JP2005287436A - Method for producing l-tyrosine - Google Patents
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Abstract
Description
本発明はＬ−チロシンの製造法に関するものである。 The present invention relates to a method for producing L-tyrosine.
Ｌ−チロシンは医薬品の原料、アミノ酸輸液、経口・経腸栄養剤の成分、健康食品などの栄養強化等に多量に利用されている。 L-tyrosine is used in large amounts for raw materials of pharmaceuticals, amino acid infusions, components of oral and enteral nutrients, and fortification of health foods.
従来、Ｌ−チロシンの製造法としては大豆たんぱく質、動物由来物質の加水分解物から抽出等により安価に製造されてきたが、近年のＢＳＥ問題等で抽出品に替わる安価な合成Ｌ−チロシンが期待されている。 Conventionally, as a method for producing L-tyrosine, it has been produced at low cost by extraction from soybean protein or a hydrolyzate of animal-derived substances, but inexpensive synthetic L-tyrosine is expected to replace the extract due to recent BSE problems. Has been.
チロシンフェノールリアーゼ（β−チロシナーゼ）存在下、フェノールとＬ−セリンまたはピルビン酸と反応させＬ−チロシンを製造する方法はすでに知られている。該酵素反応は反応基質であるフェノールがチロシンフェノールリアーゼの活性を阻害して反応収率を低下させる事が知られている。フェノールによる酵素阻害を回避するために、連続的に間欠的にフェノールを添加する方法、分割フィードする方法にて酵素反応中のフェノール濃度を低濃度下に管理して反応を行うことが開示されている（特許文献１、非特許文献２参照）。
しかしながら、前記の背景技術に記載された方法では酵素反応中のフェノール濃度の管理が難しく煩雑である。また反応収率低下も招き易く酵素反応も長時間となり、必ずしも工業的に満足する製造法とは言い難い。 However, in the method described in the background art, it is difficult and complicated to control the phenol concentration during the enzyme reaction. In addition, the reaction yield tends to decrease, and the enzyme reaction also takes a long time, which is not necessarily an industrially satisfactory production method.
したがって、本発明の目的は、反応時間を短縮でき、かつ高収率でＬ−チロシンを製造する方法を提供することを目的とする。 Therefore, an object of the present invention is to provide a method for producing L-tyrosine with a high yield, which can shorten the reaction time.
本発明者等は上記課題を解決する為、鋭意検討した結果、フェノールが有機溶媒層と水層間で分配することを利用して、反応中の水層中のフェノール濃度を酵素活性の阻害濃度以下に制御することによりＬ−チロシンを効率的に製造できることを見出し、本発明を完成するに至った。 As a result of intensive investigations to solve the above problems, the present inventors have made use of the fact that phenol is distributed between the organic solvent layer and the water layer, and the phenol concentration in the aqueous layer during the reaction is below the inhibitory concentration of the enzyme activity. As a result, it was found that L-tyrosine can be efficiently produced by controlling to the above, and the present invention has been completed.
即ち、本発明は、チロシンフェノールリアーゼの存在下、フェノールとセリン、または、フェノールと、ピルビン酸またはその塩と、アンモニアまたはその塩からＬ−チロシンを製造する方法において、水と混和しないがフェノールと混和する有機溶媒の存在下で反応させることを特徴とする、Ｌ−チロシンの製造法に関するものである。 That is, the present invention relates to a process for producing L-tyrosine from phenol and serine, or phenol, pyruvic acid or a salt thereof, and ammonia or a salt thereof in the presence of tyrosine phenol lyase, but not miscible with water, but with phenol. The present invention relates to a method for producing L-tyrosine, characterized by reacting in the presence of a miscible organic solvent.
本発明によれば、煩雑な操作により水層中のフェノール濃度を制御する必要がなく、短時間でかつ高収率でＬ−チロシンを製造することができる。 According to the present invention, it is not necessary to control the phenol concentration in the aqueous layer by a complicated operation, and L-tyrosine can be produced in a short time and in a high yield.
本発明は、チロシンフェノールリアーゼの存在下、フェノールとセリン、または、フェノールと、ピルビン酸またはその塩と、アンモニアまたはその塩からＬ−チロシンを製造する方法において、水と混和しないがフェノールと混和する有機溶媒を存在させることにより有機溶媒層と水層の二層系を形成させ、水層中のチロシンフェノールリアーゼの酵素活性を低下させないように水層中のフェノール濃度を制御させながら反応させてＬ−チロシンを製造することを特徴とするものである。 The present invention relates to a method for producing L-tyrosine from phenol and serine, or phenol, pyruvic acid or a salt thereof, and ammonia or a salt thereof in the presence of tyrosine phenol lyase, which is immiscible with water but miscible with phenol. In the presence of an organic solvent, a two-layer system of an organic solvent layer and an aqueous layer is formed, and the reaction is carried out while controlling the phenol concentration in the aqueous layer so as not to reduce the enzyme activity of tyrosine phenol lyase in the aqueous layer. -Tyrosine is produced.
チロシンフェノールリアーゼとしては、市販されているチロシンフェノールリアーゼ以外に、チロシンフェノールリアーゼを含有する微生物またはこの微生物に後記の処理を施した処理物（以下、「チロシンフェノールリアーゼの供給源」と略記する。）を用いることもできる。 As a tyrosine phenol lyase, in addition to a commercially available tyrosine phenol lyase, a microorganism containing tyrosine phenol lyase or a processed product obtained by subjecting this microorganism to the treatment described below (hereinafter abbreviated as “source of tyrosine phenol lyase”). ) Can also be used.
チロシンフェノールリアーゼを含有する微生物としては、例えば、エシェリヒア属、シトロバクター属、アエロバクター属、プロテウス属、エルビニア属など（山田ら（1975）Adv.Appl.Microbiol.,Vol.19,pp.249-288）、これらの微生物からチロシンフェノールリアーゼ遺伝子を含むDNA断片を単離し、遺伝子組換え技術を応用することにより、チロシンフェノールリアーゼを高発現させたＦＥＲＭ Ｐ−１１３８６の微生物（特許2999005、WO 90/04031）を挙げることができる。 Examples of microorganisms containing tyrosine phenol lyase include, for example, the genus Escherichia, Citrobacter, Aerobacter, Proteus, Erbinia (Yamada et al. (1975) Adv. Appl. Microbiol., Vol. 19, pp. 249- 288), a FERM P-11386 microorganism in which tyrosine phenol lyase is highly expressed by isolating a DNA fragment containing a tyrosine phenol lyase gene from these microorganisms and applying a gene recombination technique (Patent 2999005, WO 90 / 04031).
このような微生物は、培養液としてそのまま用いてもよいし、凍結保存した培養液を融解して用いてもよい。また、培養液から膜分離、遠心分離等で回収した微生物菌体を用いることもできる。
また、さらに、微生物菌体を超音波処理等で破砕した破砕物、またはその破砕物をさらに水等で抽出した抽出液、または該抽出液からさらに硫安分画、有機溶媒沈殿、カラム精製等の精製操作により粗精製したチロシンフェノールリアーゼを用いることもできる。
Such a microorganism may be used as it is as a culture solution, or may be used after thawing a cryopreserved culture solution. In addition, microbial cells recovered from the culture solution by membrane separation, centrifugation, or the like can also be used.
Furthermore, a crushed product obtained by crushing microbial cells by ultrasonic treatment or the like, or an extract obtained by further extracting the crushed product with water or the like, or an ammonium sulfate fraction, organic solvent precipitation, column purification, etc. from the extract. It is also possible to use tyrosine phenol lyase that has been roughly purified by a purification operation.
セリンを用いる場合、セリンとしては、Ｌ−セリン、Ｄ−セリン、ＤＬ−セリンが用いられる。 When using serine, as serine, L-serine, D-serine, and DL-serine are used.
ピルビン酸またはその塩を用いる場合、ピルビン酸、またはピルビン酸のアルカリ金属塩、例えば、ナトリウム塩、カリウム塩、リチウム塩の一水和物、ピルビン酸アンモニウム等の塩類が用いられる。 When pyruvic acid or a salt thereof is used, pyruvic acid or an alkali metal salt of pyruvic acid, for example, sodium salt, potassium salt, monohydrate of lithium salt, ammonium pyruvate or the like is used.
セリンまたはピルビン酸の使用量は、フェノールに対してそれぞれ０．１〜１．５当量が好ましく、より好ましくは０．７〜１．１当量である。０．１当量以下では生産効率が悪く好ましくない。１．５当量以上ではセリン、ピルビン酸が高価であり経済的見地から好ましくない。 The amount of serine or pyruvic acid used is preferably 0.1 to 1.5 equivalents, more preferably 0.7 to 1.1 equivalents, relative to phenol. If it is 0.1 equivalent or less, the production efficiency is poor, which is not preferable. If it is 1.5 equivalents or more, serine and pyruvic acid are expensive, which is not preferable from an economical viewpoint.
セリン、またはピルビン酸の水層中の濃度は、それぞれ１〜５０ｗｔ％が好ましく、より好ましくは５〜２０ｗｔ％である。１ｗｔ％以下でも特に問題はないが、容積効率の観点から１ｗｔ％以上が好ましい。５０ｗｔ％以下であると反応中に生成したＬ−チロシンが粘ちょうにならず、反応が充分に進行し収率低下を防ぐことができるため好ましい。 The concentration of serine or pyruvic acid in the aqueous layer is preferably 1 to 50 wt%, more preferably 5 to 20 wt%. Even if it is 1 wt% or less, there is no particular problem, but 1 wt% or more is preferable from the viewpoint of volumetric efficiency. The L-tyrosine produced during the reaction is preferably 50 wt% or less because it does not become sticky and the reaction proceeds sufficiently to prevent a decrease in yield.
アンモニウム塩としては塩化アンモニウム、硫酸アンモニウム、酢酸アンモニウム、炭酸水素アンモニウム、炭酸アンモニウムなどが挙げられる。
アンモニアもしくはアンモニウム塩の使用量としてはセリン、ピルビン酸に対して０．１〜５．０当量が好ましく、より好ましくは０．２〜３．０当量である。０．１当量以下では酵素反応中のｐＨ維持管理が難しい。また５．０当量以上でも問題はないが生成したＬ−チロシンと塩との分離精製が煩雑になり好ましくない。
Examples of the ammonium salt include ammonium chloride, ammonium sulfate, ammonium acetate, ammonium hydrogen carbonate, ammonium carbonate, and the like.
As usage-amount of ammonia or ammonium salt, 0.1-5.0 equivalent is preferable with respect to serine and pyruvic acid, More preferably, it is 0.2-3.0 equivalent. If it is 0.1 equivalent or less, it is difficult to maintain and maintain the pH during the enzyme reaction. Moreover, although there is no problem even if it is 5.0 equivalents or more, separation and purification of the produced L-tyrosine and the salt become complicated, which is not preferable.
チロシンフェノールリアーゼの補酵素として用いられるピリドキサールりん酸の量は特に限定されるものではないが、あまり少ないと酵素反応収率が低下し好ましくない。通常、セリン、ピルビン酸に対して０．０２重量倍以上が好ましい。 The amount of pyridoxal phosphate used as a coenzyme for tyrosine phenol lyase is not particularly limited, but if it is too small, the enzyme reaction yield is unfavorable. Usually, 0.02 weight times or more is preferable with respect to serine and pyruvic acid.
水層のｐＨは、通常の酵素反応と同様にｐＨ６〜１０の範囲が好ましい。より好ましくはｐＨ７．５〜９．５の範囲である。ｐＨ６以下、ｐＨ１０以上では酵素が失活し易く、反応が十分に進行せず好ましくない。 The pH of the aqueous layer is preferably in the range of pH 6 to 10 as in the usual enzyme reaction. More preferably, the pH is in the range of 7.5 to 9.5. At pH 6 or lower and pH 10 or higher, the enzyme is easily deactivated, and the reaction does not proceed sufficiently, which is not preferable.
水層のｐＨ調製は、アンモニア、水酸化ナトリウム、水酸化カリム、塩酸、硫酸などの水溶液で調整すればよい。 The pH of the aqueous layer may be adjusted with an aqueous solution of ammonia, sodium hydroxide, kalim hydroxide, hydrochloric acid, sulfuric acid or the like.
水と混和しないがフェノールと混和する有機溶媒としては、フェノールの水層と有機溶媒層への分配係数が３以上である有機溶媒であれば特に制限されない。分配係数があまり大きな有機溶媒を使用すると、反応初期の水層中のフェノール濃度が低いために反応速度が遅くなる傾向がある。そのため分配係数が３〜２１である有機溶媒を使用するのが好ましい。分配係数が３〜２１である有機溶媒としては、例えば、メチルイソブチルケトン（以下、「ＭＩＢＫ」と略記する。）、アセトフェノン、酢酸エチル、酢酸ブチル、イソプロピルエーテル、１−ペンタノール、オクタノール、デカノール、トルエンなどの有機溶媒が挙げられる。
なお、分配係数３以上の有機溶媒を使用すると容積効率が向上する点でも好ましい。
The organic solvent that is not miscible with water but miscible with phenol is not particularly limited as long as it is an organic solvent having a partition coefficient of 3 or more between the phenol aqueous layer and the organic solvent layer. When an organic solvent having a very large partition coefficient is used, the reaction rate tends to be slow because the phenol concentration in the aqueous layer at the initial stage of the reaction is low. Therefore, it is preferable to use an organic solvent having a distribution coefficient of 3 to 21. Examples of the organic solvent having a partition coefficient of 3 to 21 include methyl isobutyl ketone (hereinafter abbreviated as “MIBK”), acetophenone, ethyl acetate, butyl acetate, isopropyl ether, 1-pentanol, octanol, decanol, An organic solvent such as toluene may be mentioned.
Note that the use of an organic solvent having a distribution coefficient of 3 or more is also preferable from the viewpoint of improving volumetric efficiency.
フェノールの水層と有機溶媒層への分配係数は丸善株式会社発行の化学反応工学Ｉ、化学工業計算法 第６版を参考に測定することができる。具体的には、有機溶媒１００ｍｌにフェノール７．０ｇを溶解させ、純水１００ｍｌを加えて、３２℃で３０分間攪拌する。３２℃で１時間静置した後、有機溶媒層と水層中のフェノール濃度（ｗｔ％）を高速液体クロマトグラフィーにて分析し有機層／水層の濃度比から分配係数を算出することができる。 The distribution coefficient of phenol to the aqueous layer and the organic solvent layer can be measured with reference to Chemical Reaction Engineering I, Chemical Industry Calculation Method 6th edition published by Maruzen Co., Ltd. Specifically, 7.0 g of phenol is dissolved in 100 ml of an organic solvent, 100 ml of pure water is added, and the mixture is stirred at 32 ° C. for 30 minutes. After standing at 32 ° C. for 1 hour, the phenol concentration (wt%) in the organic solvent layer and the aqueous layer is analyzed by high performance liquid chromatography, and the distribution coefficient can be calculated from the concentration ratio of the organic layer / aqueous layer. .
有機溶媒の使用量としては、フェノールの水層と有機溶媒への分配係数を利用し、酵素反応開始時の水層中フェノール濃度が１％以下になるように用いれば良い。有機溶媒の使用量は水層に対して０．２〜１０重量倍が好ましく、０．４〜５重量倍がより好ましい。０．２重量倍以下では反応が粘ちょうになり充分に反応が進行せず好ましくない。１０重量倍以上では容積効率が悪く経済的な見地から好ましくない。 The amount of the organic solvent used may be such that the phenol concentration in the aqueous layer at the start of the enzymatic reaction is 1% or less by using the partition coefficient of the phenol to the aqueous layer and the organic solvent. The amount of the organic solvent used is preferably 0.2 to 10 times by weight, more preferably 0.4 to 5 times by weight with respect to the aqueous layer. Less than 0.2 times by weight is not preferable because the reaction becomes viscous and the reaction does not proceed sufficiently. If it is 10 weight times or more, the volumetric efficiency is poor and it is not preferable from an economical viewpoint.
反応温度としては１０℃〜５０℃が好ましく、より好ましくは２５〜４５℃である。１０℃以下では反応速度が遅く好ましくない。５０℃以上では酵素が失活して好ましくない。 As reaction temperature, 10 to 50 degreeC is preferable, More preferably, it is 25 to 45 degreeC. If it is 10 degrees C or less, reaction rate is slow and is not preferable. Above 50 ° C, the enzyme is inactivated, which is not preferable.
チロシンフェノールリアーゼおよびチロシンフェノールリアーゼの供給源の使用量は特に限定されるものではないが、あまり少ないと反応時間が長く反応が完結せず好ましくない。あまり多いと経済的見地から好ましくない。 The amount of tyrosine phenol lyase and the tyrosine phenol lyase source used is not particularly limited, but if it is too small, the reaction time is long and the reaction is not completed, which is not preferable. Too much is not preferable from an economic point of view.
本発明の製造法の実施態様を具体的に例示するとすれば、フェノールを溶解させた有機溶媒を、フェノールを除くチロシンフェノールリアーゼまたはチロシンフェノールリアーゼの供給源等の原材料を含有する水層に一括して添加して撹拌させながら反応させる方法、チロシンフェノールリアーゼまたはチロシンフェノールリアーゼの供給源を除く原材料およびフェノールを溶解させた有機溶媒の混合物を０．５〜１．０時間程度攪拌した後、チロシンフェノールリアーゼまたはチロシンフェノールリアーゼの供給源をこれに添加して反応させる方法が挙げられる。 If the embodiment of the production method of the present invention is specifically exemplified, an organic solvent in which phenol is dissolved is collectively contained in an aqueous layer containing raw materials such as tyrosine phenol lyase excluding phenol or a source of tyrosine phenol lyase. After stirring the mixture of the raw material excluding the tyrosine phenol lyase or the source of tyrosine phenol lyase and the organic solvent in which the phenol is dissolved for about 0.5 to 1.0 hour, the tyrosine phenol is added. A method may be mentioned in which a source of lyase or tyrosine phenol lyase is added and reacted.
反応は数時間で完結する。反応終了後は通常の脱溶媒、菌体分離、晶析、ろ過して高品質のＬ−チロシンを得る事ができる。 The reaction is complete in a few hours. After completion of the reaction, high-quality L-tyrosine can be obtained by ordinary solvent removal, bacterial cell separation, crystallization, and filtration.
以下、実施例によって本発明を更に詳細に説明するが、本発明はこれらに限定されるものではない。
なお、フェノールの分配率の算出、生成したＬ−チロシンの定量は高速液体クロマトグラフィー（以下、「ＨＰＬＣ」と略記する。）分析によった。分析条件は以下の通りである。
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these.
In addition, the calculation of the partition rate of phenol and the quantification of the generated L-tyrosine were performed by high performance liquid chromatography (hereinafter abbreviated as “HPLC”) analysis. The analysis conditions are as follows.
・ カラム Ｄｅｖｅｒｏｓｉｌ ＯＤＳ−ＭＧ−５
・ 移動相 ０．０５％ＴＦＡ：ＭｅＯＨ＝９０：１０
・ 流 速 １．０ｍｌ／ｍｉｎ．
・ カラム温度 ４０℃
・ 検出波長 ＵＶ２８０ｎｍ
・ 内部標準物質 ３，５−ジヒドロキシ安息香酸
・ Column Deverosil ODS-MG-5
Mobile phase 0.05% TFA: MeOH = 90: 10
・ Flow velocity: 1.0 ml / min.
・ Column temperature 40 ℃
・ Detection wavelength UV280nm
・ Internal standard 3,5-dihydroxybenzoic acid
実施例１
純水８６２．３ｇにＬ−セリン１０５．１ｇ（１．０００ｍｏｌ）、塩化アンモニウム２．５ｇ（０．０４７ｍｏｌ）、ピリドキサル０．０４ｇを溶解させた後、２５％アンモニア水にて該溶解液のｐＨを８．８に調整した。ｐＨ調整液にメチルイソブチルケトン３３５．８ｇと フェノール９８．８ｇを加え３０分間室温下で攪拌した。３０分後に４０℃まで昇温し、チロシンフェノールリアーゼ含有微生物としてＦＥＲＭ Ｐ−１１３８６の微生物を培養して得られた菌体５２．０ｇ（固形分５．７ｇ）を装入し酵素反応を開始した。５時間後に脱溶媒し、３５％塩酸水にて結晶を溶解しＬ−チロシン水溶液１３９８．４ｇを得た。ＨＰＬＣにてＬ−チロシンを分析した結果、１２．８２ｗｔ％、Ｌ−チロシン量は１７９．３ｇ（０．９９０ｍｏｌ）であった。Ｌ−セリンに対する収率は９９．０ｍｏｌ％で有った。
Example 1
After dissolving 105.1 g (1.000 mol) of L-serine, 2.5 g (0.047 mol) of ammonium chloride, and 0.04 g of pyridoxal in 862.3 g of pure water, the pH of the solution was adjusted with 25% aqueous ammonia. Was adjusted to 8.8. 335.8 g of methyl isobutyl ketone and 98.8 g of phenol were added to the pH adjusting solution, and the mixture was stirred at room temperature for 30 minutes. After 30 minutes, the temperature was raised to 40 ° C., and 52.0 g (solid content 5.7 g) obtained by culturing the microorganism of FERM P-11386 as a tyrosine phenol lyase-containing microorganism was charged to start the enzyme reaction. . After 5 hours, the solvent was removed, and the crystals were dissolved in 35% aqueous hydrochloric acid to obtain 1398.4 g of an aqueous L-tyrosine solution. As a result of analyzing L-tyrosine by HPLC, it was 12.82 wt% and the amount of L-tyrosine was 179.3 g (0.990 mol). The yield based on L-serine was 99.0 mol%.
実施例２
純水８６２．３ｇにＬ−セリン１０５．１ｇ（１．０００ｍｏｌ）、塩化アンモニウム２．５ｇ（０．０４７ｍｏｌ）、ピリドキサル０．０４ｇを溶解させた後、２５％アンモニア水にて該溶解液のｐＨを８．８に調整した。ｐＨ調整液に酢酸ブチル３３５．８ｇと フェノール９８．８ｇを加え３０分間室温下で攪拌した。３０分後に４０℃まで昇温してチロシンフェノールリアーゼ含有微生物としてＦＥＲＭ Ｐ−１１３８６の微生物を培養して得られた５２．０ｇ（固形分５．７ｇ）を装入し酵素反応開始した。５時間後に脱溶媒し、３５％塩酸水にて結晶を溶解しＬ−チロシン水溶液１４０１．３ｇを得た。ＨＰＬＣにてＬ−チロシンを分析した結果、１２．６０ｗｔ％、Ｌ−チロシン量は１７６．７ｇ（０．９７５ｍｏｌ）であった。Ｌ−セリンに対する収率は９７．５ｍｏｌ％で有った。
Example 2
After dissolving 105.1 g (1.000 mol) of L-serine, 2.5 g (0.047 mol) of ammonium chloride, and 0.04 g of pyridoxal in 862.3 g of pure water, the pH of the solution was adjusted with 25% aqueous ammonia. Was adjusted to 8.8. To the pH adjusting solution were added 335.8 g of butyl acetate and 98.8 g of phenol, and the mixture was stirred at room temperature for 30 minutes. After 30 minutes, the temperature was raised to 40 ° C., and 52.0 g (solid content 5.7 g) obtained by culturing the FERM P-11386 microorganism as a tyrosine phenol lyase-containing microorganism was charged to start the enzyme reaction. After 5 hours, the solvent was removed, and the crystals were dissolved in 35% aqueous hydrochloric acid to obtain 1401.3 g of an L-tyrosine aqueous solution. As a result of analyzing L-tyrosine by HPLC, it was 12.60 wt% and the amount of L-tyrosine was 176.7 g (0.975 mol). The yield based on L-serine was 97.5 mol%.
実施例３
純水８６２．３ｇにＬ−セリン１０５．１ｇ（１．０００ｍｏｌ）、塩化アンモニウム２．５ｇ（０．０４７ｍｏｌ）、ピリドキサル０．０４ｇを溶解させた後、２５％アンモニア水にて該溶解液のｐＨを８．８に調整した。ｐＨ調整液にオクタノール３３５．８ｇと フェノール９８．８ｇを加え３０分間室温下で攪拌した。３０分後に４０℃まで昇温し、チロシンフェノールリアーゼ含有微生物としてＦＥＲＭ Ｐ−１１３８６を培養して得られた菌体５２．０ｇ（固形分５．７ｇ）を装入した。５時間後に脱溶媒し、３５％塩酸水にて溶解しＬ−チロシン水溶液１４０６．１ｇを得た。ＨＰＬＣにてＬ−チロシンを分析した結果、１１．４０ｗｔ％、Ｌ−チロシン量は１６０．３ｇ（０．８８５ｍｏｌ）であった。Ｌ−セリンに対する収率は８８．５ｍｏｌ％で有った。
Example 3
After dissolving 105.1 g (1.000 mol) of L-serine, 2.5 g (0.047 mol) of ammonium chloride, and 0.04 g of pyridoxal in 862.3 g of pure water, the pH of the solution was adjusted with 25% aqueous ammonia. Was adjusted to 8.8. To the pH adjusting solution, 335.8 g of octanol and 98.8 g of phenol were added and stirred for 30 minutes at room temperature. After 30 minutes, the temperature was raised to 40 ° C., and 52.0 g (solid content 5.7 g) of cells obtained by culturing FERM P-11386 as a tyrosine phenol lyase-containing microorganism was charged. After 5 hours, the solvent was removed, and the residue was dissolved in 35% aqueous hydrochloric acid to obtain 1406.1 g of an aqueous L-tyrosine solution. As a result of analyzing L-tyrosine by HPLC, it was 11.40 wt% and the amount of L-tyrosine was 160.3 g (0.885 mol). The yield based on L-serine was 88.5 mol%.
実施例４
純水８６２．３５ｇにピルビン酸８８．１ｇ（１．０００ｍｏｌ）、塩化アンモニウム６４．２０ｇ（１．２００ｍｏｌ）、ピリドキサル０．０４ｇを溶解させた後、２５％アンモニア水にて該溶解液のｐＨを８．３に調整した。ｐＨ調整液にメチルイソブチルケトン３３５．８ｇと フェノール９８．８ｇを加え３０分間室温下で攪拌した。３０分後に４０℃まで昇温し、チロシンフェノールリアーゼ含有微生物としてＦＥＲＭ Ｐ−１１３８６を培養して得られた菌体５２．０ｇ（固形分５．７３ｇ）を装入し酵素反応を開始した。１０時間後に脱溶媒し、３５％塩酸水にて結晶を溶解しＬ−チロシン水溶液１４０６．１ｇを得た。ＨＰＬＣにてＬ−チロシンを分析した結果、１９．３７ｗｔ％、Ｌ−チロシン量は１５４．９ｇ（０．８５５ｍｏｌ）であった。ピルビン酸に対する収率は８５．５ｍｏｌ％で有った。
Example 4
After dissolving 88.1 g (1.000 mol) of pyruvic acid, 64.20 g (1.200 mol) of ammonium chloride and 0.04 g of pyridoxal in 862.35 g of pure water, the pH of the solution was adjusted with 25% aqueous ammonia. Adjusted to 8.3. 335.8 g of methyl isobutyl ketone and 98.8 g of phenol were added to the pH adjusting solution, and the mixture was stirred at room temperature for 30 minutes. After 30 minutes, the temperature was raised to 40 ° C., and 52.0 g (solid content: 5.73 g) of cells obtained by culturing FERM P-11386 as a tyrosine phenol lyase-containing microorganism was charged to start the enzyme reaction. After 10 hours, the solvent was removed, and the crystals were dissolved in 35% aqueous hydrochloric acid to obtain 1406.1 g of an aqueous L-tyrosine solution. As a result of analyzing L-tyrosine by HPLC, it was 19.37 wt% and the amount of L-tyrosine was 154.9 g (0.855 mol). The yield based on pyruvic acid was 85.5 mol%.
実施例５
純水８６２．３ｇにＤ−セリン１０５．１ｇ（１．０００ｍｏｌ）、塩化アンモニウム２．５ｇ（０．０４７ｍｏｌ）、ピリドキサル０．０４ｇを溶解させた後、２５％アンモニア水にて該溶解液のｐＨを８．８に調整した。ｐＨ調整液にメチルイソブチルケトン３３５．８ｇと フェノール９８．８ｇを加え３０分間室温下で攪拌した。３０分後に４０℃まで昇温してチロシンフェノールリアーゼ含有微生物としてＦＥＲＭ Ｐ−１１３８６を培養して得られた菌体５２．０ｇ（固形分５．７ｇ）を装入し酵素反応を開始した。５時間後に脱溶媒し、３５％塩酸水にて結晶を溶解しＬ−チロシン水溶液１３８６．４ｇを得た。ＨＰＬＣにてＬ−チロシンを分析した結果、１２．９３ｗｔ％、Ｌ−チロシン量は１７９．３ｇ（０．９９０ｍｏｌ）であった。D−セリンに対する収率は９９．０ｍｏｌ％で有った。
Example 5
After dissolving 105.1 g (1.000 mol) of D-serine, 2.5 g (0.047 mol) of ammonium chloride and 0.04 g of pyridoxal in 862.3 g of pure water, the pH of the solution is adjusted with 25% aqueous ammonia. Was adjusted to 8.8. 335.8 g of methyl isobutyl ketone and 98.8 g of phenol were added to the pH adjusting solution, and the mixture was stirred at room temperature for 30 minutes. After 30 minutes, the temperature was raised to 40 ° C. and 52.0 g (solid content 5.7 g) of cells obtained by culturing FERM P-11386 as a tyrosine phenol lyase-containing microorganism was charged to start the enzyme reaction. After 5 hours, the solvent was removed, and the crystals were dissolved in 35% aqueous hydrochloric acid to obtain 1386.4 g of an aqueous L-tyrosine solution. As a result of analyzing L-tyrosine by HPLC, it was 12.93 wt% and the amount of L-tyrosine was 179.3 g (0.990 mol). The yield based on D-serine was 99.0 mol%.
実施例６
純水８６．２ｇにL−セリン１０．５ｇ（０．１０００ｍｏｌ）、塩化アンモニウム０．２５ｇ（０．００５ｍｏｌ）、ピリドキサル０．００４ｇを溶解させた後、２５％アンモニア水にて該溶解液のｐＨを８．８に調整した。ｐＨ調整液にトルエン４２０．０ｇと フェノール９．９ｇを加え３０分間室温下で攪拌した。３０分後に４０℃まで昇温してチロシンフェノールリアーゼ含有微生物としてＦＥＲＭ Ｐ−１１３８６を培養して得られた菌体５．２ｇ（固形分０．５７ｇ）を装入し酵素反応を開始した。５時間後に脱溶媒し、３５％塩酸水にて結晶を溶解しＬ−チロシン水溶液１１６．９ｇを得た。ＨＰＬＣにてＬ−チロシンを分析した結果、１４．７０ｗｔ％、Ｌ−チロシン量は１７．２ｇ（０．０９５ｍｏｌ）であった。Ｌ−セリンに対する収率は９５．２ｍｏｌ％で有った。
Example 6
After dissolving 10.5 g (0.1000 mol) of L-serine, 0.25 g (0.005 mol) of ammonium chloride and 0.004 g of pyridoxal in 86.2 g of pure water, the pH of the solution with 25% ammonia water is dissolved. Was adjusted to 8.8. To the pH adjusting solution, 420.0 g of toluene and 9.9 g of phenol were added and stirred for 30 minutes at room temperature. After 30 minutes, the temperature was raised to 40 ° C., and 5.2 g (solid content: 0.57 g) obtained by culturing FERM P-11386 as a tyrosine phenol lyase-containing microorganism was charged to start the enzyme reaction. After 5 hours, the solvent was removed, and the crystals were dissolved in 35% aqueous hydrochloric acid to obtain 116.9 g of an L-tyrosine aqueous solution. As a result of analyzing L-tyrosine by HPLC, it was 14.70 wt% and the amount of L-tyrosine was 17.2 g (0.095 mol). The yield based on L-serine was 95.2 mol%.
本発明は、医薬品の原料、アミノ酸輸液、経口・経腸栄養剤の成分、健康食品などの栄養強化の分野において大量に利用されているＬ−チロシンの製造方法として有用である。 INDUSTRIAL APPLICABILITY The present invention is useful as a method for producing L-tyrosine that is used in large quantities in the field of nutrition enhancement such as pharmaceutical raw materials, amino acid infusions, oral and enteral nutrient components, and health foods.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107532184A (en) * | 2015-03-10 | 2018-01-02 | 格拉茨大学 | A kind of method for preparing 4-Vinyl phenol |
| CN109929889A (en) * | 2019-03-29 | 2019-06-25 | 安徽华恒生物科技股份有限公司 | A kind of preparation method of l-tyrosine |
| CN110541310A (en) * | 2019-09-17 | 2019-12-06 | 江南大学 | Preparation method of conductive real silk based on enzymatic conversion and catalytic oxidation |
| CN114560783A (en) * | 2022-02-22 | 2022-05-31 | 福建科宏生物工程股份有限公司 | Method for extracting L-tyrosine from conversion solution |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107532184A (en) * | 2015-03-10 | 2018-01-02 | 格拉茨大学 | A kind of method for preparing 4-Vinyl phenol |
| CN109929889A (en) * | 2019-03-29 | 2019-06-25 | 安徽华恒生物科技股份有限公司 | A kind of preparation method of l-tyrosine |
| CN110541310A (en) * | 2019-09-17 | 2019-12-06 | 江南大学 | Preparation method of conductive real silk based on enzymatic conversion and catalytic oxidation |
| CN110541310B (en) * | 2019-09-17 | 2021-08-31 | 江南大学 | Preparation method of conductive real silk based on enzymatic conversion and catalytic oxidation |
| CN114560783A (en) * | 2022-02-22 | 2022-05-31 | 福建科宏生物工程股份有限公司 | Method for extracting L-tyrosine from conversion solution |
| CN114560783B (en) * | 2022-02-22 | 2024-04-30 | 福建科宏生物工程股份有限公司 | Extraction method of L-tyrosine in conversion solution |
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