added, and particularly good results can be obtained by additionally adding ACP. E. coli itself has strong acetate kinase activity, so there is no need to add vinegar #I, which is necessary for the ATP regeneration system.
固定化菌体を充てんしたカラムで連続的にオ７タルミン
酸を生産する場合はカラムを２０°〜４０℃（好適には
３７℃）に保温し、上記原料液を空間速度（Ｓ、Ｖ）
０．１〜２．０ （好適には０．５）テ通塔することに
よって目的が達せられる。When producing 7-thalumic acid continuously using a column filled with immobilized bacterial cells, the column is kept at a temperature of 20° to 40°C (preferably 37°C), and the raw material solution is heated at a space velocity (S, V).
The objective is achieved by passing the temperature between 0.1 and 2.0 (preferably 0.5).
以上の反応により生成されたオフタルミン酸を反応液中
よシ採取する方法はイオン交換樹脂を用いる等の通常の
方法で行なうことができる。The ophthalmic acid produced by the above reaction can be collected from the reaction solution by a conventional method such as using an ion exchange resin.
（実施例〕
以下実施例をもって本発明を具体的に説明するが本発明
はこれに限定されるものではない。(Examples) The present invention will be specifically described below with reference to Examples, but the present invention is not limited thereto.
実施例１
１、微生物の培養
Ｉｌ！轟シペゾトン】Ｏｙ、酵母工中ス１０Ｉ！、肉エ
キス５９、グルコース１０９、Ｍｇ８０４・７７Ｈ２Ｏ
１、ＫＨ２ｐｏ４５ ／ｉを含みｐ）１７．０に調節し
た培地５００ｔ／１−５Ｊの三角フラスコに入れ、加熱
滅菌した。これに予めブイヨン培地にて前培養したエシ
ェリヒア・コリＡＴＣＣ１１３０３ｅ接種し、３０℃に
て３０時間振トウ培養した。一方３０！容ジャ７アーメ
ンターに上記と同じ組成の培地１０１金入れ殺菌後、上
記フラスコの培養液を入れた。これを好気条件下で３０
℃で３０時間培養を行った。Example 1 1. Cultivation of microorganisms Il! Todoroki Sipezoton】Oy, yeast engineering school 10I! , meat extract 59, glucose 109, Mg804.77H2O
1. A medium containing KH2po45/i and adjusted to p) 17.0 was placed in a 500 t/1-5 J Erlenmeyer flask and sterilized by heat. This was inoculated with Escherichia coli ATCC 11303e, which had been precultured in a bouillon medium, and shake cultured at 30°C for 30 hours. 30 on the other hand! After sterilization, a culture medium with the same composition as above was placed in a 7-armenter, and the culture solution from the flask was then placed therein. Under aerobic conditions, this
Culture was performed at ℃ for 30 hours.
この様にして得られ次培養液を６，０００ ｒｐｒｎで
遠心分離し、湿重量３６０ｇの菌体を得た。The subculture solution thus obtained was centrifuged at 6,000 rprn to obtain bacterial cells with a wet weight of 360 g.
２、トルエン処理菌体の調製
得られた菌体２４０ｇを生理食塩水で洗浄後、ｐＨ７，
０の５ｍＭ）リス塩酸溶液に０．５ｍＭとなる様にシス
ティンを加えた緩備液６０１１７に懸濁する。2. Preparation of toluene-treated bacterial cells After washing 240 g of the obtained bacterial cells with physiological saline, pH 7,
Suspend in buffer solution 60117, which is obtained by adding cysteine to a 0.5 mM lithium-hydrochloric acid solution.
この懸濁液にトルエンを７８１７添加し、３０℃で激し
く攪拌する。１時間攪拌後遠心分離によシトルエン処理
菌体２００ｇを得た。To this suspension, 7817 g of toluene is added and stirred vigorously at 30°C. After stirring for 1 hour, 200 g of citoluene-treated bacterial cells were obtained by centrifugation.
３、オフタルミン酸生戊反応
第−表に示す組成の反応液１００ｄｉ２００１ｄ容ビー
カーに入れ３７℃に保ちつつマグネチックスターラーで
１０ Ｏｒｐｍにて攪拌し、１０時間反応を行う。その
結果２．１ ｍＭ （０，６ｍ９７ｄ ）のオフタルミ
ン酸が生成した。なお、反応液にＡＣＰｔ−添加しなか
った場合、オ７タルミン酸の生成量は０．３ ｍＭであ
った。生成したオフタルミン酸の量はアミノ酸アナライ
ザーで定量を行った。3. Phthalmic acid production reaction The reaction solution having the composition shown in Table 1 is placed in a 100 x 2001 d beaker and stirred at 10 Orpm with a magnetic stirrer while maintaining the temperature at 37°C, and the reaction is carried out for 10 hours. As a result, 2.1 mM (0.6m97d) of ophthalmic acid was produced. Note that when ACPt- was not added to the reaction solution, the amount of otalminic acid produced was 0.3 mM. The amount of ophthalmic acid produced was determined using an amino acid analyzer.
第−表
成 分 量リン酸カリウム
緩衝液（ｐＨ７，ｏ ） ２５１ＴＩＭＬ−
グルタミン酸ナトリウム ８０ｍＭＤ、Ｌ
−α−アミノ酪酸 ４０ｍＭグリシン
２０ｍＭ塩化マグネ
シウム ２５ｍＭＡＴＰ
１ｍＭＡＣＰ
６０ｍＭトルエン処理菌体
（湿重量）濃度 １ｏ ０ＩＩＩｐ／ｒｔｔｌ笑
施例２
実施例１と同じ組成の培地に加熱＃ＣＣ後後０り４とな
る様にクロラムフェニコールを添加し几培地でエシェリ
ヒア・コリ微工研菌寄第６７３４号を実施例１と同様に
培養して、湿重量３００ＩＩの菌体を得ｔ、、得られた
菌体２４０Ｉを実施例１と同様に処理してトルエン処理
菌体２００ｇを得比。Table - Ingredients Quantity Potassium phosphate buffer (pH 7, o) 251TIML-
Sodium glutamate 80mMD, L
-α-aminobutyric acid 40mM glycine 20mM magnesium chloride 25mMATP
1mM ACP
60mM toluene treated bacterial cells (wet weight) concentration 1o0IIIp/rttlExample 2 Chloramphenicol was added to a medium with the same composition as in Example 1 so that the concentration was 4 after heating #CC, and Escherichia was cultured in a diluted medium.・Culture No. 6734 of Koli Microtechnology Laboratory in the same manner as in Example 1 to obtain bacterial cells with a wet weight of 300 II.The obtained bacterial cells of 240 I were treated in the same manner as in Example 1 and treated with toluene. Obtained 200g of bacterial cells.
得られたトルエン処理菌体を用いて実施例１と同様第−
表に示し几反応組成の反応液１１で１０時間反応を行っ
た結果１６ｍＭ （４，６１／ ｌ ）のオフタルミン
酸が生成した。Using the obtained toluene-treated bacterial cells, the same procedure as in Example 1 was carried out.
As a result of 10 hours of reaction with reaction solution 11 having the reaction composition shown in the table, 16 mM (4,61/l) of ophthalmic acid was produced.
得られ几反応液１ノを塩酸でｐＨ１，５に調整した後、
セパビーズ８Ｐ２０７（三菱化成工業ｍ）Ｉｌを充填し
たカラムに通塔しオフタルミン酸を吸着させ水洗後１０
％メタノールで溶出することでオフタルミノ酸を単離し
、その溶離液を儀縮後結晶化し、３Ｉのオ７タルミン酸
の結晶を得た。又得られた結晶を加水分解してグルタミ
ン酸、α−アミノ酪酸、グリシンが生ずることをアミノ
酸アナライザーにて確認し友。After adjusting the obtained reaction solution to pH 1.5 with hydrochloric acid,
Sepabeads 8P207 (Mitsubishi Chemical Industries m) was passed through a column filled with Il to adsorb ophthalmic acid and washed with water for 10 minutes.
Ophthalminoic acid was isolated by elution with % methanol, and the eluate was evaporated and crystallized to obtain crystals of 3I ophthalmic acid. We also confirmed using an amino acid analyzer that the resulting crystals were hydrolyzed to produce glutamic acid, α-aminobutyric acid, and glycine.
紋千床令
実施例３
実施例２で得たエッシェリヒア・コリ微工研菌寄第６７
３４号の湿菌体２０．９’ｉ生理食塩水で洗浄後生理食
塩水２０ＩＩＬｔに懸濁し、４０℃に保っ０あらかじめ
１．２４Ｉ！のに一カライーナンを６ｏＩＩＬｌの生理
食塩水に溶解し、４０℃に保ったに一カラギーナン溶液
に上記菌体懸濁液を添加混合する。この混合液を０．３
Ｍ塩化カリウム水溶液中に滴下して球状の固定化菌体を
得る。この固定化菌体を２チの塩化カリウム、０．０８
Ｍのへキサメチレンジアミン及び１％のグルタルアルデ
ヒドを含む０．３３Ｍのリン酸カリ９ム緩衝液（ｐＨ７
，０）４００ゴに移し、５℃で１時間ゆるやかに攪拌し
て硬化処理を行う。硬化処理を行った固定化菌体を生理
食塩水で洗浄後０．５ｍＭのシスティンを含む５ｍＭ）
リス−塩酸緩衝液（ｐＨ７，０）に入れて２５℃で１時
間攪拌する。この後トルエン処理を行っ念固定化菌体８
０ｇを生理食塩水で洗浄後ジャケット付のカラム（φ２
．７ｃｒＩＬＸ １４ｃｒｎ ）に充てんする。この固
定化菌体を充てんしたカラムを、ジャケットに温水を通
して３７℃に保ち第１表に示した組成の原料液（但し、
トルエン処理菌体を除く）全毎時４０ゴの流速で通塔さ
せる。この操作を１０日間連続して行い、通過液中に生
成してくるオ７タルミン酸を実施例１と同様の方法で単
離結晶化する。Monsentoko Rei Example 3 Escherichia coli obtained in Example 2 No. 67
Wet bacterial cells of No. 34 were washed with 20.9'i physiological saline, suspended in 20 IIL of physiological saline, kept at 40°C, and pre-incubated with 1.24 I! Dissolve carrageenan in 60ml of physiological saline, and add and mix the above cell suspension to the carrageenan solution kept at 40°C. Add this mixture to 0.3
It is dropped into an aqueous M potassium chloride solution to obtain spherical immobilized bacterial cells. This immobilized bacterial body was mixed with 2 g of potassium chloride, 0.08
0.33 M potassium phosphate buffer (pH 7) containing M hexamethylene diamine and 1% glutaraldehyde.
, 0) Transfer to a 400-degree oven and perform hardening treatment by gently stirring at 5° C. for 1 hour. After washing the hardened and immobilized bacterial cells with physiological saline, 5mM containing 0.5mM cysteine)
The mixture was added to Lis-HCl buffer (pH 7,0) and stirred at 25°C for 1 hour. After this, toluene treatment was carried out to ensure immobilization of bacterial cells 8.
After washing 0g with physiological saline, add a jacketed column (φ2
．． 7crILX 14crn). The column filled with the immobilized bacteria was kept at 37°C by passing warm water through the jacket, and the raw material solution had the composition shown in Table 1 (however,
(Excluding toluene-treated bacterial cells) The total flow rate is 40 g/hour. This operation is carried out continuously for 10 days, and the otthalminic acid produced in the passed liquid is isolated and crystallized in the same manner as in Example 1.
その結果１０日間の連続生産で合計２４．５ｇの結晶を
得た。又取得した結晶の同定は実施例１と同様の方法で
行なった。As a result, a total of 24.5 g of crystals was obtained in 10 days of continuous production. The obtained crystals were identified in the same manner as in Example 1.
（作用・効果）
本発明に訃いてＬ−グルタミン酸、Ｌ−α−アミノ酪酸
とグリシンにＧＳＨＩとＧＳＨ■の２酵素活性物質を同
時に作用させるが、このときＧＳＨＩはＬ−グルタミン
酸とＬ−α−アミノ酪酸を結合し、ＧＳＨＩＩはその結
合物にグリシンを結合しオフタルミン酸を与える。これ
らの反応は同一系内で併行して行われ互いに反応を阻害
せずかえって全体として反応の進行が促進される。工程
が少い分高収率が達成される。アセテートキナーゼによ
るＡＴＰ再生系を利用することによって、安価なＡＣＰ
＠供給するだけで、これと、ＡＴＰが反応に消費され
たときに生じるＡＤＰとの反応によｊ９ ＡＴＰ ｉ再
生でき反応に順次利用される。従って高価なＡＴＰをエ
ネルギー源として多量に添加し消費することなくオフタ
ルミン酸が酵素的に合成される。菌体又は菌体固定化物
を反応に用いるとき菌体のトルエンなど有機溶媒又は界
面活性剤による処理は細胞膜の基質に対する透過性を改
良する。菌体固定化物を充填したカラムを利用した場合
オフタルミン酸の数十日間の連続生産が可能であり、菌
体な扱い回数が少くて済み工程が単純である点工業的に
有利である。(Action/Effect) According to the present invention, two enzymatic active substances, GSHI and GSH, are simultaneously applied to L-glutamic acid, L-α-aminobutyric acid, and glycine. GSHII binds aminobutyric acid and GSHII binds glycine to the conjugate to give ophthalmic acid. These reactions are carried out in parallel in the same system, and do not inhibit each other, but rather promote the progress of the reaction as a whole. High yields are achieved due to fewer steps. By utilizing the ATP regeneration system using acetate kinase, inexpensive ACP can be produced.
By simply supplying @, j9 ATP i can be regenerated by the reaction between this and ADP, which is generated when ATP is consumed in the reaction, and is sequentially used in the reaction. Therefore, ophthalmic acid can be enzymatically synthesized without adding or consuming a large amount of expensive ATP as an energy source. When bacterial cells or immobilized bacterial cells are used in a reaction, treatment of the bacterial cells with an organic solvent such as toluene or a surfactant improves the permeability of cell membranes to substrates. When a column filled with immobilized bacterial cells is used, it is possible to continuously produce ophthalmic acid for several tens of days, which is industrially advantageous in that the number of times the bacterial cells are handled is small and the process is simple.
Claims (3)
−α−アミノ酪酸、グリシン及びアデノシン三リン酸を
含む水溶液にγ−グルタミルシステインシンセターゼ、
グルタチオンシンセターゼ、及びアセテートキナーゼ活
性を有する大腸菌又はその処理物を接触させるγ−Ｌ−
グルタミル−Ｌ−α−アミノ−ｎ−ブチリルグリシンの
製造方法。(1) In the presence of acetyl phosphate, L-glutamic acid, L
- γ-glutamylcysteine synthetase in an aqueous solution containing α-aminobutyric acid, glycine and adenosine triphosphate;
γ-L- in which Escherichia coli having glutathione synthetase and acetate kinase activities or a treated product thereof is contacted.
A method for producing glutamyl-L-α-amino-n-butyrylglycine.
ステインシンセターゼならびにグルタチオンシンセター
ゼを増大させた大腸菌である特許請求の範囲第１項のγ
−Ｌ−グルタミル−Ｌ−α−アミノ−ｎ−ブチリルグリ
シンの製造方法。(2) The γ of claim 1, wherein the E. coli is E. coli that has been genetically modified to increase γ-glutamylcysteine synthetase and glutathione synthetase.
- A method for producing L-glutamyl-L-α-amino-n-butyrylglycine.
あるいは界面活性剤処理した細胞の懸濁液、固定化され
た菌体又は酵素のいずれかである特許請求の範囲第１項
のＬ−グルタミル−Ｌ−α−アミノ−ｎ−ブチリルグリ
シンの製造方法。(3) L of claim 1, in which the E. coli-treated product is any of a cell-free extract of E. coli, a suspension of cells treated with an organic solvent or a surfactant, immobilized bacterial cells, or an enzyme. - A method for producing glutamyl-L-α-amino-n-butyrylglycine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61003682A JPH0634744B2 (en) | 1986-01-13 | 1986-01-13 | Method for producing γ-L-glutamyl-L-α-amino-n-butyrylglycine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61003682A JPH0634744B2 (en) | 1986-01-13 | 1986-01-13 | Method for producing γ-L-glutamyl-L-α-amino-n-butyrylglycine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS62163698A true JPS62163698A (en) | 1987-07-20 |
| JPH0634744B2 JPH0634744B2 (en) | 1994-05-11 |
Family
ID=11564171
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61003682A Expired - Lifetime JPH0634744B2 (en) | 1986-01-13 | 1986-01-13 | Method for producing γ-L-glutamyl-L-α-amino-n-butyrylglycine |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0634744B2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2016042846A (en) * | 2014-08-25 | 2016-04-04 | 国立大学法人名古屋大学 | Method for production of ophthalmic acid |
| JP2019014663A (en) * | 2017-07-04 | 2019-01-31 | 天津科技大学 | Peptide synthesis method |
| JP2021151256A (en) * | 2017-09-29 | 2021-09-30 | 三菱ケミカル株式会社 | Method for producing nicotinamide mononucleotide and transformant used in the method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59192088A (en) * | 1982-09-29 | 1984-10-31 | Hikari Kimura | Plasmid formed by gene recombination and preparation of gluthathione by it |
-
1986
- 1986-01-13 JP JP61003682A patent/JPH0634744B2/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59192088A (en) * | 1982-09-29 | 1984-10-31 | Hikari Kimura | Plasmid formed by gene recombination and preparation of gluthathione by it |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2016042846A (en) * | 2014-08-25 | 2016-04-04 | 国立大学法人名古屋大学 | Method for production of ophthalmic acid |
| JP2019014663A (en) * | 2017-07-04 | 2019-01-31 | 天津科技大学 | Peptide synthesis method |
| JP2021151256A (en) * | 2017-09-29 | 2021-09-30 | 三菱ケミカル株式会社 | Method for producing nicotinamide mononucleotide and transformant used in the method |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0634744B2 (en) | 1994-05-11 |
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